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NOVEL NANOSCALE PLATFORMS FOR

THE ISOLATION AND ULTRA-TRACE

DETECTION OF BIOACTIVE MOLECULES

Waleed Ahmed Mostafa Ahmed Hassanain

MSc Analytical Chemistry

A thesis Submitted for fulfilment of the requirements for

the degree of Doctor of Philosophy

School of Chemistry, Physics and Mechanical Engineering

Faculty of Science and Engineering

Queensland University of Technology

2018

Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules i

Keywords

Bioactive molecules, Surface enhanced Raman spectroscopy (SERS),

Electrochemical detection, Differential pulse voltammetry (DPV), Ultra-sensitive

detection, Biological fluids, Disposable substrates, Recyclable bio-nanosensors,

Molecular diagnostics, Points of care (POC), Drug metabolites, Environmental

toxins, Protein biomarkers, Sofosbuvir metabolite, Microcystin LR, Cystatin C,

Erythropoietin.

ii Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules

Abstract

The term "Bioactive molecules" describes many compounds of different

origins such as plant, animal and man-made origins. These compounds include small

drug molecules, environmental toxins and proteins. Due to their critical effects on

human life, there is an ongoing need to monitor their levels in biological fluids and

environmental samples.

This research presents proof – of – concept and basic validation study for the

development of novel functionalized nanomaterials and nanosensors for the selective

and ultra-sensitive bio-analysis of different bioactive molecules using surface

enhanced Raman spectroscopy (SERS) and electrochemical detection methods.

A disposable paper SERS substrate was synthesized for the detection of a drug

metabolite, sofosbuvir metabolite, after its chromatographic separation from blood

plasma. The developed substrate showed a good sensitivity with a quantification

limit of 50 pM. The cost-effectiveness, ease of manufacture and good sensitivity of

the new paper SERS substrate encouraged its utilization for the detection of the

environmental toxin, microcystin – LR. A recyclable gold nanomaterial was first

fabricated to selectively isolate the toxin from biological fluids. The fabricated

nanomaterial was developed by coating magnetic gold nanoparticles with target

specific antibody fragments. The developed nanomaterial showed an excellent

selectivity towards the isolation of the toxin from the biological fluids. After its

isolation, the toxin was detected by using gold coated silicon nanopillar and paper

SERS substrates. The toxin was quantified down to 10 fM using the gold coated

nanopillar substrate and a handheld Raman spectrometer. The developed method

demonstrated the potential for rapid protein nanosensing in biomedical and

environmental applications.

The metallic nature of the gold coated nanopillar substrate promoted its

utilization as a dual nanosensor for the combined SERS and electrochemical

detection of bioactive compounds. Therefore, it was used for the detection of the

protein biomarker, cystatin C, in human blood by both SERS and differential pulse

voltammetry (DPV). To selectively extract the protein from the plasma, a recyclable

2D extractor chip was functionalized with cystatin C antibody fragments. For the

first time, the protein’s own disulphide bond structure was utilized for its dual

Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules iii

nanosensing by SERS and DPV. The molecular structure of the isolated protein was

chemically reduced to break its disulfide bonds and allow its unified orientation onto

the conductive SERS substrate via formation of gold – sulphur bonds. Using this

approach, cystatin C was quantified by SERS and DPV down to 1 pM and 62.5 nM,

respectively.

The thiol chemistry of biomolecules was also utilized for the detection of

another protein biomarker, recombinant human erythropoietin (rhuEPO), in blood

plasma by a simple and direct electrochemical method. For the label-free

electrochemical quantification of the protein, it was first extracted using antibody-

functionalized magnetic beads. The isolated protein was then electrochemically

reduced by chronoamperometry and assembled onto the nanostructured gold

electrode via formation of gold – sulphur bonds. The protein was then quantified by

reductive desorption using DPV. This electrochemical reduction allowed for the

detection of the recombinant human erythropoietin down to 1 pM without signal

amplification.

This research has established novel approaches for the label-free detection of

biomolecules in biological and environmental matrices using SERS and

electrochemical methods. This research demonstrated a novel use of the thiol

chemistry of biomolecules for the detection of environmental toxins and protein

biomarkers. In addition, this research has developed new knowledge within the field

of functionalized nanomaterials and their application in analytical science for the

detection of bioactive molecules. The nanosensors and methodologies developed in

this research show strong potential for the rapid and recyclable determination of

bioactive molecules in the field and at pathology labs.

iv Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules

List of Publications and Presentations

Published journal articles

1- W.A. Hassanain, E.L. Izake, A. Sivanesan, G.A. Ayoko, Towards interference

free HPLC-SERS for the trace analysis of drug metabolites in biological fluids,

Journal of Pharmaceutical and Biomedical Analysis, 2017, 136, 38-43. (IF. 2.83, Q1)

2- W.A. Hassanain, E.L. Izake, M.S. Schmidt, G.A. Ayoko, Gold nanomaterials for

the selective capturing and SERS diagnosis of toxins in aqueous and biological

fluids, Biosensors and Bioelectronics, 2017, 91, 664-672. (IF. 8.17, Q1)

3- W.A. Hassanain, E.L. Izake, G.A. Ayoko, Spectroelectrochemical Nanosensor for

the Determination of Cystatin C in Human Blood, Analytical Chemistry, 2018, 90,

10843-10850. (IF 6.04, Q1)

4- W.A. Hassanain, A. Sivanesan, E.L. Izake, G.A. Ayoko, An electrochemical

biosensor for the rapid detection of erythropoietin in blood, Talanta, 2018, 189, 636-

640. (IF. 4.24, Q1)

Presentations

1- SERS Detection of environmental toxin in Plasma by Smart Nanomaterials, the

international conference of bionano innovation (ICBNI 2017), 24-27 September

2017, Brisbane, Australia, ORAL.

2- Novel nanosensors for the combined isolation and detection of proteins, RACI

centenary congress 2017, 23-28 July 2017, Melbourne, Australia, ORAL.

3- Detection of Sofosbuvir Metabolite in Biological Samples by HPLC-SERS method,

the 1st Queensland annual chemistry symposium (QACS 2016), 25 November 2016,

Brisbane, Australia, ORAL.

4- Rapid Determination of Sofosbuvir Active Metabolite in Plasma by Paper-Based

HPLC-SERS Method, the 2016 RACI analytical and environmental division national

symposium (RACI Anachem 2016), 18-20 July 2016, Adelaide, Australia, POSTER.

5- SERS detection of bio-active molecules in body fluids, the 3rd annual

nanotechnology and molecular science HDR symposium at Queensland University of

Technology, 16-17 February 2016, Brisbane, Australia, ORAL.

Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules v

Table of Contents

Keywords..............................................................................................................................i

Abstract ...............................................................................................................................ii

List of Publications ............................................................................................................. iv

List of Figures ..................................................................................................................... ix

List of Tables ..................................................................................................................... xii

List of Abbreviations.........................................................................................................xiii

Statement of Original Authorship ....................................................................................... xv

Aknowledgements............................................................................................................. xvi

Significance of the Study ................................................................................................. xvii

Thesis Structure ................................................................................................................ xix

Chapter 1: Introduction and literature review ...................................................... 1

1.1. Introduction ................................................................................................................... 1

1.2. Background of Raman spectroscopy .............................................................................. 3

1.3. Theoritical background of SERS.................................................................................... 4

1.4. SERS substrates ............................................................................................................ 6

1.5. Types of SERS .............................................................................................................. 8

1.6. SERS applications ....................................................................................................... 10

1.6.1. Protein analysis...................................................................................................... 10

1.6.2. Pharmaceuticals analysis........................................................................................ 13

1.7. Electrochemical nanosensors for biomedical applications ............................................ 16

1.7.1. Electrochemical sensing and biosensors ................................................................. 16

1.7.2. Types of electrochemical biosensors ...................................................................... 18

1.7.3. Electrochemical detection applications ................................................................... 18

1.7.4. Combined optical-electrochemical methods for the detection of bioactive

molecules........................................................................................................................ 20

1.8. Concluding remarks and outlook ................................................................................. 23

1.9. Research objectives ..................................................................................................... 25

1.10. Biactive molecules involved in this research .............................................................. 27

1.10.1. Drug metabolite (sofosbuvir metabolite) .............................................................. 27

1.10.2. Environmental toxin (microcystin LR) ................................................................. 28

1.10.3. Protein biomarkers (cystatin C and erythropoietin) ............................................... 28

Chapter 2: Disposable nanosensor for the detection of drug metabolite in blood

plasma ................................................................................................................... 30

Statement of Contribution of Co-Authors for Thesis by Published Paper ............................ 31

2.1. Preface ........................................................................................................................ 32

vi Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules

2.2. Abstract ...................................................................................................................... 33

2.3. Keywords.................................................................................................................... 33

2.4. Graphical abstract ....................................................................................................... 34

2.5. Introduction.. .............................................................................................................. 35

2.6. Materials and methods ................................................................................................ 37

2.6.1. Chemicals and materials ........................................................................................ 37

2.6.2. Preparation of standard solution and spiked plasma samples .................................. 37

2.6.3. Characterization of PSI-6206 by direct Raman spectroscopy .................................. 38

2.6.4. Determination of PSI-6206 in blood plasma by surface enhanced Raman spectroscopy (SERS) ....................................................................................................... 38

2.6.5. Determination of PSI-6206 in blood plasma by HPLC-DAD.................................. 38

2.6.6. Detection of PSI-6206 by LC-MS .......................................................................... 39

2.6.7. Instrumentation ..................................................................................................... 39

2.7. Results and discussion ................................................................................................. 41

2.7.1. Raman spectrometry of PSI-6206 .......................................................................... 41

2.7.2. SERS measurements of PSI-6206 on gold nanopillars substrate ............................. 43

2.7.3. Paper-based substrate for the determination of PSI-6206 by SERS ......................... 43

2.7.4. Quantification of PSI-6206 by paper-based SERS substrate ................................... 45

2.7.5. Determination of PSI-6206 in spiked plasma samples by HPLC-DAD, HPLC-SERS and HPLC-MS ................................................................................................................ 46

2.8. Conclusions ................................................................................................................ 50

2.9. Supplementary material ............................................................................................... 51

2.9.1. Synthesis of gold nanoparticles .............................................................................. 51

2.9.2. Manufacture of paper SERS substrate .................................................................... 51

Chapter 3: Recyclable functionalized nanomaterials for SERS detection of

environmental toxin in biological samples ........................................................... 55


3.1. Preface ........................................................................................................................ 57

3.2. Abstract ...................................................................................................................... 58

3.3. Keywords.................................................................................................................... 58

3.4. Graphical abstract ....................................................................................................... 59

3.5. Introduction.. .............................................................................................................. 60

3.6. Material and methods .................................................................................................. 63

3.6.1. Chemicals and reagents ......................................................................................... 63

3.6.2. Preparation of MC-LR standard solutions .............................................................. 63

3.6.3. Detection of MC-LR by SERS ............................................................................... 64

3.6.4. Preparation of functionalized gold coated magnetic nanoparticles .......................... 64

3.6.5. Selective capture of MC-LR from blood plasma and SERS monitoring of antibody-antigen interaction ........................................................................................................... 65

Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules vii

3.6.6. Direct SERS detection of purified MC-LR and cross-validation by ELISA ............. 66

3.6.7. Manufacture of paper-based SERS substrate .......................................................... 66


3.7.1. Synthesis of MC-LR antibody Fab' fragments and surface functionalization of gold

coated magnetic nanoparticles ......................................................................................... 68

3.7.2. SERS spectrum of MC-LR..................................................................................... 68

3.7.3. Selective capture of MC-LR and SERS monitoring of the toxin binding to the

functionalized nanoparticles............................................................................................. 71

3.7.4. Direct SERS detection of MC-LR in aqueous solution ........................................... 73

3.7.5. SERS quantification of MC-LR ............................................................................. 75

3.7.6. Direct SERS Detection of MC-LR in blood plasma and cross-validation against

ELISA. ............................................................................................................................ 76

3.7.7. Direct SERS detection of MC-LR in spiked blood plasma by paper based substrate and handheld Raman spectrometer ................................................................................... 77

3.7.8. Reproducibility of the SERS measurements by the paper-based substrate ............... 77

3.7.9. Recycling of functionalized nanoparticles .............................................................. 77

3.8. Conclusion .................................................................................................................. 79

3.9. Supplementary material ............................................................................................... 80

Chapter 4: Dual biosensing of protein biomarkers in human blood by recyclable

plasmonic probes .................................................................................................. 86


4.1. Preface ........................................................................................................................ 88

4.2. Abstract ....................................................................................................................... 89

4.3. Keywords .................................................................................................................... 89

4.4. Graphical abstract ........................................................................................................ 90

4.5. Introduction ................................................................................................................. 91

4.6. Materials and methods ................................................................................................. 93

4.6.1. Synthesis of antibody fragments and functionalization of the extractor chip ........... 93

4.6.2. Isolation and purification of CST-C from blood plasma.......................................... 93

4.6.3. Control tests .......................................................................................................... 93

4.6.4. Determination of CST-C in human blood plasma by SERS and DPV ..................... 94

4.6.5. Recycle of functionalized extractor chip by cyclic voltammetry (CV) .................... 94


4.7.1. SERS spectrum of authentic and reduced CST-C ................................................... 96

4.7.2. Manufacture of CST-C extractor chip .................................................................... 99

4.7.3. Isolation of CST-C from blood plasma and control tests ......................................... 99

4.7.4. SERS quantification of CST-C ............................................................................. 101

4.7.5. Electrochemical quantification of CST-C ............................................................. 102

4.7.6. Determination of CST-C in human plasma ........................................................... 104

viii Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules

4.7.7. Recycle of the extractor chip ............................................................................... 105

4.8. Conclusion ................................................................................................................ 107

4.9. Supplementary material ............................................................................................. 108

4.9.1. Instrumentation ................................................................................................... 108

4.9.2. Chemicals and reagents ....................................................................................... 108

4.9.3. Preparation of CST-C standard solution ............................................................... 109

4.9.4. SERS spectra of authentic and reduced CST-C protein ........................................ 109

4.9.5. SERS quantification of reduced CST-C ............................................................... 109

4.9.6. Electrochemical quantification of reduced CST-C by DPV .................................. 109

4.9.7. Cross-validation by ELISA .................................................................................. 110

Chapter 5: Electrochemical pathway for rapid and label-free detection of

protein biomarkers in blood ................................................................................ 117

Statement of Contribution of Co-Authors for Thesis by Published Paper .......................... 118

5.1. Preface ...................................................................................................................... 119

5.2. Abstract .................................................................................................................... 120

5.3. Keywords.................................................................................................................. 120

5.4. Graphical abstract ..................................................................................................... 121

5.5. Introduction.. ............................................................................................................ 122

5.6. Materials and methods .............................................................................................. 124

5.6.1. Chemicals and materials ...................................................................................... 124

5.6.2. Instruments.......................................................................................................... 124

5.6.3. Preparation of rhuEPO standard solutions ............................................................ 125

5.6.4. Preparation of gold nanostructured electrode for DPV detection........................... 125

5.6.5. Synthesis of antibody-conjugated magnetic beads ................................................ 125

5.6.6. Selective extraction of rhuEPO ............................................................................ 125

5.6.7. Electrochemical reduction and quantification of rhuEPO ..................................... 126

5.6.8. ELISA cross-validation ....................................................................................... 126

5.7. Results and discussion ............................................................................................... 127

5.7.1. Selective extraction of rhuEPO from blood plasma .............................................. 127

5.7.2. Electrochemical reduction and assembly of rhuEPO on nanostructured gold

electrode ....................................................................................................................... 127

5.7.3. DPV quantification of rhuEPO in blood plasma and cross-validation by ELISA ... 130

5.8. Conclusion.................................................................................................................... . 132

5.9. Supplementary material ............................................................................................. 133

Chapter 6: Conclusion and future work ............................................................. 136

Appendix ........................................................................................................................ 141

References ...................................................................................................................... 142

Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules ix

List of Figures

Figure 1.1: Energy level diagram of different possibilities of light scattering ....................... 3

Figure 1.2: SERS enhancement diagram for an analyte within metal nanostructures hot

spots .................................................................................................................................... 4

Figure 1.3: Schematic representation of (a) preparation of paper SERS substrate, (b) gold

coated silicon nanopillar SERS substrate design ................................................................... 7

Figure 1.4: Illustration of different types of SERS detection. (a) Direct SERS detection of

the analyte, (b) indirect SERS detection of the analyte by monitoring the SERS signal of

Raman label ......................................................................................................................... 8

Figure 1.5: Schematic representation for the design of electrochemical biosensor integrated

with analyte recognition molecules ..................................................................................... 17

Figure 1.6: Molecular structures of PSI-6206, MC-LR, CST-C and rhuEPO with the key

spectral Raman bands of PSI-6206, MC-LR and CST-C ..................................................... 27

Figure 2.1: Structural formula of PSI-6206 ........................................................................ 41

Figure 2.2: (a) Raman spectrum of standard PSI-6206 by Renishaw Invia Raman

microscope, (b) Raman spectrum of standard PSI-6206 by handheld ID Raman mini

spectrometer ...................................................................................................................... 42

Figure 2.3: (a) SERS spectrum of standard PSI-6206 on gold coated silicon nanopillars

substrate, (b) SERS spectrum of standard PSI-6206 on paper SERS substrate ..................... 44

Figure 2.4: (a) HPLC chromatogram of 7.5x10−5 M PSI-6206 in methanol, (b) HPLC chromatogram of control plasma sample, (c) HPLC chromatogram of 5x10−6 M PSI-6206

spiked plasma sample. ........................................................................................................ 47

Figure 2.5: SERS spectrum of PSI-6206 after HPLC separation from spiked horse

plasma ............................................................................................................................... 48

Figure 2.6: (a) MS fragmentation of PSI-6206 standard after chromatographic separation,

(b) MS fragmentation of PSI-6206 in spiked blood plasma after chromatographic

separation........................................................................................................................... 49

Figure S2.1: UV spectrum of standard PSI-6206 in methanol............................................. 52

Figure S2.2: UV spectrum of gold nanoparticles colloid (SERS ink) .................................. 52

Figure S2.3: SEM image of (a) blank white A4 paper, (b) A4 paper after coating with gold nanoparticles, (c) magnification of deposited nanoparticles on paper substrate after coating

with gold nanoparticles ...................................................................................................... 53

Figure S2.4: Raman spectra of A4 paper before and after coating with gold nanoparticles

(SERS ink) ......................................................................................................................... 53

Figure 3.1: SERS spectra of standard MC-LR on gold coated silicon nanopillar substrate

using (a) Renishaw Raman microscope and (b) handheld Raman spectrometer ................... 69

Figure 3.2: SERS spectra of the functionalized gold coated magnetic nanoparticles, (a) before interaction with MC-LR and (b) after the capture of MC-LR. The spectra were

collected using the InVia Raman spectrometer ................................................................... 72

Figure 3.3: SERS spectra of (a) MC-LR on gold coated nanopillar substrate by the InVia Raman microscope, (b) by the handheld Raman spectrometer. The green spectrum represents

a blank water sample (negative control) and the red spectrum represents a spiked milk

sample (positive control) .................................................................................................... 74

x Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules

Figure 3.4: (a) The relationship between log the concentration of MC-LR and the SERS

intensity at 1441 cm-1, (b) the change in the Raman signal intensity at 1441 cm-1 with the

concentration of MC-LR .................................................................................................... 75

Figure 3.5: SERS spectrum of MC-LR after extraction from spiked horse blood plasma

sample using gold coated silicon nanopillar substrate and handheld Raman spectrometer ... 76

Figure 3.6: SERS spectra of (a) standard MC-LR, (b) MC-LR after extraction from spiked horse blood plasma sample, (c) Nine SERS measurements of MC-LR from spiked blood

plasma, (d) MC-LR after extraction from a second spiked sample by the recycled

nanoparticles. The spectra were collected using paper-SERS substrate and handheld Raman

spectrometer ...................................................................................................................... 78

Figure S3.1: Structure of MC-LR ...................................................................................... 80

Figure S3.2: SERS spectrum of intact anti MC-LR antibody on SERS nanopillar substrate

using handheld Raman spectrometer .................................................................................. 81

Figure S3.3: SERS spectrum of antibody F(ab')2 fragments on SERS nanopillar substrate

using handheld Raman spectrometer .................................................................................. 81

Figure S3.4: ELISA measurements and calibration curve of MC-LR ................................. 82

Figure S3.5: UV spectrum of nanoparticle ink used for the preparation of paper-based SERS

substrate ............................................................................................................................ 82

Figure S3.6: Images of (a) the nanoparticle colloid, (b) SERS ink, (c) squares of A4 paper

after coating with SERS ink ............................................................................................... 83

Figure S3.7: SEM images of (a) paper before coating, (b) nanoparticles on A4 paper

substrate, (c) A4 paper after coating with gold nanoparticles .............................................. 83

Figure S3.8: SERS spectra of the functionalized gold coated magnetic nanoparticles, (a) before interaction with MC-LR and (b) after the capture of MC-LR. The spectra were

collected using the handheld Raman spectrometer .............................................................. 84

Figure 4.1: Raman spectra of (a) standard CST-C (the red line depicts the Raman spectrum of the bare substrate), (b) different scans of standard CST-C, (c) CST-C standard after

reduction with TCEP, and (d) 17 measurements of seventeen reduced CST-C samples in the

concentration range 10-7 to 10-12 M on 17 independent gold coated silicon nanopillar

substrates.. ......................................................................................................................... 97

Figure 4.2: Raman spectra of (i) extracted and reduced CST-C from human blood plasma,

(ii) positive control, (iii) negative control, (iv) insulin, and (v) phenylalanine on gold coated

silicon nanopillar substrate ............................................................................................... 100

Figure 4.3: (a) Raman band intensity of reduced CST-C at 1363 cm-1 in the concentration

range of 100 nM to 1 pM, and (b) SERS calibration curve of reduced CST-C within the same

concentration range .......................................................................................................... 101

Figure 4.4: (a) Electrochemical desorption of CST-C by DPV (the black arrow depicts the

desorption potential of CST-C at -0.92 V. The red line depicts the DPV of a blank substrate),

(b) DPV pulse of reduced CST-C in the concentration range 0.0625 μm to 1 μm, and (c)

electrochemical calibration curve of reduced CST-C by DPV ........................................... 103

Figure 4.5: Raman spectra of (i) CST-C antibody fragments on the extractor chip, (ii)

extractor chip after electrochemical desorption of antibody fragments (after 130 CV cycles),

and (iii) fresh CST-C antibody fragments on recycled extractor chip after the electrochemical

treatment (4.5 ii) .............................................................................................................. 106

Figure S4.1: Raman spectrum of CST-C by the InVia Raman microscope ....................... 113

Figure S4.2: SEM image of commercial silicon nanopillar SERS substrate ...................... 113

Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules xi

Figure S4.3: Chemisorption of reduced CST-C onto gold coated silicon nanopillar substrate

via Au-S bond .................................................................................................................. 114

Figure S4.4: ELISA calibration curve of CST-C .............................................................. 114

Figure S4.5: Raman spectra of CST-C antibody fragments on extractor chip (a) after 30 CV

cycles, (b) after 60 CV cycles, and (c) after 90 CV cycles................................................. 115

Figure S4.6: (a) Electrochemical desorption of the CST-C antibody fragments from the surface of the extractor chip by cyclic voltammetry (the black arrow depicts the desorption

potential of the antibody fragments at -0.8 V), and (b) voltammogram of un-functionalized

gold-coated silicon nanopillar chip ................................................................................... 115

Figure 5.1: DPV of rhuEPO on a polished gold electrode (blue line) and on a nanostructured

gold electrode (red line) ................................................................................................... 128

Figure 5.2: DPV of reduced EPO on a nanostructured gold electrode, the red dotted line

denoted the blank nanostructured electrode ...................................................................... 129

Figure 5.3: (a) DPV of reduced rhuEPO on nanostructured gold electrode in the

concentration range 1 – 1000 pM, (b) corresponding calibration curve in the same

concentration range. The inset depicts the linear relationship in the concentration range 1–

250 pM ............................................................................................................................ 130

Figure S5.1: The electrochemical cell assembly ............................................................... 133

Figure S5.2: ELISA calibration curve of rhuEPO ............................................................. 133

Figure S5.3: DPV current of reduced rhuEPO at -1.19 V after 10, 20, 25, 30 and 35 minutes

of chronoamperometry reduction ...................................................................................... 134

Figure A.1: SERS spectra of different concentrations of PSI-6206 on paper SERS substrate

using handheld Raman spectrometer................................................................................. 134

xii Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules

List of Tables

Table 3.1: Band assignment of the SERS spectra of MC-LR (the * symbol denotes the SERS

bands detected by the handheld Raman spectrometer) ........................................................ 70

Table 3.2: Comparison of various analytical methods for the detection of MC-LR ............. 75

Table S4.1: Band assignment of the Raman spectrum of CST-C on gold coated silicon

nanopillar substrate .......................................................................................................... 111

Table S4.2: Comparison of different methods used for the detection of CST-C ................ 112

Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules xiii

List of Abbreviations

2D Two dimensional

3D Three dimensional

Ag Silver

Au Gold

Au-S Gold–sulphur bond

BSA Bovine serum albumin

CRP C-reactive protein

CST-C Cystatin C

CV Cyclic voltammetry

DNA Deoxyribonucleic acid

DPV Differential pulse voltammetry

EF Enhancement factor

ELISA Enzyme linked immunoassay

Fab Fragment antigen-binding

FDA Food and Drug Administration

HCV Hepatitis C virus

HOMO Highest occupied molecular orbital

HPLC High performance liquid chromatography

DAD Diode array detection

IgG Immunoglobulin G

LC-MS Liquid chromatography-mass spectroscopy

LIF Laser induced fluorescence

LOD Limit of detection

LOQ Limit of quantification

LSPR Localized surface plasmon resonance

LUMO Lowest unoccupied molecular orbital

MC-LR Microcystin LR

MRI Magnetic resonance imaging

NIR Near-infrared

NMR Nuclear Magnetic Resonance

OD Optical density

PBS Phosphate buffered saline

PCR Polymerase chain reaction

xiv Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules

PENIA Particle-enhanced nephelometric immunoassay

PETIA Particle enhanced turbidimetric immunoassay

PIS Product ion scan

POC Point of care

PPI assay Phosphatase inhibition assay

PSI-6206 Sofosbuvir metabolite

RNA Ribonucleic acid

ROS Raster orbital scanning

RSD Relative standard deviation

SEM Scanning electron microscope

SERS Surface enhanced Raman spectroscopy

SF Sofosbuvir

SH Sulfhydryl

SPR Surface plasmon resonance

TCEP Tris(2-carboxyethyl) phosphine

TLC Thin liquid chromatography

UV/Vis Ultra violet/visible

WHO World health organization

Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules xv

Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet

requirements for an award at this or any other higher education institution. To the

best of my knowledge and belief, the thesis contains no material previously

published or written by another person except where due reference is made.

Signature:

Date: 23-12-2018

QUT Verified Signature

xvi Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules

Acknowledgements

Firstly, all praise be to ALLAH, the Almighty, with whose gracious help it was

possible to accomplish this work. It was a long and tough journey and I am feeling

grateful to God to reach this step in my life.

I would like to express my deep appreciations to my principal supervisor, Dr. Emad

Kiriakous. I am truly grateful for his prudent supervision, valuable advices and

continuous support during my candidature. His constructive comments and

suggestions through the experimental work and thesis writing have been invaluable.

Also, I wish to express my gratitude to my associate supervisor Prof. Godwin Ayoko

for his support, kind guidance and fruitful criticism. Many thanks for all the help

towards the completion of my research.

I would like also to thank Queensland University of Technology (QUT) for granting

me the QUT Postgraduate Research Award (QUTPRA) scholarship and QUT

International HDR tuition fee sponsorship. I would like also to acknowledge the

generous funding from the Science and Engineering Faculty at QUT to support the

access to the Central Analytical Research Facility (CARF) at QUT.

I am also grateful for the all assistance and knowledge that I received from the QUT

technical and support staff. In addition, I wish deeply to thank my colleagues in the

Raman research group for their continuous help, productive cooperation and

sympathetic feelings.

Finally, I dedicate this thesis to my parents, my wife and my beloved kids. Mum and

dad; your love, motivation, encouragement and non-stopping prayers kept me going

despite the many difficulties I faced. My darling wife and friend, Amira; all of this

wouldn’t be done without you. You kept pushing me forward and you sacrificed with

many things to keep me up when I was down. Really, words can’t satisfy what you

did for me.

Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules xvii

Significance of the Study

The ultra-trace detection of bioactive molecules within biological fluids has a

significant impact on a range of applications including clinical diagnosis and

environmental monitoring. The current routine analysis protocol of bioactive

molecules includes: liquid chromatography – mass spectrometry (LC-MS),

electrophoresis, polymerase chain reaction (PCR) and immunoassay detection

methods such as ELISA. These techniques are highly reliable and widely acceptable.

However, they are still challenging due to time consuming, costly instrumentation

and the requirement of highly trained operators. Therefore, the development of rapid,

sensitive and cheap detection method will positively impact human life quality,

particularly in terms of health, environmental and financial impacts.

This research demonstrates novel, sensitive, selective and recyclable

nanosensors that can be used for the combined isolation and detection of bioactive

molecules in complex biological matrices. The developed nanosensors were

combined with portable, label-free SERS and electrochemical detection modes.

These combined platforms have the potential to enhance sensitive, selective,

recyclable and in-field analysis of bioactive molecules in a rapid way. The developed

platforms can be easily adapted to target any biomolecule, provided a suitable

recognition molecule or separation method is available.

In terms of sensitivity, sample handling, analysis time and cost of analysis, the

presented novel nanosensing methodologies should have significant applications in

pharmaceutical analysis, environmental monitoring, analytical toxicology and

molecular diagnostics. The presented work is multidisciplinary, so it would be of

interest to a wide section of the scientific community in sensors, material science

applications, Raman spectroscopy, electrochemistry and analytical chemistry.

xviii Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules

Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules xix

Thesis Structure

Novel Nanoscale Platforms for the Isolation and Ultra-Trace

Detection of Bioactive Molecules

Chapter 1

Introduction and literature review

Chapter 2

Disposable nanosensor for the detection of drug

metabolites in blood plasma Towards interference free HPLC-SERS for the trace analysis

of drug metabolites in biological fluids, Journal of Pharmaceutical and Biomedical Analysis, 2017, 136, 38-43

Chapter 3

Recyclable functionalized nanomaterials for SERS

detection of environmental toxin in biological

samples Gold nanomaterials for the selective capturing and SERS

diagnosis of toxins in aqueous and biological fluids, Biosensors and Bioelectronics, 2017, 91, 664-672

Chapter 4

Dual biosensing of protein biomarkers in human

blood by recyclable plasmonic probes Spectroelectrochemical Nanosensor for the Determination

of Cystatin C in Human Blood, Analytical Chemistry, 2018, 90, 10843-10850

Chapter 5

Electrochemical pathway for rapid and label-free

detection of protein biomarkers in blood An electrochemical biosensor for the rapid detection of erythropoietin in blood, Talanta, 2018, 189, 636-640

Chapter 6

Conclusion and future work

xx Novel Nanoscale Platforms For The Isolation and Ultra-Trace Detection of Bioactive Molecules

Introduction and literature review 1

Chapter 1: Introduction and literature

review

1.1. Introduction

Bioactive molecules include wide range of compounds that have biological

effects on the human body, from small molecules such as glucose, amino acids and

pharmaceuticals to larger biomolecules as proteins, lipids and DNA. They play a

vital role in the diagnosis, monitoring and control of many diseases that can affect

human health. Therefore, it is very important to develop a quick and reliable

technique for the in vivo, in vitro and/or ex vivo detection of bioactive molecules.

Enzyme-linked immunoassay (ELISA) [1], western blot [2], fluorescence [3], flow

cytometry [4], mass spectrometry [5] and electrophoresis [6] are well established

bioanalytical techniques and have been commonly used for the detection of bioactive

molecules. Polymerase chain reaction (PCR) is being also utilized as a first step for

the amplification of the target analytes (DNA, protein) prior to their detection by

various methods [7]. However, these methods suffer from unsatisfactory limitations

such as: low sensitivity, sophisticated lengthy protocols, high cost of analysis and a

high risk of contaminations [8].

Surface enhanced Raman spectroscopy (SERS) is a powerful technique that

can be used for the ultra-sensitive detection of bioactive molecules. It offers high

detection sensitivity with good specificity, as well as multiplexing ability [9]. After

four decades of development, SERS has now become one of the most valuable

methods for the ultra-trace detection and analysis of bioactive compounds in research

and practical applications [8, 10]. The distinct advantages of SERS over the other

analytical techniques are:

(1) Its ability to achieve ultra-sensitive detection for the target analytes [11].

(2) Its capacity to provide rich spectral information on the molecular structure of the

analyte [12-13].

Despite its high sensitivity, SERS has some drawbacks, such as:

(1) The lack of selectivity [14-16] as SERS cannot be used independently for the

2 Introduction and literature review

selective detection of a target analyte in a mixture contains other interfering

molecules.

(2) SERS variability, in term of band positions and signal intensity [17-18]. This

effect can be attributed, in part, to the random adsorption and different orientations of

the analyte molecules onto the SERS substrate [19].

(3) The spontaneous fluorescence radiation can mask the SERS signals [20].

SERS can be combined with other analytical techniques for the confirmatory

analysis of compounds. This is favoured for bioanalytical practices such as the

determination of diagnostic protein biomarkers in biological fluids. Electrochemical

methods have already been applied for the detection of bioactive compounds. This is

due to their simplicity, relative cost-effectiveness, reasonable sensitivity and capacity

for miniaturization in portable analytical platforms [21-22]. In addition, there are

many sub-modes of detection that can provide great quantitative information about

the analyte such as amperometry [23] potentiometry [24] and voltammetry [25].

Since SERS substrates are usually fabricated using materials that have a conductive

property (e.g. gold and silver) [26], they can also be used as a combined platform for

the Raman and electrochemical detection of biomolecules

This chapter will aim to introduce SERS theory with a special focus on

biochemical and biomedical applications of SERS. In addition, we also briefly

discuss the electrochemical detection of such molecules.


1.2. Background of Raman spectroscopy

Raman scattering was first reported by Raman and Krishnan in 1928 [27]. It is

an inelastic scattering of photons that exploits the fact that individual bonds give rise

to unique vibrations, resulting in molecularly specific spectra [27]. When photons of

incident light interact with an analyte, a portion of their energy is adsorbed and

causes the excitation of the analyte molecules to a virtual state. The majority of the

excited molecules release back the energy and relax to their original ground state

(Rayleigh scattering, Figure 1.1).

A very small number of the excited molecules relax back to a different ground /

virtual state, thus giving Raman radiation. Therefore, Raman is considered as weak

and inelastic scattering [28]. In the 1970s, Fleischmann et al. [29] reported strong

Raman spectra of pyridine molecule that was adsorbed onto a silver electrode. This

phenomenon was further investigated by Jeanmaire and Van Duyne [30], where it

was found that the adsorption of molecules onto a roughened noble metal surface can

significantly enhance the Raman signal intensity by orders of magnitude. This was

attributed to electromagnetic and chemical enhancement mechanisms and established

the technique that is now known as SERS.

Figure 1.1: Energy level diagram of different possibilities of light scattering.


1.3. Theoretical background of SERS

SERS is the sensitive mode of Raman spectroscopy and it can be used for the

ultrasensitive detection of the target analytes. The exact mechanism of SERS is not

fully understood. However, the electromagnetic enhancement is believed to play a

major role in the Raman signal enhancement observed in SERS [31]. The

nanostructures of a plasmonic metal surface exhibit unique optical properties where

the surface electrons of the metal couple with the photons of incident light thus

forming a quasi-particle called the surface polariton (SP). This quasi-particle

oscillates at a frequency that is referred to as the localized surface plasmon resonance

(LSPR). When analyte molecules are adsorbed on a nanostructured plasmonic

surface and become excited with incident light, the emerging Raman photons couple

to the LSPR of the nanostructured plasmonic surface [32]. This results in a massive

increase (106-1014) of the Raman signal intensity [12, 33]. The Raman signal

enhancement by the large electromagnetic fields in the small gaps between

plasmonic nanostructures, known as hotspots (Figure 1.2), allows SERS to detect

ultra-low concentration of analytes [34-35]. The hotspots are commonly found in the

gaps between nanoparticle aggregates. Therefore, when two nanoparticles are

brought close enough to each other, they can create a large SERS enhancement in the

gap and exhibits extremely high electromagnetic field enhancement.

Figure 1.2: SERS enhancement diagram for an analyte within metal nanostructure

hot spots.


The most commonly used SERS active metals are silver and gold, although

copper, palladium, or platinum can be also used. The magnitude of the SPR effect is

influenced strongly by the size, surface structure, shape, and proximity to other

SERS-active surfaces.

The Raman signals can also be enhanced by the formation of charge transfer

complexes between the roughened metal surface and the analyte molecules. This

mode of enhancement is known as chemical enhancement and is analyte dependent,

whereby the polarizability change that leads to an enhanced Raman signal relies

upon the adsorption process [36].

Consequently, SERS enhancement can be considered as a joint effect of

electromagnetic and chemical enhancement. However, the electromagnetic

enhancement is the predominant contributor to the observed enhancement on

metallic surfaces.


1.4. SERS substrates

In order to amplify the SERS signal of the analyte, a sensitive SERS substrate

should be used as a platform to adsorb the sample. The ideal substrates for SERS

should provide (1) high signal enhancement, (2) reproducible and uniform response,

and (3) can be fabricated using cost-effective methods [37-38]. Coinage metals such

as gold and silver are usually used to fabricate nanostructured SERS substrates due to

their capacity to provide a high SERS enhancement factor. The surface plasmon

resonance absorption bands of these metals lie within the visible region and,

therefore, they can be excited by readily available lasers. Silver nanostructures give

strong SERS signals. However, they can be unstable due to their rapid oxidation by

the surrounding environment [39-40]. Gold nanostructures are inert and have high

stability. In addition, they can be easily functionalized and/or bio-conjugated using

established reactions [41]. Furthermore, the gold nanostructures support LSPR in the

near-infrared (NIR) and visible regions which make gold substrates suitable for

SERS measurements by NIR lasers where the fluorescence background is minimized.

Within the last decade, there has been rapid progress in the development of

new SERS substrates for the detection of different analytes including proteins, DNA,

toxins, bacteria, viruses, cells and pharmaceuticals [42]. Generally, SERS substrates

can be classified to (a) 3D colloidal nano structures and, (b) 2D solid substrates.

(a) 3D colloidal nanostructures

These substrates can be easily synthesised by multiple chemical methods such

as the citrate reduction method [43-46]. The aggregation of colloidal plasmonic

nanostructures causes their surface plasmon to couple and lead to strong local

electromagnetic field enactment [43, 47]. In addition, the 3D geometry of colloidal

nanostructures allows for their efficient interaction with target analytes [46, 48].

(b) 2D solid substrates

These substrates are manufactured using two main approaches. The first

approach utilizes wet chemistry methods to synthesis colloidal metallic

nanostructures that are then deposited onto the substrate surface [49-50]. This

approach produces substrates that have non-patterned geometry such as the paper-

based substrate which usually has random distribution of plasmonic nanostructures

(Figure 1.3a). Despite being simple and inexpensive, this approach requires elaborate


design and careful operation. The second approach utilizes lithographic and/or ion

etching methods where the nanostructures are directly fabricated onto the solid

substrate [51-52]. This approach produces highly patterned and uniform

nanostructure assemblies onto the surface of the substrates. However, it requires

expensive instrumentation and trained operators. Gold coated silicon nanopillar

SERS substrate is an example of this type (vide infra), where the substrate is

fabricated by using reactive ion etching process to obtain patterned nanostructures of

high aspect ratios distributed homogeneously on a fused silica wafer (Figure 1.3b).

Paper-based substrates have been developed as cost-effective 2D SERS

substrate [53-54]. The paper SERS substrate exhibits excellent sensitivity that is

comparable to other 2D substrates [55]. However, the reproducibility of the SERS

signal still needs further optimization. 2D SERS substrates have been also used as

sensing elements in microfluidic devices to develop SERS detecting systems for the

rapid trace-level biological and environmental analysis [56-57].

Figure 1.3: Schematic representation of (a) preparation of paper SERS substrate, (b)

gold coated silicon nanopillar SERS substrate design.


1.5. Types of SERS

Generally, the SERS detection of biomolecules can be classified into two main

categories, (1) label-free (direct) detection and (2) labelled (indirect) detection. In the

label-free method, the bioactive molecule is brought to the enhanced electromagnetic

field of plasmonic nanostructures resulting in SERS signals of the analyte itself

(Figure 1.4a). This method takes the full advantage of the fingerprint information of

Raman spectroscopy over the other method. This approach assumes that only a single

compound exists in the sample matrix. However, in complex matrices that contain

multiple components, the non-selective adsorption of these compounds onto the

SERS substrate would cause the Raman fingerprint of the target analyte to be

obscured by the Raman signals contribution from other compounds. Therefore,

extensive isolation and clean up procedures are required prior to the direct SERS

detection of the target analyte.

Figure 1.4: Illustration of different types of SERS detection. (a) Direct SERS

detection of the analyte, (b) indirect SERS detection of the analyte by monitoring the

SERS signal of the Raman label.


The labelled method involves modification of the metal surface with an

external Raman label through the use of aggregating agents and/or a recognition

layer to create hot spots. After coupling the sample to the surface, the Raman

intensity of the label will change. Hence, this platform is used to indirectly detect the

target analyte by monitoring the alteration in the SERS signal intensity of the

conjugated Raman label (Figure 1.4b). This method is used commonly for the

detection of biomolecules in molecular diagnosis and biomedical applications and it

has achieved much lower detection limits than fluorometric methods [58]. However,

this method does not detect the target analyte itself, but depicts the SERS spectrum

of the Raman label.

4-mercaptobenzoic acid [59], 4-nitrobenzenethiol [60], p-aminothiophenol [61]

and malachite green isothiocyanate [62] are among the most commonly used Raman

labels in SERS applications. The ideal Raman reporter should:

(1) Maintain high stability during tagging and/or SERS measurement.

(2) Produce a consistent and robust Raman spectrum under the measurements

conditions.

Compared to labelled SERS method, label-free SERS detection procedure is

much more simple and straightforward. However, it requires a highly efficient SERS

substrate and strong nanostructures – analyte binding.


1.6. SERS applications

SERS has been widely used for the detection of different bioactive molecules,

such as: proteins, DNA, viruses, cells and toxins [8-9]. SERS has also been used for

the detection and monitoring of pharmaceuticals levels [63]. This section will outline

a summary of the recent SERS bioanalytical sensing methods that are alreday used

for the detection of proteins and pharmaceuticals.

1.6.1. Protein analysis

The accurate monitoring of protein biomarker levels in biological systems is

mandatory for the clinical diagnosis of many medical conditions. In addition, it is

essential to understand the role of these proteins in the genetic modifications and

cellular, physiological and pathological changes that are associated with diseases.

Many methods have been developed for the detection of proteins, such as: ELISA,

mass spectroscopy, electrophoresis and polymerase chain reaction (PCR) [64-67].

SERS can be considered a promising alternative to these techniques for protein

analysis due to its high sensitivity, multiplexing ability, capability of rapid

measurements and ability to be integrated in small packages for measurements in the

field or at the point of care. There are numerous SERS methods that have been

reported for protein detection. These methods can be sub-classified into two main

categories (1) label-free (direct) detection and (2) labelled (indirect) [9].

(a) Label-free SERS detection of proteins

In this method the target protein molecules are directly adsorbed onto the

surface of the substrate and screened by SERS without the use of a Raman probe.

A novel SERS method has been developed for the monitoring of colorectal

cancer progression. The SERS spectra of serum protein from cancer patients were

compared with those of healthy volunteers. The accuracy of the obtained results was

assessed using chemometrics methods [68]. A novel drop-coating deposition SERS

diagnostic method was performed for the early detection of adenoviral conjunctivitis

in human tears using a portable Raman spectrometer. The method used trace sample

volume (2 µL) and showed high SERS spectra reproducibility [69]. C-reactive

protein (CRP), the infection and inflammation biological biomarker, was detected

directly by a label-free method using silver nanoparticles functionalized with

phosphocholine-terminated monolayer [70]. The functionalized nanoparticles


selectively captured CRP and enhanced the SERS intensity of the protein. The short

distance between the nanoparticles and CRP (less than 4 nm) was responsible for the

sensitive limit of detection (100 fM).

An effective strategy to enhance the sensitivity in SERS measurements is to

induce nanoparticles aggregation in the presence of the target analyte. Kahraman et

al. [71] applied this approach for the label-free SERS detection of six proteins,

namely; bovine serum albumin, haemoglobin, thrombin, avidin, cytochrome c, and

lysozyme. Their method depended on the self-assembly and aggregation of

negatively charged Ag nanoparticles in the presence of the target proteins. The same

group also used 2D substrates that were patterned with Cr and Ag nanostructures to

create nanovoids for the detection of the same proteins [72]. Using this approach,

they were able to detect 0.05 µg/mL of the target proteins. A recent label-free SERS

method for the detection of bovine serum albumin (50 nM) and lysozyme (100 nM)

in liquid was reported by Fazio et al. [73]. The method used radiation to form gold

aggregates for the direct detection of purified proteins by SERS.

Other label-free SERS methods monitored the Raman shifts in the Raman

spectrum of target-specific plasmonic nanostructures upon binding with the target

proteins [74-75].

(b) Labelled SERS detection of proteins

In this method, a Raman probe is used to label the target protein and the SERS

spectrum of the Raman probe is monitored for the detection of the protein molecules.

This approach allows for highly sensitive SERS measurements of proteins and very

useful for monitoring protein interactions.

An on/off SERS aggregation system has been designed to investigate the

interaction between lactose-functionalized nanoparticles and lectin protein ConA in

the presence of benzotriazole as a Raman probe. The functionalized nanoparticles

have four binding sites for efficient multiple interaction with protein molecules. The

aggregation of the Ag nanoparticles occurs upon binding the target protein and leads

to an increase in the SERS signal intensity of the Raman probe. This led to a very

low detection limit down to 40 pM [76]. Based on the same nanoparticles assembly

approach, Robson et al. [77] developed a method for the detection of mouse double

minute protein. The method used silver nanoparticles that were functionalized with


the peptide mimic of the tumour suppression protein to enhance the nanoparticles

aggregation in the presence of benzotriazole dye.

A labelled SERS method for the detection of cholera toxin B-subunit (CTB)

from synthetic freshwater sample was recently developed by Simpson et al. [78]

where they used functionalized silver glyconanoparticles that aggregate upon their

interaction with CTB. The aggregation leads to an increase in the signal intensity of

the Raman probe that is attached to the nanoparticles surface. Using their

functionalized and Raman-tagged nanoparticles, the authors reported detection limit

of 56 ng/mL for CTB.

SERS immunoassay was first demonstrated for the detection of proteins in

1999 [79]. To create selectivity in SERS immunoassay, target-specific recognition

molecules are usually immobilized onto a nanostructured substrate and used for the

selective extraction of target analyte from complex matrices such as biological fluids.

Due to their high affinity towards their target analyte, antibodies and aptamers are

usually used as recognition molecules in SERS immunoassay. The nanostructured

substrate may be functionalized with a Raman probe that is used to detect the binding

of the target protein by SERS. Alternatively, the change in the Raman fingerprint of

the recognition molecules can be monitored by SERS to indicate the binding of the

target analyte. In a third scenario, the captured protein molecules can be released

from the functionalized nanostructures and directly detected by SERS without the

use of a Raman label.

SERS-based immunoassay methods have been utilized for the molecular

diagnosis of many protein biomarkers. For example, prostate cancer biomarker

(PSA) was evaluated in biological fluids down to 10-9 ng/mL using antibody-

functionalized Ag nanoparticles [80]. This method relies on the aggregation of silver

nanoparticles which allows for multistage signal amplifications. Zhou et al. [81]

applied a sandwich-type SERS immunoassay for the sensitive detection of PSA (1.79

fg/mL), AFP (0.46 fg/mL) and CA19-9 (1.3x10-3 U/mL) in human blood serum. In

this method, antibody-functionalized immunosensor and silicon nanoparticles were

used to form a sandwich complex in the presence of the target protein. This led to the

immobilization of the functionalized silver nanoparticles onto the immunosensor

substrate and, in effect, a significant increase of the intensity of their SERS signal.

Another sandwich-type immunoassay was developed for the detection of the tumor


marker VEGF (vascular endothelial growth factor) using DNA aptamers [82]. The

detection limit of the method (1 pg/mL) was higher by 2-3 orders of magnitudes than

the conventional ELISA. Wu et al. [83] designed aptamer–Ag–Au nanostructures for

the specific detection of breast cancer biomarker MUC1 in human cells using

rhodamine 6G as a Raman label. The resulting nanostructures were also utilized for

the photo thermal therapy of breast cancer due to their high capacity to adsorb NIR

irradiation and re-transmit it as heat thus causing the death of the cancer cells.

One of the significant advantages of SERS is its ability to perform a

simultaneous detection of multiple protein biomarkers in mixtures. The sharp Raman

bands make SERS the ideal technique for the multiplex detection. Xu et al. [84]

reported a labelled sensitive multiplex assay for three different protein biomarkers;

PSA, thrombin and mucin-1 using silver pyramids and DNA aptamers as recognition

molecules. Upon their interaction with the target antigens, an alteration in the 3D

geometry of the silver pyramids takes place and enhances the Raman signal intensity

which leads to ultrasensitive multiplexed SERS detection of the biomarkers down to

attomolar levels. In another immunoassay, SERS and 3D barcode chip have been

used in a microfluidic platform for the multiplexed and high-throughput analysis of

IgG molecules from human, mouse and rabbit sources [85]. In a recent study, Alpha-

fetoprotein (AFP) and carcinoembryonic antigen (CEA) tumor markers have been

detected by dual encoding in a multiplex bioassay [86]. In this method, photonic

crystal carrier beads and SERS nanotags were used for the dual encoding of antigens.

The reported limit of detection was 9.2 pg/mL.

1.6.2. Pharmaceuticals analysis

The rapid and sensitive determination of pharmaceuticals in formulations and

biological matrices is of significant value for the pharmaceutical industry and for the

therapeutic drug monitoring. The use of SERS in the pharmaceuticals analysis would

complement the currently used methods such as gas chromatography and mass

spectrometry which are expensive and time-consuming. Considering its many

advantages, SERS has become a valuable analytical tool in pharmaceuticals research.

The high sensitivity of the SERS technique also qualifies it for quality control

screening in the pharmaceutical industry, such as: the detection of trace impurities in

drug formulations [87].


SERS has been applied for the detection of drug metabolites, illicit substances,

and doping agents [88-91]. For example, Wu and Cunningham [92] designed a

highly sensitive in-line SERS active plasmonic nanodome array (SERS enhancement

factor of 8.51x107) for the real-time monitoring of the chemical contents of

intravenous (IV) fluids. Paper SERS substrate was also applied in other applications

as a sensitive and cost-effective substrate for the detection of pharmaceuticals in

serum [93] and illicit drugs [94].

To overcome the inherent non-selectivity within SERS, it has been coupled

with chromatographic separation methods for the detection of pharmaceuticals in

mixtures and biological samples. A TLC-SERS method was developed for the

detection of four anti-diabetic drugs used as adulterants in botanical dietary

supplements [95]. TLC plates were used for the separation of the compounds. The

separated zones were then sprayed with silver colloid and screened by SERS within

the concentration range 0.0005 to 0.1 μg/mL. Hidi et al. [96] reported the use of lab

on a chip with SERS (LoC-SERS) for the detection of the antibiotic levofloxacin in

human urine using a portable Raman system. The analysis results demonstrated the

robustness of the new bioanalytical platform for urine analysis in clinical

applications. SERS was also combined with multiplicative effects model for the

quantitative analysis of antihypertension drug, captopril, in blood plasma samples

[97]. The multiplicative effects model was used to address the heterogeneity in the

physical properties of the enhancing substrate. The method had detection and

quantifications limits of 0.149 and 0.451 μM, respectively, and was cross-validated

against LC-MS.

Andreou et al. [98] designed a microfluidic device for the detection of a potent

central nervous system stimulant compound, methamphetamine, in saliva at

concentration 10 nM using SERS. In this method, the device was used to allow the

analyte to adsorb onto silver nanoparticles, and salt ions are introduced to cause the

silver nanoparticles to aggregate, thus creating species with strong SERS signal. In

another study, methamphetamine was quantified in human urine using new 3D SERS

hotspots with an emulsion of silver nanoparticles. The compound was separated from

urine within 5 minutes using cyclohexane extraction. The developed SERS platform

showed a detection limit of 10 ppb [99]. Recently, SERS was used to distinguish

between a set of structurally similar synthetic cannabinoids in biological fluids. In


this method, the analytes were mixed with gold nanoparticles prepared in alkali or

alkaline earth salt solutions. The salts enhance the aggregation of the gold

nanoparticles and so enhance the SERS signal. The reported detection limit was 18

ng/mL [100].

In a recent study, SERS was combined with multivariate data analysis in order

to develop on-site detection method for the β-blocker agent, propranolol, in human

biological fluids [101]. A more recent study developed multiplex SERS detection

method for fluoroquinolone and its metabolites (enrofloxacin and ciprofloxacin) with

a limit of quantification in ppm level [102]. In this study, periodic metallic

nanostructures were fabricated using laser interference lithography and used as SERS

substrates with less than 1% variation.


1.7. Electrochemical nanosensors for biomedical applications

1.7.1. Electrochemical sensing and biosensors

Electrochemical sensing techniques have the potential for the rapid and

sensitive detection of bioactive molecules relevant to the diagnosis and treatment of

different clinical conditions. There are numerous electrochemical sub-methods that

can be used with different electrochemical biosensors to improve the sensitivity of

the detection. The most common techniques include differential pulse voltammetry,

cyclic voltammetry, chronoamperometry, chronopotentiometry and electrochemical

impedance spectroscopy.

Electrochemical biosensors have attracted a considerable attention as an

analytical tool for the detection of different biomolecules. They have the ability to

convert the biological events to an electronic signal output. Currently, many

electrochemical biosensors are used for the sensitive detection of large variety of

biomolecules including biological fluids, food samples, cell cultures and

environmental samples [103-104].

The use of nanomaterials, such as: metallic nanostructures, metal oxide

semiconductors and carbon nanotubes/nanosheets, in the manufacture of the

electrochemical biosensors has improved their conductivity, biocompatibility and

high sensitivity [105-107]. In addition to the sensitivity, the advance in the

manufacturing technology allowed for the development of user friendly - portable

electrochemical systems which make the technique suitable for POC applications

[107]. The significant advances in the construction of functional electrode materials

coupled with numerous electrochemical detection methods have broadened the use of

the technique in biochemical and biomedical applications [108].

One of the limitation that hinders the use of electrochemical sensors is their

lack of selectivity. This limitation was addressed in research by the advance in the

integration of target-specific recognition molecules (antibody fragments, aptamers,

enzymes, receptors, lectins) onto the transducer surface within the sensors to develop

highly specific electrochemical biosensors and enhance their selective detection

towards the target analyte (Figure 1.5). Electrochemical biosensors have also some

limitations, such as: the effect of the change in the temperature and pH of biological

fluids on the biosensors’ response [109]. The sample temperature should be in a


narrow or limited range during the analysis which is practically unsuitable especially

for in-field analysis.

Figure 1.5: Schematic representation for the design of electrochemical biosensor

integrated with analyte recognition molecules.

Generally, the ideal electrochemical biosensor should have the following

properties:

(1) Highly sensitive, cheap, portable and user-friendly.


(2) Highly selective to the target analyte.

(3) Stable under normal storage condition with low variation between assays.

(4) Non-toxic and biocompatible.

1.7.2. Types of electrochemical biosensors

Electrochemical biosensors can be categorized into four main types:

(a) Voltammetric / amperometric biosensors

Voltammetric and amperometric biosensors apply a potential to the working

electrode to measure the electrochemical current versus the reference electrode. The

current is developed either by electrochemical oxidation or reduction events at the

working electrode and is dependent on the transport rate of the reactant molecules

from the solution to the electrode interface [110].

(b) Impedimetric biosensors

Electrochemical impedance spectroscopy biosensors are used to measure the

resistance properties of the molecules when applying a small amplitude AC

excitation signal (typically of 2–10 mV). The frequency is changed over a wide

range to obtain an impedance spectrum [110].

(c) Conductometric biosensors

Conductometric biosensors are used to measure the changes in the electrical

conductivity of the analyte solution due to the change in the solution composition

during the reaction [110].

(d) Potentiometric biosensors

Potentiometric biosensors are used to measure the electrochemical potential of

an electrochemical cell while no or negligible current is present [110].

1.7.3. Electrochemical detection applications

Electrochemical methods have been extensively described before for the detection of

different bioactive molecules using different electrochemical techniques with various

designs of the used biosensors [105]. In this section, we report some electrochemical

applications for the detection of proteins and pharmaceuticals.


(a) Protein analysis

Electrochemical biosensors for protein detection have shown significant

advantages compared to traditional techniques in clinical laboratory diagnosis.

Different fabrication and signal amplification strategies have been designed for fast

quantitative detection of proteins with high sensitivity and selectivity.

A portable electrochemical aptasensor was designed for the rapid and direct

detection of hepatocellular exosomes [111]. The developed method incorporated

expanded nucleotide-containing aptamer into a DNA nanotetrahedron structure for

the sensitive electrochemical detection of cancerous exosomes using square wave

voltammetry. The tetrahedrally oriented immobilized aptamers improved the

electrochemical detection sensitivity and capturing performance of the exosomes.

The reported limit of detection was 2.09x104 / mL. Another selective amperometric

method for the ultrasensitive detection of cancer biomarkers proteins (PSA and

PSMA) in serum was described by Sharafeldin et al. [112]. In this method,

Fe3O4 nanoparticles were assembled onto graphene oxide nanosheets and coated with

selective antibodies to extract the target protein biomarkers from serum. This was

followed by ultrasensitive electrochemical detection using the intrinsic peroxidase

activity. The reported limits of detection were 15 and 4 fg/mL for PSA and PSMA,

respectively.

A recent method described the design of dual-modality electrochemical

biosensor for the simultaneous detection of two cancer biomarkers in human serum

by differential pulse voltammetry [113]. This biosensor was developed by coating

graphene oxide / single strand DNA onto gold electrode for VEGF detection, and

incorporated with poly-L-lactide nanoparticles for signal amplification and PSA

detection. The detection limits for the VEGF and PSA were 50 pg/mL and 1 ng/mL,

respectively. Another selective recognition molecule for the sensitive

electrochemical detection of protein biomarker was used by Ribeiro et al. [114] for

the detection of cardiac biomarker myoglobin in serum with high specificity. In this

method, a molecularly imprinted polymer (MIP) -synthetic protein receptor-

biosensor was designed by electropolymerization of phenol, via cyclic voltammetry,

onto a gold screen printed electrode in the presence of the analyte. The reported limit

of detection using square wave voltammetry was 14 pg/mL.

https://www.sciencedirect.com/topics/chemistry/cyclic-voltammetry


(b) Pharmaceuticals analysis

The high sensitivity and relatively low-cost has made electrochemical

biosensors a suitable alternative for the high cost methods of pharmaceuticals

analysis, especially for monitoring the pharmaceuticals’ therapeutic window.

A gold nanocluster was incorporated into a glassy carbon electrode modified

with a 3-amino-5-mercapto-1,2,4-triazole film and used for the simultaneous DPV

detection of dopamine, ascorbic acid, uric acid and nitrite in spiked urine and serum

samples [115]. The developed nanosensor showed high electrocatalytic activities

towards the oxidation of these molecules. The reported limits of detection were 1.1

µM, 50 nM, 80 nM and 0.89 µM, respectively. Another nanosensor was developed

for the determination of the anti-HIV drug, deferiprone, in spiked serum and urine

samples down to 0.005 µM [116]. To develop the nanosensor, gold nanorods were

deposited onto pencil graphite electrode and used for the drug detection by

impedimetric technique in a short response time (within 15 seconds).

A recent method has been described for the determination of anti-cancer drug,

flutamide, in spiked serum sample using graphene oxide modified glassy carbon

electrode as an electrochemical sensor [117]. The modified glassy carbon electrode

was used to study the electrochemical performance of flutamide by cyclic

voltammetry and linear sweep voltammetry. The electrochemical sensor exhibited

strong electrocatalytic activity towards the reduction of flutamide with a limit of

detection of 6 nM. Another recent electrode design was described by Mahmoud et al.

[118] for the determination of acetaminophen in the presence of its common

interference isoniazid in human serum, urine, saliva, and tablet samples. In this

method, hydrothermal synthesis of bismuth oxide nanorods was developed and cast

onto disposable graphite screen-printed electrodes. This design allowed for

ultrasensitive detection of acetaminophen and isoniazid with detection limits of 30

nM and 1.85 μM, respectively.

1.7.4. Combined optical – electrochemical methods for the detection of bioactive

molecules

The electrochemical methods can be combined with other techniques such as

optical and chromatographic techniques for the simultaneous and confirmatory

detection of an analyte [119-120]. The use of optical sensing techniques as SERS


with electrochemical techniques can allow for real-time, non-destructive and label-

free analysis of biomolecules [121]. Applying such combinations in biochemical and

biomedical applications can provide additional information for the complex

biosystems and make full use of both techniques advantage, improved selectivity and

enhanced the limit of detection.

The use of conductive nanostructured substrates allows for the dual spectro and

electrochemical detection of analytes, thus providing molecular structure

identification and rapid quantification of analytes [122-123]. Therefore, a metallic

nanostructured SERS substrate can be used as a dual nanosensor for the spectro-

electrochemical detection of the analytes.

The application of dual SERS-electrochemical platform offers some

advantages over the use of single technique. For example, (1) the shared high

sensitivity of both techniques can provide a richer set of initial data in addition to the

benefit of increased control over the sensing environment. It can provide spectral

information about the molecular structure of the analyte beside the complementary

rapid quantification. (2) The dual assay offers a confirmatory and accurate analysis

results with a wide detection range, over either technique indecently, which is useful

in the detection of different blood biomarkers at POC units.

The use of combined SERS and electrochemical biosensors in biomedical

applications has been reported in the literature. A SERS method has been developed

for the discrimination between different DNA mutations. The method used an

electrochemical electrode to apply a negative potential that denatures the DNA

double strand. The changes caused to the DNA were monitored by SERS [124].

Another biosensor was developed for screening the chemotherapeutic drug,

doxorubicin, and to aid in the assessment of DNA modification after interaction with

the drug using SERS and electrochemical methods [125]. The sensor was designed

by coating a gold-disk electrode with a reduced graphene oxide monolayer and

coating it with magnetic gold nanoparticles. The electrode was then functionalized

with the double-stranded DNA sequence of the breast cancer gene BRCA1. The

biosensor was then exposed to the drug and the modification that occurred within the

DNA probe was correlated to the dose of the drug.

Another application for the use of dual SERS-electrochemical modes in the

drugs detection was presented by Bailey et al. [26] for the detection of riboflavin


(vitamin B2). They used hydrodynamic focusing to load riboflavin onto a SERS

electrode. The vitamin was then screened by SERS and amperometry down to 1 and

100 nM, respectively. A simultaneous immunoassay for lung cancer protein

biomarkers, carcinoembryonic antigen (CEA) and cytokeratin-19 (CK-19), was

developed using a combined electrochemical and SERS immunoassay [126]. The

method used resin microspheres decorated with a Raman dye which shows both

strong SERS signals and electrochemical redox characteristic peaks upon its

interaction with the target protein biomarkers. The reported limits of detection were

0.01 ng/mL and 0.04 ng/mL for CEA and CK-19, respectively.


1.8. Concluding remarks and outlook

SERS offers many advantages over the other techniques for the detection of

bioactive molecules. These include sensitivity, structural identification, minimal

sample preparation, potential miniaturization and multiplex analysis. SERS can also

be combined with other detection techniques as fluorescence, SPR, MRI and

electrochemistry for the multimodal detection of bioactive molecules. The

development of new sensitive SERS substrates paves the way for the detection

bioactive molecules at the ultra-trace level. To address the inherent non-selectivity in

SERS, the substrate should be functionalized with target specific molecules that only

bind the target analyte. To maximize the selectivity of the substrate, it should also be

backfilled with a passive monolayer to prevent non-specific binding of interfering

molecules.

The use of SERS for biomedical applications still faces some challenges that

need to be overcome before it can advance from a proof – of – concept research to

real life applications. Significant efforts are still needed to adapt SERS to the POC

analysis of biomolecules. The ideal SERS platform for POC should be recyclable,

rapid, and easily miniaturized into portable analytical platform. Most importantly, the

low reproducibility in SERS measurements must be addressed. This can be achieved

by immobilizing the analyte molecules in a unified orientation onto sensitive SERS

substrate that have high and uniform density of hotspots. In addition, efficient SERS

immunoassay platforms should take the advantage of the conductive property of the

SERS sensor to allow for the multimodal determination of biomolecules by Raman

and electrochemical methods.

In addition to SERS, electrochemical biosensors are becoming increasingly

important for the detection of different bioactive molecules due to their rapid

response, small sample volume requirement and potential for miniaturization into

POC devices. Despite the recent advances in the design of electrochemical

biosensors, achieving sensitivity levels comparable to that of optical sensors is still

challenging. This limitation can be addressed by the use of derivatization reagents

and nanomaterials to enhance the sensitivity of the electrochemical sensing. In

addition, the manufacture of sensitive and selective biosensors requires multiple

expensive recognition molecules and complex signal amplification protocols to

detect the target analyte at low concentration levels. Therefore, more researches are


still required for the development of simple, selective, sensitive and cost-effective

biosensors for biomedical and biochemical applications.


1.9. Research objectives

This research aims to develop novel nanoscale platforms for the isolation and

ultra-trace detection of bioactive molecules by label-free SERS and electrochemical

methods. In order to fulfil this aim, we will undertake the following steps:

(1) Develop and utilize novel cost-effective nanostructured substrates for the SERS

detection of bioactive molecules such as drug metabolites, environmental toxins and

protein biomarkers in biological fluids.

(2) Develop a novel approach for the fabrication of recyclable target-specific

extractor nanomaterials, using antibodies and/or their fragments as selective

recognition molecules, for the selective extraction of proteins and environmental

toxins from blood plasma.

(3) Address the problem of signal irreproducibility in SERS by developing a novel

approach for the oriented immobilization of biomolecules through the modification

of their thiol chemistry.

(4) Develop and cross-validate a label-free approach for the dual SERS and

electrochemical nanosensing of bioactive molecules in biological fluids.

As indicated above, the prime aim of this research is to establish a proof – of –

concept on novel label-free optical-electrochemical nanosensing techniques for the

sensitive and selective detection of bioactive molecules (drug metabolite,

environmental toxin and protein biomarkers) in environmental and biological

matrices. For this purpose, this research will develop recyclable selective

nanomaterials for the selective extraction of the target bioactive molecules from

complex matrices. The research will also utilize dual-function substrates for the

ultra-sensitive SERS/DPV detection of the extracted analytes. In this study,

chromatographic and immunoassay methods will be used to cross-validate the

developed nanosensing methodologies. Due to the high cost of the commercial

substrates, a full validation study would be beyond the scope of this proof – of –

concept research. Therefore, the LOD, LOQ, uncertainty, confidence intervals for the

slope and intercept, % error between measurements, day to day and batch to batch

RSD measurements of the developed nanosensing methods will be only investigated

in this work. The new nanosensing approaches would have a significant application


in pharmaceutical analysis, environmental monitoring, analytical toxicology and

molecular diagnostics.

The experimental work in this research is outlined in the following steps:

(1) A cost-effective gold nanostructured paper-based SERS nanosensor will be

fabricated and combined with chromatographic separation technique for the detection

of the drug metabolite in blood plasma. The developed SERS substrate will be

characterized by UV and SEM scans and its SERS enhancement factor will be

quantified.

(2) Functionalized nanomaterials will be developed for the rapid and selective

extraction of the environmental toxin and protein biomarkers from complex matrices.

This will be achieved by the oriented immobilization of target-specific antibody

fragments, as recognition molecules, onto gold nanomaterials.

(3) To ensure selectivity toward the target analyte, any bare sites on the

functionalized nanomaterials surface will be backfilled with alkanethiol layer to

prevent non-specific binding by other interfering molecules.

(4) After capturing the target analyte, the functionalized nanomaterials will be treated

with a suitable buffer to release and retrieve the isolated biomolecules analyte from

the extractor nanomaterials.

(5) The functionalized nanomaterials will be regenerated using chemical and

electrochemical recycling methods for repeated use.

(6) For the environmental toxin, the purified biomolecule will be loaded onto a

plasmonic substrate and screened by SERS for label-free detection and

quantification.

(7) For the protein biomarkers analysis, the purified protein molecules structure will

be chemically and/or electrochemically modified for their oriented immobilization

onto a conductive and plasmonic substrate for dual sensing by SERS and DPV.

(8) The screening results will be cross-validated against chromatographic and/or

ELISA methods.

(9) To utilize the new nanosensing methodology for field analysis, handheld Raman

spectrometer will be utilized for the SERS detection of the nominated analytes in

blood plasma.


1.10. Bioactive molecules involved in this research

In this research we applied the proposed nanosensing approach for the

detection of three different categories of bioactive molecules in biological samples;

(1) drug metabolite sofosbuvir metabolite (PSI-6206), (2) environmental toxin

microcystin LR (MC-LR) and (3) protein biomarkers cystatin C (CST-C) and

recombinant human erythropoietin (rhuEPO). The molecular structures of theses

bioactive molecules and the key spectral Raman bands of PSI-6206, MC-LR and

CST-C are depicted in Figure 1.6.

Figure 1.6: Molecular structures of PSI-6206, MC-LR, CST-C and rhuEPO with the

key spectral Raman bands of PSI-6206, MC-LR and CST-C.

1.10.1. Drug metabolite (sofosbuvir metabolite)

Sofosbuvir is a highly effective drug against hepatitis C viral infection.

Sofosbuvir undergoes metabolism and phosphorylation inside liver cells by enzymes

to form a metabolite which is the active form of the drug. This metabolite affects the

viral replication step by terminating the growth of the virus RNA chain.


1.10.2. Environmental toxin (microcystin LR)

Microcystin LR is an environmental toxin that can contaminate water and food.

It can cause severe liver damage, gastroenteritis, malignant liver tumors and impair

vital immune response. The World Health Organization has recommended a

concentration of 1 nM as a cut-off limit for microcystin-LR in the drinking water.

1.10.3. Protein biomarkers (cystatin C and recombinant human erythropoietin)

Cystatin C is an important biomarker for renal function and Alzheimer’s

disease. Also, it can be used as a useful biomarker for cancer and type-2 diabetes.

Erythropoietin is a naturally occurring hormone that is used for the regulation of

erythropoiesis and as a signalling protein in the production of red blood cells in bone

marrow. Sports doping with exogenous erythropoietin increases oxygen capacity and

leads to enhanced aerobic performance by athletes.

30 Disposable nanosensor for the detection of drug metabolites in blood plasma

Chapter 2: Disposable nanosensor for the

detection of drug metabolites in

blood plasma

This chapter is made up of the following journal article published in the Journal of

Pharmaceutical and Biomedical Analysis.

Towards interference free HPLC-SERS for the trace analysis of drug

metabolites in biological fluids

Waleed A. Hassanain, Emad L. Izake, Arumugam Sivanesan, Godwin A. Ayoko

DOI: 10.1016/j.jpba.2016.12.019

https://www.sciencedirect.com/science/article/pii/S0731708516307555?via%3Dihub

Disposable nanosensor for the detection of drug metabolites in blood plasma 31

Statement of Contribution of Co-Authors for

Thesis by Published Paper

The following is the suggested format for the required declaration provided at the

start of any thesis chapter which includes a co-authored publication.

The authors listed below have certified that:

1. They meet the criteria for authorship in that they have participated in the conception,

execution, or interpretation, of at least that part of the publication in their field of

expertise;

2. They take public responsibility for their part of the publication, except for the

responsible author who accepts overall responsibility for the publication;

3. There are no other authors of the publication according to these criteria;

4. Potential conflicts of interest have been disclosed to (a) granting bodies, (b) the

editor or publisher of journals or other publications, and (c) the head of the

responsible academic unit, and

5. They agree to the use of the publication in the student’s thesis and its

publication on the QUT’s ePrints site consistent with any limitations set by

publisher requirements.

In the case of this chapter:

W.A. Hassanain, E.L. Izake, A. Sivanesan, G.A. Ayoko, Towards interference free HPLC-

SERS for the trace analysis of drug metabolites in biological fluids, Journal of Pharmaceutical and Biomedical Analysis, 2017, 136, 38-43.

Principal Supervisor Confirmation

I have sighted email or other correspondence from all Co-authors confirming their certifying

authorship.

Emad Kiriakous 23-12-2018

Name Signature Date

Contributor Statement of contribution*

Waleed A. Hassanain

Conducted experiments, data analysis, and primary

manuscript authorship. Date 23-12-2018

Emad L. Izake Supervision and major editing.

Arumugam Sivanesan Contributed to research discussions.

Godwin A. Ayoko Contributed to research discussions and editing.



http://www.mopp.qut.edu.au/F/F_01_03.jsp


2.1. Preface

In this chapter, a sensitive, cost-effective and disposable handmade paper

substrate was fabricated and utilized for the quantification of sofosbuvir metabolite

for the first time in blood plasma sample by SERS. The compound was first

separated from the plasma by an HPLC method. The eluted compound was then

deposited onto the paper-based substrate and detected directly by SERS using a

handheld Raman spectrometer.

The at-line HPLC – SERS approach allowed for the elimination of the memory

effect which can cause false detection result. The developed at-line HPLC – paper

SERS platform with the handheld Raman spectrometer present great potential for the

sensitive, low cost and in-field detection of drugs and their metabolites in biological

fluids.

The method was cross validated by LC-MS and the main predominant

protonated precursor [M + H]+ ion at 261.1 m/z of the eluted drug matched that of

the standard drug that reported by literature and manufacturer analysis certificate.


2.2. Abstract

Sofosbuvir metabolite, 2'-deoxy-2'-fluoro-2'-C-methyluridine (PSI-6206) was

studied for the first time by surface enhanced Raman spectroscopy (SERS) using the

paper-based SERS substrate. The quantification limit of PSI-6206 by SERS was

found to be 13 ng L-1 (R2 value = 0.959, RSD =5.23%). For the structural and

quantitative analysis of PSI-6206 in blood plasma, an interference-free HPLC-SERS

method was developed and compared to HPLC-DAD and HPLC-MS methods. The

SERS quantification of the drug by the paper substrate was 4 orders of magnitude

more sensitive than that by the diode array detector. In addition, the SERS detection

provided unique structural identification of the drug in blood plasma, similar to Mass

spectroscopy detector. Due to the disposable nature of the SERS substrate, the new

method does not suffer from the known “memory effect” which is known to lead to

false positive identification in traditional HPLC-SERS methods. Therefore, the

presented HPLC-paper SERS platform holds great potential for the sensitive and

cost-effective determination of drugs and their metabolites in biological fluids.

2.3. Keywords

Paper SERS substrate, HPLC-SERS, Biological fluids, PSI-6206 nucleoside,

Infectious diseases.


2.4. Graphical abstract


2.5. Introduction

Chromatographic methods are widely used for the determination of drug

metabolites in biological samples [127]. HPLC methods usually require the

utilization of UV and/or mass spectrometric (MS) detection. The major disadvantage

of HPLC-UV even with diode array detection (DAD) is its limitation to give

comprehensive structural information and the situation becomes even worse when

analytes co-eluted [128]. Mass spectrometry provides confirmatory structural

information for the accurate identification of the separated compounds. However, the

instrumentation for mass detection are expensive and of large footprint. In addition,

the use of mass detectors is limited by the choice of compatible solvents and suitable

chromatographic parameters.

Within the last decade, surface-enhanced Raman spectroscopy (SERS) has

strongly developed into a highly sensitive tool for the detection of Raman-active

molecules down to the single molecule level [129-130]. SERS can provide rich

structural information for definitive analyte identification similar to Infra-red and

Mass spectroscopy [130]. In SERS, a nanostructured rough metal surface is used as

the substrate. When incident light of a proper wavelength interacts with the surface

plasmon of the metal, a large enhancement of the local electromagnetic field occurs

at the gapped metal regions, creating hot spots [131]. When an analyte molecule is

trapped within or near the hot spots, its Raman vibrations become greatly enhanced.

In addition, the analyte molecule may form a charge transfer complex with the metal

surface. The formation of a charge transfer complex leads to chemical enhancement

and contributes to the enhancement of the Raman spectrum.

Many researchers attempted to interface SERS with chromatographic

separation. Coupling SERS with thin layer chromatography (TLC) suffers from

adsorption-induced spectral distortions of Raman spectra due to the interaction of the

analytes with the silica gel TLC substrate [129, 132]. The spectral distortions lead to

a significant decrease of the signal resolution and compromise the quality of the

spectral identification. SERS was also used as a detector for in HPLC analysis [133-

134]. HPLC-SERS suffers from interference that is caused by the memory effect.

This effect occurs when colloidal nanoparticles are used as the SERS substrate for

detection where the deposition of contaminated nanoparticles onto the inner walls of

the flow cell leads to false positive identifications [133-134]. In addition, the use of


colloidal nanoparticles leads to poor reproducibility of the SERS spectra due to the

uncontrolled aggregation of the nanoparticles [51]. To eliminate the memory effect

and the subsequent false identification in HPLC-SERS, a disposable SERS substrate

is required [135]. The substrate should have high sensitivity, long shelf life and can

be manufactured by low cost synthesis process.

In this work, we developed SERS substrate on A4 paper and used it for the

HPLC-SERS detection of 2'-deoxy-2'-fluoro-2'-C-methyluridine (PSI-6206) [136],

the active metabolite of the hepatitis C drug sofosbuvir (SF). Hepatitis C virus

(HCV) infection is a very dangerous disease. About 2% to 3% of the world’s

population is infected with HCV. Every year more than 350,000 patients die due to

HCV-related conditions, including cirrhosis and hepatocellular carcinoma [137]. SF

is highly effective against HCV genotypes 1-4 by inhibiting non-structural 5B

polymerase [137]. SF undergoes metabolism and phosphorylation inside liver cells

by the human cathepsin A and/or carboxylesterase 1 to form PSI-6206 triphosphate,

which affects the viral replication step by terminating the growing of HCV RNA

chain [138]. In June 2016, PSI-6206 has been indicated as a promising lead candidate

for further development of specific antivirals against Zika virus [139]. Therefore, the

rapid, sensitive and simple determination of PSI-6206 in biological fluids is

important to clinical pharmacology and pharmacokinetic studies as well as the

pharmaceutical analysis of sofosbuvir [140-141]. PSI-6206 has never been studied by

Raman spectroscopy and its detection by HPLC-SERS has not been demonstrated.

Herein, we report, for the first time, the Raman spectroscopy of PSI-6206 and

demonstrate a highly sensitive detection method for its determination in blood

plasma by HPLC-SERS and handheld Raman spectrometer.


2.6. Materials and methods

2.6.1. Chemicals and materials

Hydrogen tetrachloroaurate (HAuCl4), trisodium citrate dihydrate, methanol,

acetonitrile and ortho-phosphoric acid were purchased from Sigma Aldrich (USA).

0.1% formic acid (FA) was purchased from Fisher Scientific (USA). 2'-deoxy-2'-

fluoro-2'-C-methyluridine (PSI-6206, Sofosbuvir metabolite) standard was purchased

from Abexbio (USA). Deionized water (18.2 MΩ cm) was used for all aqueous

preparations. All other chemicals were of analytical grade and used without further

purification. 22 μm nylon filters were purchased from Thomas Scientific, USA.

Kinetex 5µ XB-C18 (250 x 4.6 mm) and Kinetex 2.6 µm EVO C18 (100 x 2.1 mm)

reverse phase columns were purchased from Phenomenex, USA. 1.5 mL LoBind

centrifuge tubes were purchased from Eppendorf AG, Germany. Blank horse plasma

was donated by Dr. Rohan Steel, Project Leader, Biological Research Unit, Racing

Analytical Services Ltd, Melbourne, Australia. The horse plasma was collected and

shipped to QUT under the Melbourne lab protocols, ethical clearances and

arrangements for shipping biological specimens. A4 paper and soft brush pen were

purchased from Australian domestic suppliers.

2.6.2. Preparation of standard solution and spiked plasma samples

1x10-3 M PSI-6206 stock solution was prepared by dissolving 26 x 10-4 g of

PSI-6206 standard in 10 mL methanol. Serial dilutions, using methanol, were carried

out to prepare PSI-6206 concentrations in the range 5x10-5 to 5x10-11 M.

Horse blood plasma was used as a matrix to demonstrate the potential of the

HPLC-SERS methodology for the sensitive detection of PSI-6206 in complex

biological fluids. 113 µL of blank horse plasma were mixed in LoBind tube with 150

µL of 5x10-5 M PSI-6206 solution. The volume was completed to 1.5 mL using

methanol to give a final concentration of 5x10-6 M PSI-6206 in the sample. The

spiked plasma sample was then centrifuged for 20 minutes at 10000 rpm and finally

filtered using a 22 μm nylon filter. To prepare a control plasma sample, 113 µL of

blank horse plasma was diluted to 1.5 mL with methanol in LoBind tube, centrifuged

for 20 minutes at 10000 rpm and filtered using 22 μm nylon filter. All prepared

solutions were kept in the fridge at 4° C.


2.6.3. Characterization of PSI-6206 by direct Raman spectroscopy

Certified PSI-6206 reference material was screened by Raman spectroscopy

and the acquired spectrum used as the reference Raman spectrum of the drug.

2.6.4. Determination of PSI-6206 in blood plasma by surface enhanced Raman

spectroscopy (SERS)

PSI-6206 standard solutions in the concentration range 5x10-6 to 5x10-11 M

were used to construct a calibration curve for PSI-6206 by SERS. 80 µL of each

concentration was loaded onto a clean paper SERS substrate and the SERS intensity

of the Raman band at 1576 cm-1 was measured and plotted against log the

concentration of the PSI-6206 standard.

For the SERS detection of PSI-6206 in biological fluid, 10 µL of spiked blood

plasma sample (5x10-6 M) was injected for chromatographic separation by HPLC

(vide infra). The eluate, at the retention time of the drug, was deposited onto a clean

paper-based SERS substrate and screened by handheld Raman spectrometer. For

control test, 10 µL of blank blood plasma were injected under the same

chromatographic parameters (vide infra) and the eluate at retention time of PSI-6206

was deposited on paper-based SERS substrate and screened by handheld Raman

spectrometer.

2.6.5. Determination of PSI-6206 in blood plasma by HPLC-DAD

The determination of PSI-6206 by HPLC was carried out using XB-C18

column. The mobile phase consisted of 0.1% aqueous ortho-phosphoric acid

(component A) and acetonitrile (component B). A linear gradient was used for the

chromatographic separation of the drug where the percentage of component B

changed from zero to 35% over 7 minutes and from 35% to 95% over 8 minutes. The

flow rate of the mobile phase was kept at 0.5 mL min-1. 10 µL of 5x10-6 M PSI-6206

solution were injected at ambient temperature. The detection was carried out at 254

nm using diode array detector.

For the quantification of PS-6206 by HPLC-DAD, a calibration curve was

established by injecting 10 µL aliquots of PSI-6206 standard solutions (7.5x10-5 -

5x10-7 M) and plotting the area under the peak, at the retention time of the drug,

against the corresponding concentration.


For the detection of PSI-6206 in biological fluid by HPLC-DAD, 10 µL of

spiked blood plasma sample (5x10-6 M) was injected and the area under the peak, at

the retention time of the drug, was detected by DAD.

2.6.6. Detection of PSI-6206 by LC-MS

To cross-validate the SERS determination of PSI-6206, a reverse phase LC-MS

method for the detection of the drug was setup using EVO C18 column. The column

temperature was maintained at 40°C. The mobile phase consisted of 0.1% aqueous

FA (component A) and acetonitrile (component B). A linear gradient was utilized

where the percentage of component B changed from 3% to 95% in 4 minutes at a

flow rate of 0.3 mL min-1. The pressure of the system was kept at 6000 psi. The

nebulizing gas flow was 2 L min-1. The interface voltage was kept at 4 kV and its

temperature was maintained at 300 °C. The collision energy was set at 45 V. 10 µL

of the PSI-6206 standard solutions in the concentration range 5x10-6 to 5x10-11 M

were screened. The ion fragments of m/z 55.2, m/z 70.2, m/z 113.1 and m/z 261.1

were monitored by product ion scan (PIS) in the positive ionization mode.

For the detection of PSI-6206 in biological fluids by HPLC-MS, a spiked blood

plasma sample (5x10-6 M) was screened by HPLC-MS and the ions fragments of m/z

55.2, m/z 70, m/z 113.1 and m/z 261.1 were monitored by product ion scan (PIS) in

the positive ionization mode.

2.6.7. Instrumentation

UV/Vis measurements were carried out in the wavelength range 300-800 nm

using Varian Cary 50 probe UV/Vis spectrophotometer (USA). SEM measures were

performed using a Zeiss Sigma VP Field Emission Scanning Electron Microscope

with an accelerating voltage of 2 kV under high vacuum (Germany). HPLC

measurements were carried out on HPLC Agilent 1100 series (Agilent Technologies,

USA). Chromatographic data were processed using Agilent chemstation for LC

software. Raman spectrum of authentic PSI-6206 was performed using the Renishaw

InVia Raman microscope using a 785 nm excitation source at 25% of its 450 mW

laser power. Spectra were collected over the wavelength range 400 to 4000 cm-1.

Four accumulations with a total acquisition time of 10s were used to acquire the

Raman spectrum of the drug. All SERS measurements were performed using

handheld ID Raman mini spectrometer (Ocean Optics, USA). SERS measurements


were carried out using the raster orbital scanning mode over the wavelength range

400 cm-1 to 2000 cm-1 using a 785 nm excitation source of 4 mW laser power. One

accumulation (acquisition time = 1 second) was carried out to collect the SERS

spectra. The spectra were automatically corrected for background noise and

florescence by the instrument built-in algorithm. LC-MS measurements were

performed on Shimadzu LCMS-8050 (USA) that is equipped with “LabSolutions

5.65” software to control the mass spectrometer working parameters.


2.7. Results and discussion

2.7.1. Raman spectrometry of PSI-6206

Except for the methyl and fluorine substitutions at the C2' carbon atom of the

ribose ring, the molecular structure of PSI-6206 is similar to that of deoxyuridine

(Figure 2.1). Due to the absence of a reference Raman spectrum of PSI-6206 in the

literature, we measured the normal Raman spectrum of authentic PSI-6206 (Figure 2.

2a) and compared it to the SERS spectrum of the compound (vide infra).

Figure 2.1: Structural formula of PSI-6206.

The Raman spectra of uridine derivatives are characterized with contributions

from the uracil and sugar moieties in their structures [142]. As indicated by Figure

2.2a,b, the Raman spectrum of PSI-6206 shows vibrational modes at 546, 621, 783,

1229, 1402, 1668, 2893, 2949, 2990 and 3113 cm-1. The bands at 546 and 621 cm-1

are attributed to the in-plane bending motions of the uracil ring and C=O out-of-

plane deformation modes, respectively [143]. The band at 783 cm-1 is attributed to

the uracil ring breathing motion [142-143]. The band at 1229 cm-1 may be attributed

to the concerted stretching vibration of the uracil ring as well as the C-N, C-F and

C6-H stretching vibrations [142, 144]. The band at 1402 cm-1 is attributed to C-N

stretching and N3-H deformation modes [142, 144]. The strong Raman band at 1668

cm-1 are assigned to symmetric C=O and C5=C6 stretching modes in N1-substituted

uracil derivatives [143]. The Raman lines at 2893 cm-1 and 2949 cm-1 are attributed

to CH3 stretching mode [145]. The bands at 2990 and 3113 cm-1 are assigned to

antisymmetric CH2 and aromatic CH stretching vibrations, respectively [145]. The


Raman spectra of PSI-6206 that were acquired by the Raman microscope and the

handheld device were significantly similar in the range 400 cm-1 to 2000 cm-1 (Figure

2.2a,b). Therefore, we utilized the handheld Raman spectrometer for the rest of the

study.

Figure 2.2: (a) Raman spectrum of standard PSI-6206 by Renishaw Invia Raman

microscope, (b) Raman spectrum of standard PSI-6206 by handheld ID Raman mini

spectrometer.


2.7.2. SERS measurements of PSI-6206 on gold nanopillars substrate

Although Raman spectrometry can detect compounds with high specificity, it

cannot be used for ultra-trace analysis [146]. Therefore, for the detection of ultra-

trace concentrations of PSI-6206 in biological fluids, we utilized SERS as the most

sensitive mode of Raman spectrometry.

The SERS spectrum of PSI-6206 on commercial gold coated silicon

nanopillars substrate [147] is given in Figure 2.3a and shows some variations from

its Raman spectra in Figure 2.2a,b. These variations may be attributed to the

interactions between the adsorbed drug molecules and the substrate surface that may

cause peak shifts, suppression and/or emergence of spectral lines as well as changes

to peak intensities [148-150]. The SERS spectrum in Figure 2.3a shows spectral lines

at 998, 1243, 1366 cm-1 and 1545 cm-1. The band at 998 cm-1 may be attributed to the

N1-C2, N3-C4 and C4-C5 stretching vibrations as well as the ribose ring breathing in

PSI-6206 [143, 148]. The band at 1243 cm-1 may be assigned to the uracil and ribose

rings breathing and C-F stretching mode [142-143]. A new Raman band appears at

1366 cm-1 and may be assigned to the N1-C2, C2-N3 stretching and N3-H, C5-H

bending motions in the drug molecule [148]. Another new strong Raman band

appears at 1545 cm-1 and may be attributed to the C6-H deformation and C4-O8

stretching modes [143-144].

2.7.3. Paper-based substrate for the determination of PSI-6206 by SERS

To develop an efficient HPLC-SERS method for the determination of PSI-6206

in biological fluids, a disposable and cost-effective SERS substrate is required.

Therefore, we prepared gold nanoparticles colloid and used it as SERS ink to

develop SERS substrates on A4 paper. Figure 2.3b shows the SER spectrum of

standard PSI-6206 on the paper-based substrate. As indicated by the figure, the

acquired Raman spectrum was in general agreement with that of the drug on the gold

coated silicon nanopillars SERS substrate. The variations within the spectra in Figure

2.3a,b are attributed in part to the differences in shape and size of the gold

nanostructures on each substrate [151]. The gold nanopillars have oval shape and

unknown size while the gold nanoparticles have spherical shape and an average

diameter of 50 nm (Figure S2.1, supplementary material).


Figure 2.3: (a) SERS spectrum of standard PSI-6206 on gold coated silicon

nanopillars substrate, (b) SERS spectrum of standard PSI-6206 on paper SERS

substrate.

Figure 2.3b shows Raman bands at 510, 852, 1001, 1169, 1237, 1371 and 1576

cm-1, respectively. The band at 510 cm-1 may be attributed to the in-plane bending

motions of the uracil ring [143, 152]. The band at 852 cm-1 may be attributed to the

trigonal pyrimidine ring breathing and out of plane N3-H wagging vibration modes


[143, 148]. The Raman band at 1001 cm-1 is assigned to N1-C2, N3-C4 and C4-C5

stretching modes as well as the ribose ring breathing modes [143, 148]. The Raman

band at 1169 cm-1 may be attributed to the C-N and C6-H motions [143, 152]. The

band at 1237 cm-1 may be assigned to the uracil and ribose ring breathing modes, C3-

N4, C6-H and C6-N1 stretching modes [142-143]. The band at 1371 cm-1 is assigned

to N1-C2-C2-N3 stretching mode as well as N3-H, C5-H and C6-H bending modes

[148]. The Raman band at 1576 cm-1 may be attributed to the C4-O8, C2-O7

stretching and C6-H deformation modes [143, 144, 148].

The emergence of new bands between 1300-1600 cm-1 wavenumber value in

the Raman spectra of PSI-6206 on both of the nanopillars and paper SERS substrates

suggest that PSI-6206 in solution adsorbs to the gold substrate via a deprotonated N3

atom as well as the C4-O8, C2-O7 and the C6-H moieties of the molecule [148]. The

deprotonation of uracil at the N3 atom leads to the formation of an enol tautomer that

adsorbs favourably on the gold substrate with the uracil ring adopting a tilted

orientation on the metal surface. This orientation causes the Raman bands in the

1300-1600 cm-1 wavenumber region to become prominent due to the short distance

between the aromatic ring and the gold surface (scheme 2.1) [148, 153, 154].

Scheme 2.1: N3 deprotonation of PSI-6206 in methanol.

2.7.4. Quantification of PSI-6206 by paper-based SERS substrate

The paper-based substrate was utilized for the SERS quantification of PSI-

6206. The intensity of the Raman band at 1576 cm-1 was found responsive to the

change in the concentration of the drug and monotonically decrease with the

concentration of PSI-6206 in the range 5x10-6 M to 5x10-11 M (13 ng L-1). The

regression equation for the relationship of the band intensity at 1576 cm-1 and log the


drug concentration was found to be y = 111.29x + 1716.9 and the R2 value = 0.96.

The RSD of the regression line was found to be 5.23%. The % error within the SERS

measurements of the drug standards was 4.29%. In addition, the deviation in the

SERS quantification of 5x10-6 M solution of the drug was found to be 2.98%. For

PSI-6202 the lower limit of detection is set by FDA to be 10000 ng L-1 in blood

plasma by LC/MS/MS [155]. Therefore, the SERS quantification of the drug by the

paper-based substrate satisfies the FDA requirements.

2.7.5. Determination of PSI-6206 in spiked plasma samples by HPLC-DAD,

HPLC-SERS and HPLC-MS

Due to its low cost and disposable nature, as compared to the nanopillars

substrate, the paper-based substrate was utilized for the determination of PSI-6206 in

blood plasma by HPLC-SERS. We optimized the chromatographic conditions for the

separation of the drug standard by reverse phase HPLC-DAD. Different ratios of

0.1% aqueous ortho-phosphoric acid (component A) and acetonitrile (component B)

were tested for the isocratic elution of the drug and resulted in poor peak resolution.

Therefore, linear gradient elution was applied for the separation of the compound. By

changing the percentage of component B in the mobile phase from zero to 35% over

7 minutes and from 35% to 95% over 8 minutes, the drug was successfully separated

at 12.44 minutes. At a flow rate of 1 mL per minute, the resolution of the PSI-6206

peak was incomplete. Therefore, the flow rate was reduced to 0.5 mL per minute to

achieve maximum resolution of the drug (Figure 2.4a). The established

chromatographic parameters were used for the quantification of PSI-6206 by HPLC-

DAD. A linear relationship between the area under the peak at 12.44 minutes and the

drug concentration in the range of 7.5x10-5 M to 5x10-7 M was found to follow the

regression equation y = 10+7x – 6.9198 (R2=0.998). Therefore the minimum

quantification limit of PSI-6206 by SERS was 4 orders of magnitude less than that

by DAD. The chromatographic parameters were also applied for the detection of

PSI-6206 in spiked horse plasma sample (Figure 2.4b,c). As confirmed by Figure

2.4b there was no interference from the plasma matrix with the retention time of PSI-

6206 (Figure 2.4c).


Figure 2.4: (a) HPLC chromatogram of 7.5x10−5 M PSI-6206 in methanol, (b)

HPLC chromatogram of control plasma sample, (c) HPLC chromatogram of 5x10−6

M PSI-6206 spiked plasma sample.


For the determination of PSI-6206 in spiked horse plasma by HPLC-SERS, the

same chromatographic parameters of the HPLC-DAD separation were utilized. 10

µL of blank and spiked blood plasma samples were injected and the eluate at 12.44

minutes was screened by SERS using paper-based substrate and handheld Raman

spectrometer (Figure 2.5). As confirmed by the figure, the SERS spectrum of the

spiked blood plasma sample matched that of PSI-6206 in Figure 2.3b and the blood

plasma matrix did not interfere with the identification of the drug by SERS (Figure

2.5, red bottom spectrum). The concentration of the drug in the spiked blood plasma

was also determined by SERS and found to be 3.76 x10-6 M.

Figure 2.5: SERS spectrum of PSI-6206 after HPLC separation from spiked horse

plasma.

The SERS identification of PSI-6206 in the spiked plasma was confirmed by

HPLC-MS (Figure 2.6a). The mass fragmentation of standard PSI-6206 was in close

agreement with that reported by other research groups [140-141]. The mass

fragmentation of the drug after separation from spiked blood plasma is shown in

Figure 2.6b and it matches that of standard PSI-6206. These results confirm that the

paper-based SERS sensor has higher sensitivity than DAD and similar selectivity as

the mass spectrometer. The new HPLC-SERS method is sensitive, selective, and


cost-effective. Therefore, it has a strong potential for the rapid determination of

drugs and their metabolites in biological fluids.

Figure 2.6: (a) MS fragmentation of PSI-6206 standard after chromatographic

separation, (b) MS fragmentation of PSI-6206 in spiked blood plasma after

chromatographic separation.


2.8. Conclusions

The metabolite PSI-6206 was studied for the first time by Raman spectrometry.

The surface-enhanced Raman spectrum of PSI-6206 was in general agreement with

those of N1-substituted uracil derivatives. The appearance of Raman bands between

1300 and 1600 cm-1 in the SERS spectra of PSI-6206 indicates that the molecule

deprotonates in solution at the N3 atom and may take a tilted orientation on the metal

surface of the gold substrate. A cost-effective and disposable paper-based SERS

substrate was developed by simple chemical procedure and used for the

determination of PSI-6206 down to 13 ng L-1 which satisfies the FDA detection limit

for PSI-6206 (10000 ng L-1) by HPLC-MS-MS. An interference-free HPLC-SERS

that utilizes the disposable paper-based substrate for detection was developed and

used for the determination of the drug metabolite in blood plasma. When compared

to HPLC-DAD and HPLC-MS, the HPLC-SERS found to be more sensitive than

HPLC-DAD while providing structural identification of the analyte, similar to

HPLC-MS. The interference-free HPLC-SERS method with the disposable paper-

based substrate has a strong potential for the trace analysis of drug metabolites in

biological fluids.


2.9. Supplementary material

2.9.1. Synthesis of gold nanoparticles

All glassware was cleaned with aqua regia solution (3: 1, 32% HCl/ 68%

HNO3) and subsequent thorough washing with copious amounts of deionized water

prior to use. Au nanoparticles were synthesized according to the method by Bastus et

al. [156]. Briefly, 150 mL of 2.2 mM trisodium citrate dihydrate was refluxed to 90°

C. Three aliquots (1 mL each) of 25 mM HAuCl4 were added to the sodium citrate

solution at time intervals of 30 minutes. The solution temperature was maintained at

90° C with continuous magnetic stirring. The formation of gold nanoparticle seeds

was indicating by the development of a pink developed within 10 minutes. To

increase the size of the formed gold nanoparticles, 53 mL of the pink coloured

solution were transferred into a new clean flask and mixed with 2 mL of 60 mM

trisodium citrate dehydrate and 55 mL of deionized water. This resulting solution

was then refluxed to 90° C and 3 aliquots (1 ml each) of 25 mM HAuCl4 added at 30

minutes time intervals with continuous magnetic stirring. After the end of the

reaction, the water content of the gold nanoparticle colloid was reduced to 1.5 mL by

centrifugation to produce gold nanoparticle ink. The shape and size of the

synthesized nanoparticles were characterized using UV/Vis spectrophotometer and

scanning electron microscopy (SEM) (Figures S2.2 and S2.3). The concentrated gold

nanoparticles colloid was used as nanoparticles ink to draw on A4 paper.

2.9.2. Manufacture of paper SERS substrate

The gold nanoparticles ink was used to draw small squares (0.5x0.5 cm) onto

clean A4 paper (Figure S2.4) using a soft brush pen to load three coats of the gold

nanoparticles onto the A4 white sheet [157]. After air drying in clean room, the

drawn squares were cut of the A4 sheet and stored in a dry clean environment for

SERS measurements.


Figure S2.1: UV spectrum of standard PSI-6206 in methanol.

Figure S2.2: UV spectrum of gold nanoparticles colloid (SERS ink).


Figure S2.3: SEM image of (a) blank white A4 paper, (b) A4 paper after coating

with gold nanoparticles, (c) magnification of deposited nanoparticles on paper

substrate after coating with gold nanoparticles.

Figure S2.4: Raman spectra of A4 paper before and after coating with gold

nanoparticles (SERS ink).

Recyclable functionalized nanomaterials for SERS detection of environmental toxin in biological samples 55

Chapter 3: Recyclable functionalized

nanomaterials for SERS

detection of environmental toxin

in biological samples

This chapter is made up of the following journal article published in Biosensors and

Bioelectronics Journal.

Gold nanomaterials for the selective capturing and SERS diagnosis of toxins in

aqueous and biological fluids

Waleed A. Hassanain, Emad L. Izake, Michael S. Schmidt, Godwin A. Ayoko

DOI: 10.1016/j.bios.2017.01.032

https://www.sciencedirect.com/science/article/pii/S0956566317300325

56 Recyclable functionalized nanomaterials for SERS detection of environmental toxin in biological samples






1. they meet the criteria for authorship in that they have participated in the conception,


expertise;

2. they take public responsibility for their part of the publication, except for the


3. there are no other authors of the publication according to these criteria;

4. potential conflicts of interest have been disclosed to (a) granting bodies, (b) the



5. they agree to the use of the publication in the student’s thesis and its publication

on the QUT’s ePrints site consistent with any limitations set by publisher

requirements.


W.A. Hassanain, E.L. Izake, M.S. Schmidt, G.A. Ayoko, Gold nanomaterials for the

selective capturing and SERS diagnosis of toxins in aqueous and biological fluids, Biosensors and Bioelectronics, 2017, 91, 664-672.



authorship.


Name Signature Date


Waleed A. Hassanain




Michael S. Schmidt Supplied samples and contributed to research editing.






3.1. Preface

The previous chapter demonstrated the use of paper SERS substrate for the

sensitive and affordable SERS detection of a drug metabolite in blood plasma. In this

chapter, the use of the paper SERS substrate has been extended, for the first time, to

the direct SERS detection of the environmental toxin Microcystin-LR (MC-LR) in

water and blood plasma. The performance of the paper SERS substrate was

compared to that of a gold coated silicon nanopillar SERS substrate that is

manufactured by complex ion etching method. The results confirmed that the paper

substrate was of good sensitivity. However, it was of low cost and can be easily

prepared by a simple chemical reduction method.

This chapter also presents a novel approach for the bio-conjugation of target-

specific antibody fragments for the combined selective isolation and label-free SERS

detection of MC-LR in biological samples down to 10 fM. The new approach utilizes

the thiol chemistry of antibodies for their oriented immobilization onto magnetic

gold nanoparticles for the development of MC-LR extractor nanomaterial. The

developed functionalized nanomaterial was recycled for multiple selective

extractions of the target protein.


3.2. Abstract

A highly sensitive nanosensing method for the combined selective capture and

SERS detection of Microcystin-LR (MC-LR) in blood plasma has been developed.

The new method utilizes gold coated magnetic nanoparticles that are functionalized

with anti MC-LR antibody Fab' fragments for the selective capture of MC-LR from

aqueous media and blood plasma. Using an oriented immobilization approach, the

Fab' fragments are covalently attached to gold surface to form a monolayer with high

capture efficiency towards the toxin. After the selective capture, the purified MC-LR

molecules were released from the extractor nanoparticles within 5 minutes by

manipulating the pH environment of the nanoparticles. The regenerated extractor

nanoparticles maintained their capture efficiency and, therefore, were re-used to

capture of MC-LR from successive samples. The released purified toxin was

screened within 10 minutes on gold coated silicon nanopillars and a new paper-based

SERS substrate by handheld Raman spectrometer. The SERS enhancement factors of

the nanopillars and the new paper-based substrate were 2.5x106 and 3x105,

respectively. The lower limit of quantification (LOQ) of MC-LR by SERS on the

nanopillar substrate was 10 fM (R2 = 0.9975) which is well below the clinically

required detection limit of the toxin. The SERS determination of MC-LR was cross-

validated against ELISA. By using antibody fragments that are specific to the target

biomolecule, the new methodology can be extended to the rapid extraction and

detection of other toxins and proteins.

3.3. Keywords

Microcystin-LR, biological fluids, functionalized nanoparticles, antibody fragments,

paper SERS substrate, molecular diagnosis.


3.5. Introduction

Microcystins are a class of more than 50 structurally similar potent

hepatotoxins that are produced by the freshwater cyanobacteria [158-160]. Human

exposure to these toxins may occur through drinking water or ingesting contaminated

food. Exposure to high levels of microcystins causes severe liver damage,

cytoskeletal deformation, mitochondrial membrane rupture and intrahepatic

haemorrhage leading to death [161-166]. Chronic exposure to low concentrations of

microcystins causes gastroenteritis, hepatoenteritis, genotoxicity, kidney impairment,

liver cancer, impairment of vital immune responses and diabetes in humans [167-

178]. Microcystin-LR (MC-LR) is the most toxic member of the microcystins family

and its chemical composition involves 7 amino acids; D-alanine, L-arginine, L-

leucine, D-methyl-aspartate, D-glutamic acid, N-methyl-dehydro-alanine and ADDA

(3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid) (Figure S3.1,

supplementary material) [160, 179]. The regulatory limit for MC-LR is set by the

world health organization (WHO) at 1 µg/L in drinking water [180]. Therefore, it is

very important to develop sensitive detection methods for the rapid determination of

ultra-trace amounts of MC-LR in water and biological fluids. Various methods have

been used for the analysis of MC-LR. These methods include chromatography [181-

189], immunoassay [190-193], electrochemical biosensors [194-202], biochemical

protein phosphatase inhibition assay (PPI assay) [203-206], capillary electrophoresis

[207], surface enhanced fluorescence [208] and chemiluminescence [209].

Chromatographic methods are reliable and widely accepted. However, they are time

consuming and require extensive sample preparation procedures by trained operators

[210]. Enzyme-linked immunosorbent assay (ELISA) is also commonly used for the

detection of MC-LR in environmental and biological samples. Similar to

chromatography, ELISA requires complex procedures for the separation of the

antigen from the sample matrix. In addition, ELISA technique requires the use of

stable primary and secondary antibodies as well as labelling agent (e.g. enzyme or a

dye molecule) to bind and detect the toxin [191, 194, 211]. The chemical procedures

to label the anti-MC-LR antibody and the toxin affect the affinity interaction between

the antigen and the antibody and the sensitivity of the technique [201].

Electrochemical biosensors have been demonstrated for the trace analysis of MC-LR

in water [206]. Nevertheless, the fabrication of these sensors requires complex


procedures for electrode modifications [212-214]. PPI assay for MC-LR was also

demonstrated; however, it suffers from false positive identification of the toxin due

to its non-specific inhibitory activities towards microcystins [214]. In addition, the

phosphatase activity within the sample results in large quantitative deviations [203].

Surface-enhanced Raman spectroscopy (SERS) is a sensitive analytical

technique for the detection of proteins [215]. In SERS, the surface plasmon

resonance of a nanostructured coinage metal surface enhances the oscillating electric

field of an incident light [20]. This near-field enhancement increases the Raman

signal of those molecules residing in a close proximity to the metal surface by

several orders of magnitude [216-219]. SERS can be carried out with or without the

use of a SERS label. In label-free SERS, the target analyte is adsorbed onto the

surface of the metallic substrate and probed with a laser beam to acquire its Raman

spectrum [220-223]. This approach assumes that, there is only one compound

existing in the sample. However, for a complex sample, that contains many

compounds, the non-selective adsorption of these compounds onto the SERS

substrate causes the Raman fingerprint of the target analyte to be obscured by

contributions from the other compounds [224]. Therefore, extensive isolation and

clean up procedures are required prior to the direct SERS detection of target analytes

in real life samples [225, 226]. In labelled SERS detection, the analyte is detected

indirectly by monitoring the SERS signal of a Raman dye that is attached to the

SERS substrate [20]. Labelled SERS methods were demonstrated for the detection of

MC-LR after its extraction from the sample where gold nanostructures labelled with

4-aminothiophenol (4-ATP) were used for the indirect detection of the toxin [227-

228].

In this work, we present for the first time, a rapid selective and sensitive

method for the combined extraction and label-free SERS detection of MC-LR in

aqueous medium and blood plasma. In a previous research, we used random

immobilization of antibodies to develop extractor nanoparticles for proteins [225].

The disadvantage of random immobilization is that the antibody molecules on the

nanoparticle surface may sterically hinder its binding to the antigen. Therefore, the

capture efficiency of the functionalized nanoparticles is limited [229]. In this work,

to overcome this problem and develop extractor nanoparticles of high capture

efficiency, we immobilized MC-LR antibody Fab' fragments onto gold coated


magnetic nanoparticles in a highly oriented order and high surface coverage. The

antibody Fab' fragments were prepared by digesting the Fc region of the MC-LR

antibody and chemically reduce the disulphide bridges within their hinge region to

develop free SH groups within the fragments structure. The formed Fab' fragments

(55 KDa) were covalently bound, via Au-S bonds, to the gold coated magnetic

nanoparticles. The functionalized nanoparticles were used to selectively capture MC-

LR from aqueous and blood plasma media within 15 minutes. The extractor

nanoparticles were regenerated and used to capture MC-LR in repeated tests. The

binding between the MC-LR and the functionalized nanoparticles was monitored by

SERS. After binding the toxin, the SERS spectrum of the functionalized

nanoparticles developed Raman bands that are characteristic of MC-LR. For the

quantification of MC-LR by SERS, the captured toxin molecules were released from

the extractor nanoparticles within 5 minutes by glycine buffer. After removing the

buffer in 5 minutes by size exclusion column, the eluted purified toxin was screened

by SERS within 10 minutes. The limit of quantification (LOQ) of MC-LR by SERS

was 10 fM which is well below ELISA detection limit (0.1 ng/mL) and meets the

clinically required detection limit for chronic exposure to the toxin [230-231].

The developed method utilizes a disposable paper-based substrate and

handheld Raman spectrometer for the SERS detection of the toxin. The paper-based

substrate was prepared by simple chemical reduction method and showed good

SERS enhancement factor (EF) of 3x105. The new method has strong potential for

the rapid and sensitive screening of toxins, protein biomarkers and amino acids in

environmental and biological fluids at pathology labs within the hour.


3.6. Material and methods

3.6.1. Chemicals and reagents

Microcystin-LR (MC-LR) standard solution (10 µg/mL in methanol),

phosphate buffered saline (PBS), 1-butanethiol, trisodium citrate dihydrate,

Hydrogen tetrachloroaurate (HAuCl4), glycine, L-cysteine, hydrochloric acid, nitric

acid and methanol were purchased from Sigma Aldrich (USA). A monoclonal anti-

microcystin antibody (ab 85548) of high specificity to MC-LR was purchased from

Abcam (UK). Pierce Mouse IgG1 F(ab')2 preparation Kit (number 44980) and

reducing agent tris(2-carboxyethyl)phosphine (TCEP) solution were purchased from

Thermo Fischer Scientific (USA). Competitive MC-LR ELISA Kit (catalogue

number KA1496) was purchased from Abnova (Taiwan). Gold coated magnetic

nanoparticles (50 nm) were purchased from nanoimmunotech (Spain). Detailed

information of the synthesis and characterization of the nanoparticles is given by the

supplier website. All chemicals were of analytical grade and used without any further

purification. Gravity flow size exclusion columns (illustra NAP-5) were purchased

from GE Healthcare Life Science (AUS). Eppendorf DNA LoBind vial 1.5 mL

(Eppendorf AG, Germany).

Blank horse plasma was donated by Dr. Rohan Steel, Project Leader,

Biological Research Unit, Racing Analytical Services Ltd, Melbourne (AUS). The

samples were collected and shipped to Queensland University of Technology (QUT)

under the Melbourne lab protocols, ethical clearances and arrangements. A4 white

paper and soft brush pen were purchased from Australian local suppliers. Deionized

water (ultrapure Millipore, 18.2 MΩ.cm@25˚C) was used in all preparations. The

glassware was cleaned with aqua regia and thoroughly rinsed with deionized water

prior to use. Gold coated silicon nanopillar substrates were purchased from Silmeco,

Denmark, and used for the SERS quantification of MC-LR [147].

3.6.2. Preparation of MC-LR standard solutions

MC-LR stock solution (1x10-5 M in methanol) was used to prepare a series of

standard solutions in the concentration range of 1x10-6 to 1x10-14 M by serial dilution

using methanol.


3.6.3. Detection of MC-LR by SERS

To develop reference SERS spectra of MC-LR, 10 µL of 1x10-9 M MC-LR

solution were loaded onto gold coated silicon nanopillar and paper-based SERS

substrates. The toxin was screened using the InVia Raman microscope (Renishaw,

UK) and the handheld ID Raman mini 2 spectrometer (Ocean Optics, USA). For

SERS measurements by the InVia Raman microscope, a 785 nm laser source was

used for excitation. The excitation laser power was set at 0.5% of 450 mW. The

spectra were collected using a 50x objective lens. The measurements were carried

out in the wavelength range 400 to 1800 cm-1. Four accumulations with a total

acquisition time of 40 seconds were used. The acquired spectra were not corrected

for florescence background and reported as raw data.

For the SERS measurements by the handheld Raman spectrometer, the spectra

were collected using the raster orbital scanning (ROS) mode within the wavelength

range 400 to 1800 cm-1. A 785 nm laser source at 5 mW laser power was used for

excitation. One accumulation with a total acquisition time of 100 milliseconds was

used. The instrument software algorithm (OceanView Spectroscopy 1.5.07)

automatically average and correct the acquired spectra for florescence background.

For sensitive quantification of the toxin by SERS, 30 µL of MC-LR standard

solutions in the concentration range 1x10-6 to 1x10-14 M were loaded onto gold

coated silicon nanopillar substrates and screened by handheld Raman spectrometer.

The SERS measurement for each MC-LR concentration was repeated 3 times (n=3).

The average intensity of the Raman band at 1441cm-1 was plotted against log the

concentration of MC-LR.

3.6.4. Preparation of functionalized gold coated magnetic nanoparticles

To synthesize F(ab')2 fragments, the Fc region of the anti microcystin-LR

antibody was digested using Pierce Mouse IgG1 F(ab')2 preparation Kit as per the

supplier protocol. The fragmentation process was checked by monitoring the SERS

spectra of the intact IgG1 antibody and the produced F(ab')2 fragments (Figures S3.2

and S3.3, supplementary material). For these measurements, 10 µL aliquots of the

antibody and F(ab')2 fragments were loaded on silicon nanopillar substrates and

screened by handheld Raman spectrometer using the same parameters reported in

section 3.5.3. For the combined synthesis and immobilization of MC-LR Fab'

fragments onto gold coated magnetic nanoparticles, 90 µL of the F(ab')2 fragments


were mixed with equivalent volume of 0.2 mM TCEP and 200 µL of the

nanoparticles in a LoBind Eppendorf vial. The TCEP reduces the disulphide bonds

within hinge region of the F(ab')2 fragments and develop Fab' fragments with free

sulfhydryl groups (R-SH). The mixture was left to stand for 40 minutes at room

temperature for the complete reduction and immobilization of the Fab' fragments

onto the nanoparticles surface via Au-S bonds. The functionalized nanoparticles were

then removed from solution by magnetic separation and re-suspended into 180 µL

PBS (pH 7.4). To backfill the remaining bare sites onto the functionalized

nanoparticle surface, they were mixed with 18 µL of 1-butanethiol (10-6 M in PBS,

pH 7.4) and the mixture left to stand for 1 hour at 4 ºC for the complete attachment

of the butanethiol to the bare sites via Au-S bonds. The functionalized and backfilled

nanoparticles were then collected, using a magnetic separator, washed 3 times with

100 µL PBS (pH 7.4), re-dispersed in 100 µL PBS (pH 7.4) and stored at 4º C for

future use.

3.6.5. Selective capture of MC-LR from blood plasma and SERS monitoring of

antibody-antigen interaction

The functionalized nanoparticles were used to capture MC-LR from aqueous

and biological media. 10 µL of MC-LR (1x10-8 M in deionized water) were mixed

with 100 µL of the nanoparticles and left to stand for 15 minutes at room temperature

to bind the toxin. The toxin-bound nanoparticles were then separated by magnetic

separation and washed 3 times with 100 µL PBS (pH 7.4) to remove the unbound

MC-LR molecules. The total washing time was 21 minutes. To capture MC-LR

molecules from biological sample, 8.06 µL of spiked horse blood plasma, containing

8x10-9 M of MC-LR, was mixed with 80.65 µL of the functionalized nanoparticles

and 403.23 µL PBS buffer (pH 7.4) in the ratio of 1:1:10:50, respectively, to a total

volume of 500 µL. The final concentration of MC-LR in the mixture was 1.3x10-10

M. The mixture was allowed to stand for 15 minutes to bind the toxin molecules. The

toxin-bound nanoparticles were washed 3 times with 100 µL of PBS (pH 7.4) to

remove any unbound MC-LR molecules. To monitor the binding of the MC-LR

molecules, the SERS spectra of the functionalized nanoparticles before and after

their interaction with the spiked blood plasma were collected and compared.


3.6.6. Direct SERS detection of purified MC-LR and cross-validation by ELISA

To release the captured MC-LR, the toxin-bound nanoparticles were re-

constituted into 100 µL of 0.1 M of glycine.HCl buffer (pH 2.5) and allowed to stand

for 5 minutes at room temperature. The free functionalized nanoparticles were then

magnetically separated and stored in PBS (pH 7.4) for future re-use. The solution

phase, containing the released MC-LR molecules, was loaded onto size exclusion

column to remove the glycine buffer within 5 minutes. The toxin molecules were

eluted from the column using 500 µL of deionized water.

For the SERS detection, 30 µL aliquots of the purified MC-LR were loaded

onto gold coated silicon nanopillar and paper-based SERS substrates. After air

drying for 10 minutes, the samples were scanned by handheld Raman spectrometer.

For cross-validation purposes, an aliquot of the purified MC-LR was re-

screened by ELISA. For the quantitative SERS analysis of MC-LR, the polynomial

relationship between the average optical density (OD) at 450 nm and the MC-LR

concentration, in the range between zero to 2.5x10-9 M, was plotted. The competitive

ELISA measurement was repeated twice for each MC-LR concentration. The

regression equation for the established relationship was found to be y = 0.0246x2 –

0.2681x + 0.8148, R2 = 0.9915 (Figure S3.4, supplementary material). VERSAmax

Tunable Microplate Reader (Molecular Devices Co. USA) was used to read the

optical density at 450 nm and the software “SoftMax Pro 4.3” was used to process

the acquired signal.

3.6.7. Manufacture of paper-based SERS substrate

Gold nanoparticle colloid was synthesized by the turkevich citrate reduction

method [232]. Briefly, 30 mL of 0.01% aqueous HAuCl4 (Mwt = 339.79 g/mol) were

heated with magnetic stirring to boiling temperature. 180 µL of 1% trisodium citrate

dihydrate was then added to the solution to produce gold nanoparticle colloid. The

colloid was kept to boil for 15 minutes to the complete reduction of the gold

chloride. To prepare gold nanoparticle ink, the gold nanoparticle colloid was left to

cool down to room temperature then centrifuged to reduce its volume to 0.5 mL. The

nanoparticles concentration of the prepared SERS ink was 1.5x109 NPs mL-1 (see

supplementary material). UV–visible spectroscopy (Cary 100 spectrophotometer,

Agilent Technologies, USA) was used to detect the plasmon absorption band of the


gold nanoparticle ink (Figure S3.5, supplementary material). The gold nanoparticle

ink was used to draw small squares (0.5x0.5 cm) onto a clean A4 paper (Figure S3.6,

supplementary material). For this purpose, a soft brush pen was used to load three

coats of the gold nanoparticle onto the A4 paper. After air drying, the drawn squares

were cut and stored in a dry clean environment. The paper-based SERS substrate was

studied by scanning electron microscopy (SEM) (Zeiss Sigma VP Field Emission

Scanning Electron Microscope, Germany) to determine the size and shape of the gold

nanostructures on the surface of the paper material (Figure S3.7, supplementary

material).



3.7.1. Synthesis of MC-LR antibody Fab' fragments and surface

functionalization of gold coated magnetic nanoparticles

Gold coated magnetic nanoparticles have been employed in many applications

such as targeted drug delivery, biosensors, lateral flow tests and cancer therapy [233-

237]. Due to the magnetic property of their core, the nanoparticles can be separated

from complex matrices using a magnetic separator. The gold shell of the

nanoparticles facilitates their surface functionalization with recognition molecules

such as antibodies and aptamers [238]. In addition, the gold shell enhances the

stability of the magnetic core of the nanoparticles and provides an excellent

plasmonic surface for SERS [239-240]. Oriented immobilization of antibodies on the

surface of a substrate plays a significant role in improving the sensitivity and

specificity of the immunosensor [241]. In the present work, MC-LR IgG antibody

(150 KDa) was digested to synthesize F(ab')2 fragments (110 KDa) [242]. This was

followed by reducing the disulphide bonds within the hinge region of the F(ab')2

fragments to produce Fab' fragments (55 KDa) with free R-SH groups. The Fab'

fragments were covalently bound to gold coated magnetic nanoparticles via Au-S

bonds. In this assembly, the binding regions of the Fab' fragments take an outward

orientation onto the nanoparticle surface [243-244]. Due to their small size, the

surface coverage by the Fab' fragments on the nanoparticles is high when compared

to intact antibody [245]. The outward orientation and high surface coverage of the

Fab' fragments improve the capture efficiency of the functionalized nanoparticles

towards their target antigen [229, 244, 245, 246, 247]. To prevent the non-specific

binding of foreign analytes to the nanoparticles, all remaining bare sites on

nanoparticle surface were backfilled with 1-butanethiol. The alkanethiol strongly

binds to gold, via Au-S bond, and prevent foreign analytes from binding to the

nanoparticle surface [248].

3.7.2. SERS spectrum of MC-LR

The SERS spectra of MC-LR standard on gold coated silicon nanopillar

substrate were recorded by the InVia Raman microscope and the handheld Raman

spectrometer. The SERS spectra of the toxin (Figure 3.1a,b) were in general

agreement with its normal Raman spectrum (Table 3.1) [249-250] with some

variations. These variations can be attributed to the adsorption of the MC-LR


molecules onto the rough metallic surface of the substrate and the relaxation of the

Raman selection rules causing the appearance of new Raman vibration modes that

are normally Raman inactive [250-258].

Figure 3.1: SERS spectra of standard MC-LR on gold coated silicon nanopillar

substrate using (a) Renishaw Raman microscope and (b) handheld Raman

spectrometer.


Table 3.1: Band assignment of the SERS spectra of MC-LR (the * symbol denotes

the SERS bands detected by the handheld Raman spectrometer)

Raman Shift

(cm-1

)

Band assignment Ref.

1582, 1588* Benzene ring and COO- stretches of ADDA and L-

leucine residues.

252-253

1536, 1538* Amide II vibration modes. 225, 254, 255

1493 Symmetric NH3+ bending of D-alanine residue. 249

1460 CH3 asymmetric bending, deformation and rocking, CH

deformation, CH2 scissoring of D-alanine and L-leucine residues.

249, 250, 259

1440, 1441* CH2 bending and scissoring of D-glutamic acid residue,

N-C-N asymmetric stretch, C-N-H side chain, CH2 bending, C-H vibration of L-arginine residue.

259-261

1367, 1373* COOH stretch, CO stretching, CH3 symmetric bending

and CH2 scissoring of D-alanine, L-leucine and D-glutamic acid residues.

249, 259

1303*, 1305 Cα-C and N-C vibrations of the amide III region

(α-Helical conformation), CH2 wagging.

249, 259, 262

1261 NH3+ rocking and amide III vibrations of peptide

backbone.

249, 250, 262

1237*, 1239 NH3+ rocking and amide III vibrations of peptide

backbone.

249, 250, 262

1211 CH2 twisting and rocking vibrations of D-glutamic acid

residue.

249

1181 NH3+ rocking of L-leucine residue. 260

1124 NH3+ deformation vibrations of D-glutamic acid and

L-leucine residues. 259

1018*, 1019 Phenyl CH in plane bending and C-N stretch of peptide

bonds.

249, 250, 259

948*, 955 C-C-N stretch and C-COO- stretch of N-Methyl-D-

aspartic acid and D-glutamic acid residues.

249, 259

882 C-COO- stretch and CH2 rocking. 249-250

845 C-C stretch, CH2 rocking and CH3 rocking of D-alanine

residue.

250

753 Phenyl ring CH out-of-plane bending. COO- bending

and CH2 rocking of D-glutamic acid residue.

249-250

719 COO- deformation and C-C twisting of D-alanine and D-methyl aspartic acid residues.

249, 259

643 COO- deformation and C-C twisting of D-alanine and D-methyl aspartic acid residues.

249, 259


The notable quality of the MC-LR SERS spectrum acquired by the handheld

Raman spectrometer may be attributed to the instrument capacity to screen a large

surface area of the SERS substrate using the ROS mode [263]. The signal acquired

by this mode represents an average SERS spectrum of the probed MC-LR molecules

from various locations on the substrate’s surface [225]. On the contrary, the tightly

focused beam of the InVia Raman microscope probes only a small number of the

analyte molecules on a small area of the substrate (the spot size of the excitation laser

in the confocal optical geometry is only ~0.5 µm). In addition, the algorithm of the

handheld Raman spectrometer software automatically compensates for fluorescence

background in the SERS measurements and, therefore, the developed spectra are

highly optimized. On the contrary, the measurements by the InVia Raman

microscope require lengthy signal processing procedures to optimize the acquired

SERS spectra and remove the fluorescence background. In this work we did not

attempt any signal processing for the raw SERS spectra acquired by the InVia Raman

microscope.

3.7.3. Selective capture of MC-LR and SERS monitoring of the toxin binding to

the functionalized nanoparticles

The functionalized nanoparticles were used to capture and pre-concentrate the

toxin from aqueous medium [264]. The SERS spectra of the functionalized

nanoparticles before and after interaction with MC-LR were acquired by the InVia

(Figure 3.2a,b) and handheld (Figure S3.8a,b, supplementary material) Raman

spectrometers. The spectral resolution of the InVia and handheld Raman

spectrometers are 1 cm-1 and 12 cm-1, respectively. Therefore, the high resolution

spectra acquired in Figure 3.2 were used to monitor the binding between the toxin

and the functionalized nanoparticles.

By comparing the spectra of the functionalized nanoparticles (blue line, Figure

3.2a) to that of the bare nanoparticles (red line in Figure 3.2), many Raman bands

that are characteristic of proteins are observed. The emergence of the protein

characteristic bands within the spectrum of the functionalized nanoparticles confirm

the successful immobilization of Fab' fragments onto the nanoparticles. The band at

1607 cm-1 can be assigned to amide I (α-helix) vibrations while the band at 1574 cm-1

is attributed to the phenylalanine residues in proteins [225, 259, 265, 266]. The band

at 1563 cm-1 is assigned to amide II vibrations [254, 267]. The bands at 1446 cm-1


and 1386 cm-1 are assigned to CH2 and Cα-H bending vibrations, respectively [254,

265]. The bands at 1316 cm-1, 1243 cm-1 and 1012 cm-1 are attributed to CH2

deformation, amide III vibrations and benzene ring breathing of phenylalanine,

respectively [225, 266, 267].

Figure 3.2: SERS spectra of the functionalized gold coated magnetic nanoparticles,

(a) before interaction with MC-LR and (b) after the capture of MC-LR. The spectra

were collected using the InVia Raman spectrometer.

The spectrum in Figure 3.2b, shows several variations from that of the Fab'

fragments in Figure 3.2a. For example, the amide I band at 1607 cm-1 and the

phenylalanine band 1574 cm-1 of the Fab' fragments disappeared. These variations

are attributed to the conformational re-arrangements that take place within the Fab'

fragments upon binding the toxin molecules [225, 226]. In addition, new Raman

bands have emerged at 1543 cm-1 (amide II vibration), 1486 cm-1 (symmetric NH3+

bending of D-alanine residue), 1417 cm-1 (COO- symmetric stretch), 1363 cm-1 (CH3

symmetric bending and CH2 scissoring) and 1188 cm-1 (NH3+ rocking) [249]. These

Raman bands can be assigned to the vibration modes of MC-LR (Table 3.1) and

indicate that the toxin molecules were captured by the Fab' fragments at a close

distance from the nanoparticle surface. Therefore, spontaneous read out of the MC-

LR SERS signal after its capture by the extractor nanoparticle is possible and can be


used for the rapid screening of the toxin. Despite the lower resolution of the handheld

Raman spectrometer, the spectrum of the functionalized nanoparticles after their

binding to the toxin (Figure S3.8b, supplementary material) still show the emergence

of new emerging bands at 1570 cm-1 (benzene ring and COO- stretches), 1371 cm-1

(COOH stretch, CO stretching, CH3 symmetric bending and CH2 scissoring), 1236

cm-1 (NH3+ rocking and amide III vibrations), 892 cm-1 (C-COO- stretch and CH2

rocking), 853 cm-1 (C-C stretch, CH2 rocking and CH3 rocking) that can be assigned

to the vibration modes of MC-LR (Table 3.1).

3.7.4. Direct SERS detection of MC-LR in aqueous solution

The captured MC-LR molecules were released by dispersing the toxin-bound

nanoparticles in glycine buffer (pH 2.5) [268]. After removing the buffer, using size

exclusion column, the purified toxin was scanned on gold coated silicon nanopillar

substrate (Figure 3.3a,b). The SERS spectra in Figure 3.3a,b were in strong

agreement with the reference spectra of MC-LR by the InVia and handheld Raman

spectrometers (Figure 3.1a,b). These results confirmed that the toxin was

successfully captured by the extractor nanoparticles.

To confirm the selectivity of extractor nanoparticles towards MC-LR, we

carried out negative and positive control tests. For the negative control test, the

nanoparticles were mixed with blank water sample. After 15 minutes, the extractor

nanoparticles were collected by magnetic separation, washed with PBS (pH 7.4) and

mixed with glycine buffer (pH 2.5). After removing the buffer, the liquid phase was

loaded on gold coated nanopillar substrate and screened using handheld Raman

spectrometer. As indicated by the SERS spectrum of the treated water sample

(depicted by the green spectrum in Figure 3.3b) MC-LR was not detected in the

negative control sample. For a positive control test, the nanoparticles were reacted

with skim milk that is spiked with 5x10-6 M L-cysteine. The spiked milk sample was

prepared by adding 50 µL of 10-5 M L-cysteine (in PBS, pH 7.4) to 50 µL of skim

milk. After the glycine buffer treatment and magnetic separation of the nanoparticles,

the liquid phase was screened using SERS. As indicated by the SERS spectrum of

the spiked milk sample (depicted by the red spectrum in Figure 3.3b), no

characteristic Raman bands of L-cysteine and milk proteins were detected. The

inability of the functionalized nanoparticles to bind to foreign targets is attributed to


the highly selective interaction between the Fab' fragments and the MC-LR antigen.

These results indicate the selectivity of the new method in the detection of MC-LR.

Figure 3.3: SERS spectra of (a) MC-LR on gold coated nanopillar substrate by the

InVia Raman microscope, (b) by the handheld Raman spectrometer. The green

spectrum represents a blank water sample (negative control) and the red spectrum

represents a spiked milk sample (positive control).


3.7.5. SERS quantification of MC-LR

For the quantification of MC-LR by SERS, standard solutions of the toxin were

screened on gold coated silicon nanopillar substrate by the handheld Raman

spectrometer. The intensity of the Raman band at 1441 cm-1 was responsive to the

change in the concentration of MC-LR (Figure 3.4a,b). The relationship between the

Raman band intensity at 1441 cm-1 and log of the concentration of MC-LR was

found to follow the linear regression equation y = 134.65x + 3358.2 with a high

correlation coefficient (R2) of 0.9975 (Figure 3.4a). The LOQ of MC-LR by SERS

was 10 fM which is significantly lower than the LOQ of most methods reported in

the literature (Table 3.2) and satisfies the clinically required detection limit for

chronic exposure to the toxin.

Figure 3.4: (a) The relationship between log the concentration of MC-LR and the

SERS intensity at 1441 cm-1, (b) the change in the Raman signal intensity at 1441

cm-1 with the concentration of MC-LR.

Table 3.2: Comparison of various analytical methods for the detection of MC-LR

Analytical technique LOD Ref.

Chromatography 10 fM-3 pM 182, 183, 185, 269

Immunoassay 2 pM-30 pM 192, 195, 270, 271, 272

Electrochemistry 0.037 fM-99 pM 197, 202, 273, 274, 275, 276

Fluorescence Spectroscopy 7 pM-0.1 nM 208, 277, 278, 279

Chemiluminescence 32 pM-6 pM 280-281

SPR 73 pM 282

PPI assay 10 nM 204

SERS (indirect) 5 pM-8.6 pM 227-228

SERS (direct) 0.012 fM This study


3.7.6. Direct SERS Detection of MC-LR in blood plasma and cross-validation

against ELISA

The functionalized nanoparticles were used to capture MC-LR from a spiked

horse blood plasma sample. After interaction with the sample, the nanoparticles were

magnetically separated and the captured toxin molecules released using glycine

buffer. The purified toxin was screened by SERS on gold coated silicon nanopillar

substrate using handheld Raman spectrometer (Figure 3.5). The acquired SERS

spectrum showed notable match to that of MC-LR standard in Figure 3.1b (Table

3.1). The concentration of LC-MR in the purified sample was quantified by SERS

and found to be 1.38x10-10 M.

The SERS determination of MC-LR in the spiked blood plasma sample was

cross-validated by ELISA which confirmed the presence of MC-LR in the purified

sample at a concentration of 1.43x10-10 M (0.143 µg/L) (Figure S3.4, supplementary

material). Therefore, the % recovery obtained for the SERS quantification, as

compared to ELISA, was calculated to be 96.5 %.

Figure 3.5: SERS spectrum of MC-LR after extraction from spiked horse blood

plasma sample using gold coated silicon nanopillar substrate and handheld Raman

spectrometer.


3.7.7. Direct SERS detection of MC-LR in spiked blood plasma by paper based

substrate and handheld Raman spectrometer

To compensate for the high cost of the gold coated silicon nanopillar substrate,

we prepared gold nanoparticle ink by the turkevich citrate reduction method [232,

283] and used it to develop paper-based SERS substrate [157]. The nanoparticle ink

showed a prominent plasmon absorption band at 528 nm (Figure S3.5,

supplementary material). The average size of developed nanoparticles was

determined by SEM to be 60 nm (Figure S3.6b, supplementary material). The paper-

based substrate was utilized to the SERS detection of MC-LR (1x10-9 M) by

handheld Raman spectrometer. Figure 3.6a depicts the SERS spectrum of the toxin

on the paper-based substrate and it was in general agreement to that of the toxin on

gold coated silicon nanopillar substrate (Figure 3.1b, Table 3.1). Using MC-LR as a

probe molecule, the SERS enhancement factors (E. F.) of the gold coated silicon

nanopillar and paper-based substrates were calculated using the equation E. F. =

(ISERS/IRS) (CRS/CSERS) [157, 284], where ISERS and IRS represent the

intensities of the MC-LR Raman band at 1540 cm-1 at MC-LR concentrations of 10-9

M (CSERS) and 10-5 M (CRS), respectively. The E. F. values were found to be

2.5x106 and 3x105 for the nanopillar and paper based substrates, respectively. The

paper-based substrate was also used for the screening of MC-LR in spiked horse

blood plasma using the handheld Raman spectrometer. The SERS spectrum of the

purified MC-LR on paper-based substrate is depicted in Figure 3.6b and shows

strong agreement with the MC-LR spectrum in Figure 3.6a.

3.7.8. Reproducibility of the SERS measurements by the paper-based substrate

To test the reproducibility of the SERS detection of the toxin by the paper-

based substrate, we repeated the experiment 9 times. The spectra in Figure 3.6c

clearly indicate that the SERS detection of MC-LR by the paper-based substrate was

highly reproducible. These results indicate the strong potential of the paper-based

substrate for the SERS screening of biomolecules in biological fluids.

3.7.9. Recycling of functionalized nanoparticles

To demonstrate the potential recyclability of the functionalized nanoparticles

for repeated testes, they were re-used to capture MC-LR from a second spiked

sample. After using the functionalized nanoparticles to capture MC-LR from the first


spiked sample, they were treated with glycine buffer to release the toxin and free up

the nanoparticles for the second spiked sample. After capturing and releasing MC-

LR from the second sample, the purified toxin was screened on the paper-based

substrate by handheld Raman spectrometer. The SERS spectrum of MC-LR from the

second sample is depicted in Figure 3.6d and matches that of the toxin in Figure

3.6a,b. This result clearly indicates the successful recycling of the functionalized

nanoparticles to capture of the toxin from spiked blood plasma samples. This success

can be attributed in part to the ability of the glycine release buffer (pH 2.5) to break

the antibody – antigen weak binding without causing the MC-LR antibody Fab'

fragments to lose their activity [285-286] Therefore, the use of glycine as a release

buffer did not compromise the capture efficiency of the functionalized nanoparticles

towards the toxin.

Figure 3.6: SERS spectra of (a) standard MC-LR, (b) MC-LR after extraction from

spiked horse blood plasma sample, (c) Nine SERS measurements of MC-LR from

spiked blood plasma, (d) MC-LR after extraction from a second spiked sample by

the recycled nanoparticles. The spectra were collected using paper-SERS substrate

and handheld Raman spectrometer.


3.8. Conclusion

We demonstrated, for the first time, a combined method for the selective

extraction and sensitive label-free SERS detection of MC-LR in aqueous and

biological media within the hour. Gold coated magnetic nanoparticles were

functionalized with oriented surface layer of MC-LR Fab' fragments to capture of the

toxin. Due to the high surface coverage of the nanoparticle surface with the small-

size Fab' fragments, the captured MC-LR molecules were immobilized and

concentrated at a close distance from the nanoparticle surface. This led to the

detection of MC-LR Raman vibration modes by the functionalized nanoparticles.

Glycine buffer was used to release the captured MC-LR molecules from the

nanoparticle surface without compromising the activity of the Fab' fragments.

Consequently, the regenerated functionalized nanoparticles can be re-used to capture

of MC-LR from multiple samples. For the SERS quantification of MC-LR, the

purified toxin was screened on gold coated silicon nanopillar substrate by handheld

Raman spectrometer. The LOQ of the SERS quantification was found to be 10 fM

which is well below the clinically required detection limit for chronic exposure to the

toxin. To compensate for the high cost of the nanopillar substrate, gold nanoparticle

ink was prepared by simple reduction method and used to develop inexpensive

disposable paper-based SERS substrate. The SERS enhancement factor of the paper-

based substrate was found to be 3x105. The paper-based substrate was utilized for the

reproducible screening of MC-LR by handheld Raman spectrometer. The new

methodology was cross-validated against ELISA. The % recovery of the SERS

detection was found to be 96.5 % when compared to ELISA. The new method is

rapid and takes place within the hour. Therefore, the new method has strong potential

for the cost-effective, rapid and sensitive detection of toxins and protein biomarkers

in environmental and biomedical applications.



Calculation of nanoparticle concentartion in the SERS ink

The SEM imgaes in figuer S7 indicate that the naoparticles are spherical and their

avarage diamter = 60 nm.

The volunme of the spherical nanoparticle = 1.13x10-13 cm3

Weight of gold chloride used for the synethis of NPs = 0.003 g

Weight of pure gold in the gold chloride salt = 0.0017 g

Density of gold = 19.30 g cm-3

Equivalent volume of gold = 0.0017 / 19.30 = 8.8x10-5 cm3

Number of nanoparticles in the colloid = 8.8x10-5 / 1.13x10-13 = 7.78x108 NPs

Concentration of NPs in the SERS ink = 7.78x108 / 0.5 = 1.556x109 NPs mL-1

Figure S3.1: Structure of Microcystin-LR


Figure S3.2: SERS spectrum of intact anti MC-LR antibody on SERS nanopillar

substrate using handheld Raman spectrometer.

Figure S3.3: SERS spectrum of antibody F(ab')2 fragments on SERS nanopillar



Figure S3.4: ELISA measurements and calibration curve of MC-LR.

Figure S3.5: UV spectrum of nanoparticle ink used for the preparation of paper-

based SERS substrate.


Figure S3.6: Images of (a) the nanoparticle colloid, (b) SERS ink, (c) squares of A4

paper after coating with SERS ink.

Figure S3.7: SEM images of (a) paper before coating, (b) nanoparticles on A4 paper

substrate, (c) A4 paper after coating with gold nanoparticles.


Figure S3.8: SERS spectra of the functionalized gold coated magnetic nanoparticles,

(a) before interaction with MC-LR and (b) after the capture of MC-LR. The spectra

were collected using the handheld Raman spectrometer.

86 Dual biosensing of protein biomarkers in human blood by recyclable plasmonic probes

Chapter 4: Dual biosensing of protein

biomarkers in human blood by

recyclable plasmonic probes

This chapter is made up of the following journal article submitted for publication in

Analytical Chemistry Journal.

Spectroelectrochemical Nanosensor for the Determination of Cystatin C in

Human Blood

Waleed A. Hassanain, Emad L. Izake, Godwin A. Ayoko

DOI: 10.1021/acs.analchem.8b02121

https://pubs.acs.org/doi/abs/10.1021/acs.analchem.8b02121?mi=aayia761&af=R&AllField=nano&target=default&targetTab=std

Dual biosensing of protein biomarkers in human blood by recyclable plasmonic probes 87






1. they meet the criteria for authorship in that they have participated in the conception,


expertise;

2. they take public responsibility for their part of the publication, except for the


3. there are no other authors of the publication according to these criteria;

4. potential conflicts of interest have been disclosed to (a) granting bodies, (b) the



5. they agree to the use of the publication in the student’s thesis and its publication

on the QUT’s ePrints site consistent with any limitations set by publisher

requirements.


W.A. Hassanain, E.L. Izake, G.A. Ayoko, Spectroelectrochemical Nanosensor for the

Determination of Cystatin C in Human Blood, Analytical Chemistry, 2018, 90, 10843-10850.



authorship.


Name Signature Date


Waleed A. Hassanain









4.1. Preface

This chapter demonstrates the utilization of the optical and conductive

properties of SERS substrates for the dual Raman / electrochemical nanosensing of

proteins in human blood. Our novel oriented immobilization strategy (demonstrated

in chapter 3) was utilized to develop a highly efficient 2D extractor chip for the

selective capture of the protein biomarker cystatin C (CST-C) form human blood.

The thiol chemistry of the purified protein was chemically modified to influence its

oriented immobilization onto a conductive SERS substrate. This led to reproducible

and ultra-sensitive detection of CST-C for the first time by SERS. It also influenced

the complementary electrochemical detection of the protein by reductive desorption

method. In addition, a simple electrochemical recycling approach to regenerate the

extractor chip was also demonstrated.


4.2. Abstract

The detection of protein biomarkers for the clinical diagnosis of diseases

requires selective and sensitive methodologies and biosensors that can be easily used

at pathology labs and points of care. An ideal methodology would be able to conduct

multimode screening of low and high concentrations of proteins in biological fluids

using recyclable platforms. In this work, we demonstrate a novel nanosensing

methodology for the dual detection of cystatin C (CST-C), as a protein biomarker

model, in blood plasma by surface enhanced Raman spectroscopy and

electrochemistry. The new methodology utilizes the thiol chemistry of biomolecules

to develop target-specific and recyclable extractor chip for the rapid isolation of

protein biomarkers from blood plasma. This is followed by the rapid reduction of the

disulfide bonds within the isolated protein to influence its oriented immobilization

onto conductive gold coated silicon nanopillar substrate via stable gold–sulphur (Au-

S) bonds. The oriented immobilization led to reproducible surface enhanced Raman

spectroscopy (SERS) measurements of the reduced protein (RSD =3.8%) and

allowed for its direct electrochemical determination. After the SERS measurement,

differential pulse voltammetry (DPV) was used to desorb the analyte from the

substrate and generate a reduction current that is proportional to its concentration.

CST-C was determined down to 1 pM and 62.5 nM by SERS and DPV, respectively,

which satisfy the requirements for monitoring Alzheimer’s and kidney failure

diseases. The new dual nanosensing methodology has strong potential for

miniaturization in a lab-on-a-chip platform for the screening of many protein

biomarkers that have disulfide bond structure.

4.3. Keywords

Dual nanosensing, Recyclable chip, Molecular diagnostics, Kidney & Alzheimer’s

diseases.


4.5. Introduction

There is a growing demand within the molecular diagnostics market for rapid

and cost-effective sensing methodologies that can be miniaturized and used for

reliable screening of biomarkers in biological fluids at pathology/research labs and

points of care.

Electrochemical biosensors have been demonstrated in the literature for the

detection of protein biomarkers in biological fluids [287]. Despite their simplicity,

the selectivity of these sensors still requires improvements to allow for their

miniaturization and utilization at pathology labs and points of care [288-289].

Surface enhanced Raman spectroscopy (SERS) is a powerful analytical tool

that can provide molecular structure identification of proteins at ultra-trace

concentrations [33]. In SERS, the Raman bands intensities are significantly enhanced

when the analyte molecules become adsorbed at the gaps between the nanostructures

of a noble metal surface where the localised surface plasmon resonance (SPR) on the

neighbouring nanostructures couple and generate a large electromagnetic field that is

experienced by the analyte molecules [10, 290]. In addition, when the analyte

molecules become adsorbed on the metal substrate, their HOMO and LUMO energy

levels interact with the Fermi energy level of the metal surface and a charge transfer

complex is formed. When the substrate interacts with incident light of energy that

matches the charge transfer transition energy, electron transition occurs between the

molecular orbitals of the analyte and the Fermi level of the substrate. This causes the

molecular polarization of the analyte molecules to change and produces a chemical

enhancement [291].

The conductive property of metallic SERS substrates qualifies them as

combined SERS / electrochemical platforms to acquire spectral information about

the molecular structure and concentration of analytes [26, 292]. Despite the high

sensitivity of SERS, the measurements often suffer from low reproducibility due to

the non-uniform distribution of hotspots, and the random orientation of the analyte

molecules on the SERS substrate [215, 293, 294]. To address this problem, we

demonstrated in a previous work a highly patterned SERS substrate that has uniform

hotspots distribution for the detection of proteins by a handheld Raman spectrometer.

By using a raster orbital scanning mode (ROS), the handheld device was able to


acquire an average SERS signal from the analyte molecules on the substrate’s

surface and reduce the SERS signal variability between repeated measurements [147,

225, 295]. However, the SERS signal variability that is caused by the random

orientation of the adsorbed analyte molecules was not addressed. Therefore, in this

work, we were motivated to develop a dual biosensing method for protein

biomarkers that takes advantage of the attractive characteristics of SERS and

electrochemistry and address the problem of SERS signal variability due to random

orientation of analyte molecules.

CST-C is a low molecular weight protein that belongs to type 2 cystatin gene

family and is used as a biomarker for kidney failure disease [296-297]. CST-C is also

a biomarker for cardiovascular diseases, Alzheimer's disease, cancer and type-2

diabetes [298-302]. In addition, CST-C levels have been reported to change in

patients with thyroid dysfunction and glucocorticoid therapy [303-304]. A number of

methods have been reported for the detection of CST-C in the biological fluids such

as: chromatography, ELISA, particle enhanced turbidimetric immunoassay (PETIA),

particle-enhanced nephelometric immunoassay (PENIA), radio immunoassay,

fluorescence, NMR and NIR methods [305-316]. Despite their sensitivity, the

reported methods suffer from disadvantages that limit their use for the rapid

screening of CST-C [316-317]. Mi et al. reported a sensitive photo-electrochemical

indirect detection method for the quantification of CST-C in serum where they

monitored the change in the photocurrent of a nanobody-TiO2 nanotube array after

its immunoreaction with CST-C [318]. However, this method required lengthy

procedures for the functionalization of the TiO2 array and the binding of the protein

(~14 hours). In addition, the method did not directly detect CST-C and the developed

immunosensor was not recyclable for repeated use.

To the best of our knowledge, no Raman or electrochemical studies have been

performed for the direct detection of CST-C. In this work, a SERS/electrochemistry

dual biosensing method was developed to detect CST-C by handheld devices for the

clinical diagnosis of many diseases such as renal, heart and Alzheimer’s diseases

[319-320].



4.6.1. Synthesis of antibody fragments and functionalization of the extractor

chip

F(ab')2 antibody fragments were synthesized by digesting anti-CST-C antibody

using Pierce Mouse IgG1 F(ab')2 preparation Kit. 90 µL of the produced F(ab')2

fragments were mixed with an equal volume of 0.2 mM neutral TCEP to reduce the

disulfide bonds in their hinge region and produce thiol-ended Fab' fragments (R-SH).

The mixture was loaded onto the gold coated silicon nanopillar chip and left for 40

min at room temperature to bind thiol-ended Fab' fragments to the gold surface of the

chip via Au-S bonds. The chip was then washed six times with 200 µL of 1x PBS to

remove any unbound Fab' fragments.

To backfill the remaining bare sites on the chip surface, 20 µL of 10-6 M

butanethiol (dissolved in 1x PBS, pH 7.4) were loaded and left for 1 hour at 4 °C to

form Au-S bonds between the bare gold sites and the alkanethiol. The chip was then

washed six times with 200 µL of 1x PBS to remove the excess butanethiol. After

washing, the chip was suspended in 200 µL of 1x PBS (pH 7.4) and stored at 4 °C

for future use.

4.6.2. Isolation and purification of CST-C from blood plasma

For the selective capture of CST-C from biological fluids by the extractor chip,

a 200 µL of a human blood plasma sample was loaded onto the chip and left to stand

for 15 min. The chip was then washed 5 times with 200 µL of 1x PBS to remove any

weakly adsorbed molecules from its surface.

To release the protein from the extractor chip, it was immersed in 100 µL of

glycine.HCl buffer (pH 2.5) for 5 min. The chip was then removed and re-used to

capture CST-C from a new human blood plasma sample. To remove the glycine

buffer from the released protein sample, it was loaded onto size exclusion column

and the CST-C protein eluted within 5 min using 500 µL of 1x PBS (pH 7.4) as the

eluent.

4.6.3. Control tests

Since CST-C exists naturally in blood, it was not possible to carry out positive

and/or negative control tests using blood plasma matrix. Therefore, we carried out


the interference study using an aqueous solution of L-cysteine in BSA, blank PBS

buffer (pH 7.4), insulin and phenylalanine.

For positive control test, 200 µL of L-cysteine in 0.1% BSA (pH 7.4) were

loaded onto a clean extractor chip for 15 min. The chip was then washed 5 times with

200 µL of 1x PBS and immersed in 100 µL of glycine.HCl buffer (pH 2.5) for 5 min.

After removing the chip, the liquid phase was loaded onto a size exclusion column to

remove the buffer. Finally the column was eluted using 500 µL of 1x PBS (pH 7.4)

and the collected eluate screened by SERS. For the negative control test, 20 µL of 1x

PBS solution was loaded onto a clean extractor chip, the above procedures carried

out and the eluate from the size exclusion column screened by SERS. For the insulin

and the phenylalanine tests, the protein or amino acid solution (10-5 M in PBS, pH

7.4) was loaded onto the extractor chip and the capture/release procedures carried

out. After removing the chip, the liquid phase was loaded onto a size exclusion

column to remove the buffer. Finally the column was eluted using 500 µL of 1x PBS

(pH 7.4) and the collected eluate screened by SERS.

4.6.4. Determination of CST-C in human blood plasma by SERS and DPV

To determine CST-C in biological fluids, the protein was isolated from blood

plasma sample using the extractor chip. 20 µl of the isolated protein were chemically

reduced using TCEP onto a gold coated silicon nanopillar substrate and left to stand

for 10 min. The substrate was then washed 5 times with 200 µL of 1x PBS and

screened by SERS and the intensity of the band at 1363 cm-1 was monitored to

determine the concentration of the protein using the developed SERS calibration

curve.

For the electrochemical quantification of the protein in blood, 200 µL of a

human blood plasma sample was extracted using the extractor chip and reduced

using TCEP. 20 µl of the isolated protein were reduced by TCEP onto a clean gold

coated silicon nanopillar substrate and left to stand for 10 min. The substrate was

then washed 5 times with 200µL of 1x PBS and the protein quantified by DPV.

4.6.5. Recycle of functionalized extractor chip by cyclic voltammetry (CV)

To recycle the extractor chip, the inactive antibody fragments were

electrochemically desorbed from the gold coated silicon nanopillar substrate by CV.

The extractor chip was utilized as the working electrode in a three electrode cell.


0.1 M KOH solution was used as an electrolyte. The cell potential was swept from

0.1 V to -1.4 V using a potential step of 0.004 V and a scan rate of 0.1 VS-1. 130 CV

cycles were carried out for the removal of the antibody fragments. The recycled chip

was then washed 3 times with 1x BPS buffer, re-functionalized with fresh thiol-

ended Fab' fragments and screened by SERS to confirm the attachment of the

fragments.



4.7.1. SERS spectrum of authentic and reduced CST-C

The molecular structure of human CST-C contains 120 amino acids with four

cysteine residues connected by two disulfide bonds [321]. To analyse CST-C by

Raman spectroscopy, we acquired, for the first time, its Raman spectrum where

authentic samples of the protein were loaded onto gold coated silicon nanopillar

substrates and screened by a benchtop and handheld Raman spectrometers in the

raster orbital scanning mode (Figures S4.1, supplementary material and 4.1a,

respectively) [263]. The background noise and florescence were automatically

corrected by the spectrometer software [295]. As indicated by the figures, the Raman

spectrum collected by the benchtop instrument showed significant fluorescence

background while that collected by the handheld device has less resolution.

However, due to its portability, and ability to rapidly acquire an average SERS signal

from the entire SERS substrate, the handheld device was utilized for the rest of this

study [295, 322]. The Raman vibration modes of CST-C, as recorded by the

handheld Raman spectrometer, are depicted in Table S4.1 (supplementary material)

[68, 72, 254, 259, 260, 262, 323-330].

As indicated by Figure 4.1b, the repeated SERS screening of CST-C on the

gold coated silicon nanopillar substrate showed noticeable variations especially in

the wave number regions of 1528-1590, 1261 and 1197 cm-1 vibration modes. These

variations are attributed, in part, to the random orientation of the protein molecules

onto the substrate [215, 293, 294].

For the determination of proteins by SERS, the substrate should have uniform

distribution of hotspots, the analyte molecules should take a unified orientation on

the substrate and the SERS measurement should represent the entire sample loaded

on the substrate [215, 294, 331]. Therefore, to screen CST-C by SERS, we utilized a

highly ordered gold coated silicon nanopillar substrate that has uniform hotspot

distribution (Figure S4.2, supplementary material) [147]. We also carried out the

measurements using the handheld Raman spectrometer in the raster orbital mode to

collect an average SERS signal. To orient the protein molecules in one direction on

the substrate, we modified the molecular structure of human CST-C. Cystatin C

contains two disulfide bonds between four cysteine residues. Therefore, we reduced


the disulfide bonds in the protein structure to generate free SH terminal groups that

form Au-S bonds with the gold surface of the substrate (Figure S4.3, supplementary

material) and assemble the protein molecules in an upward orientation onto the

substrate surface [332].


Figure 4.1: Raman spectra of (a) standard CST-C (the red line depicts the Raman

spectrum of the bare substrate), (b) different scans of standard CST-C, (c) CST-C

standard after reduction with TCEP, and (d) 17 measurements of seventeen reduced

CST-C samples in the concentration range 10-7 to 10-12 M on 17 independent gold

coated silicon nanopillar substrates.

The SERS spectrum of the reduced protein showed general agreement with that

of the unreduced protein (Figures 4.1c and 4.1a), with new bands appearing at 1363,

1179, 931, and 467 cm-1 (Table S4.1, supplementary material). The new band at 467

cm-1 indicates the immobilization of the C-S bonds of the reduced protein onto the


plasmonic gold surface of the substrate, thus experiencing strong electromagnetic

enhancement [333]. The SERS measurements of the reduced protein, on seventeen

independent gold coated silicon nanopillar substrates, were highly reproducible

(Figure 4.1d), as compared to those of the unreduced protein (Figure 4.1b).

4.7.2. Manufacture of CST-C extractor chip

To manufacture an extractor chip for CST-C, thiol-ended Fab' fragments (R-

SH) of anti CST-C antibody were synthesized and immobilized onto a gold coated

chip via Au-S bonds [334]. This allows the binding sites of the immobilized antibody

fragments to align in an upward orientation and, maximizes the capturing efficiency

of the extractor chip towards the antigen by alleviating the steric hindrance that

prevents the binding process [147, 243, 247, 295, 332]. In addition, the small size of

the Fab' fragments increases their surface loading on the substrate and increases the

overall capturing capacity of the extractor chip [229].

Following its functionalization with the antibody fragments, the remaining bare

sites on the chip surface were backfilled with butanethiol to prevent nonspecific

binding events by foreign molecules. The formation of Au-S bonds between the

alkanethiol and the extractor chip alters the charge of its gold surface. This change

and leads to a charge transfer between HOMO/LUMO energy levels of butanethiol

and the Fermi energy level of the gold [291]. The change in the surface charge also

leads to the displacement of a few antibody fragment molecules from the gold

surface and the re-orientation of the lying down antibody fragment to assume an

upward position [332, 335]. This mechanical switch of the lying-down antibody

fragments boosts the capturing efficiency of the functionalized chip towards the

target protein.

4.7.3. Isolation of CST-C from blood plasma and control tests

The extractor chip was used to selectively capture CST-C from blood plasma.

We attempted to directly determine CST-C by SERS (in situ SERS detection) after

its binding to the antibody fragments on the extractor chip. However, the binding of

the protein caused minor changes to the SERS spectrum of antibody fragments. This

may be attributed to the fact that the bound protein is lying on top of the antibody

fragment at a far distance from the plasmonic surface of the extractor chip and

therefore experience only weak SERS enhancement. In addition, the antibody


fragments and the protein have some common amino acid building blocks and

therefore it is difficult to reliably quantify the protein concentration by in situ SERS

measurements. This observation has been also reported by Hassanain et al. for the

determination of microcystin LR by SERS [295]. Therefore, for the direct detection

of CST-C by SERS, we attempted the release and purification of the protein from the

chip using glycine buffer (pH 2.5) [268]. The protein was then reduced and screened

by SERS. As indicated by Figure 4.2i, the acquired spectrum of the reduced protein

was in general agreement with that of the reduced CST-C standard in Figure 4.1c.

This result indicates the selective capture of the target protein from the biological

matrix.

Figure 4.2: Raman spectra of (i) extracted and reduced CST-C from human blood

plasma, (ii) positive control, (iii) negative control, (iv) insulin, and (v) phenylalanine

on gold coated silicon nanopillar substrate.

To further demonstrate the selectivity of the extractor chip towards CST-C,

positive and negative control tests were carried out using L-cysteine in 0.1% BSA

(positive control) and blank PBS (negative control) samples. We also tested the

selectivity of the extractor chip against insulin as another protein that has have


disulfide bond structure and against phenylalanine as hydrophobic amino acid that

exists in biological fluids. After carrying out the capture and release processes, the

samples were screened by SERS. As indicated by Figure 4.2(ii-v), no Raman spectra

were detected from the positive and negative control samples as well as the insulin

and phenylalanine molecules which confirm the inability of the extractor chip to bind

biomolecules other than CST-C.

4.7.4. SERS quantification of CST-C

For the SERS quantification of CST-C (in the reduced form), the intensity of

the Raman band at 1363 cm-1 in the reduced protein spectrum was monitored at

different concentrations and found to monotonically increase with the protein

concentration in the working range of 1x10-7 M to 1x10-12 M (Figure 4.3a). The

relationship between the intensity at 1363 cm-1 Raman band and log the

concentration of the reduced CST-C was found to follow the linear regression

equation y = 93.122x + 1495.3 (Figure 4.3b). The correlation coefficient (R2) was

found to be 0.9986 while the LOQ of the SERS quantification was 1 pM.

Figure 4.3: (a) Raman band intensity of reduced CST-C at 1363 cm-1 in the

concentration range of 100 nM to 1 pM, and (b) SERS calibration curve of reduced

CST-C within the same concentration range.

The RSD in the SERS measurements was calculated using the intensity of the

Raman band at 1363 cm-1 and found to be 3.8% (n = 15). The RSD in the SERS

measurements between days was 7.45%. The wide working range and excellent


sensitivity of the SERS screening confirms its potential value in monitoring CST-C

blood levels in renal, heart and Alzheimer’s diseases [319, 336]. The uniform

distribution of hotspots on the nanopillar substrate surface has been reported to lead

to RSD values of ≤ 8% in SERS measurements that were carried out on substrate

different batches [147]. Therefore, the low RSD of 3.8% in the SERS measurements

of CST-C is attributed to the oriented immobilization of the reduced CST-C

molecules onto the uniform hotspots of the substrate.

4.7.5. Electrochemical quantification of CST-C

The electrochemical desorption of alkanethiols from a gold substrate can be

achieved by reducing the Au-S bonds between the thiol compound and the gold at a

negative potential. The magnitude of the DPV current is proportional to the

concentration of the alkanethiol molecules on the substrate [337]. The immobilized

reduced CST-C molecules on the conductive nanopillar substrate represent an

alkanethiol. Therefore, we used DPV for their reductive desorption and

electrochemical quantification after the SERS measurement. As indicated by Figures

4.4a, b, the electrochemical desorption of the reduced protein resulted in a negative

potential at -0.92 V of a magnitude that is proportional to the reduced protein

concentration in the range 6.25x10-8 M to 1x10-6 M. The relationship between the

DPV current and the concentration of reduced CST-C followed the linear regression

equation y = 4.3657x + 9.6945, R2 = 0.9932 (Figure 4.4c). The LOQ of the

electrochemical quantification of CST-C was found to be 62.5 nM which is suitable

for the determination of high levels of the protein in patients at a high risk of

developing kidney failure disease [327]. The sensitivity of the electrochemical

measurement of CST-C is attributed to the high population of gold nanostructures on

the surface of the nanopillar substrate where they act as active sites for the

electrochemical measurement of the reduced protein.

The LOQ of the SERS / DPV determinations are compared to other CST-C

detection methods in Table S4.2 (supplementary material) [309, 310, 312-316, 318,

338, 339]. As indicated by the table the developed dual sensing method compares

favourably to other detection methods in terms of simplicity, wide working range,

recyclability of the used sensors, and capacity to provide molecular structure

information of the screened biomolecule similar to Mass spectrometric detection.


Due to their 0.4 x 0.4 cm small surface area, the extractor chip and SERS

substrate can be miniaturized in “a lab on a chip” platform for molecular diagnostics

where a few microliters of blood plasma can be injected onto the extractor chip and

the captured protein released to a detection zone where it is screened onto the

conductive SERS substrate by commercial handheld Raman and potentiostat devices.

Figure 4.4: (a) Electrochemical desorption of CST-C by DPV (the black arrow

depicts the desorption potential of CST-C at -0.92 V. The red line depicts the DPV of

a blank substrate), (b) DPV pulse of reduced CST-C in the concentration range

0.0625 μm to 1 μm, and (c) electrochemical calibration curve of reduced CST-C by

DPV.


4.7.6. Determination of CST-C in human plasma

To demonstrate the potential of the new dual biosensing for molecular

diagnostics, it was applied for the quantification of CST-C in human blood by SERS

and DPV. The protein was extracted from 20 µL of human blood plasma sample,

reduced and quantified by SERS on the nanopillar substrate. The concentration of the

protein in the human plasma sample was found to be 9.66x10-8 M (n=6). Another

aliquot of the blood plasma sample was rescreened by ELISA for the cross-validation

of the SERS measurement. The concentration of CST-C in the sample by found to be

9.86x10-8 M (n=6) by the ELISA method (Figure S4.4, supplementary material).

Therefore, the average % agreement between the SERS and ELISA determinations

was 97.97 % which indicates the high capture efficiency of the extractor chip.

For the electrochemical quantification of CST-C, the protein was captured

from a second human blood plasma sample (20 µL), reduced and loaded onto a new

nanopillar substrate. The protein concentration was first screened by SERS then

desorbed by DPV. The SERS and DPV quantification of the protein in the sample

were found to be 1.42x10-7 M (n=13) and 1.61x10-7 M (n=3), respectively.

Therefore, the average % agreement between the DPV and SERS quantifications was

113.38% and 111.08 % between the DPV and ELISA quantifications.

Yang et al. recently reported an ultra-sensitive electrochemical immunoassay

for CST-C down to 75 fM in blood [339]. The method utilized 2 complementary

target-specific antibodies to bind CST-C from human serum within 120 min. The

sandwich immunoreaction induced proximity hybridization between two DNA

strands and caused the displacement of an output DNA. The output DNA was then

loaded onto a modified gold electrode and incubated for 5 hours before its

electrochemical detection by DPV. Therefore, this method utilized a complex

analytical protocol that involved the use of 8 different biomolecules for the indirect

detection of CST-C. Considering the complexity of Yang’s method and the fact that

the median concentration of CST-C in healthy subjects is ~1067 ng mL-1 (80 nM)

[320], the new dual nanosensing method presented in this work is satisfactory for the

rapid and cost-effective detection of CST-C in healthy subjects and kidney failure/

Alzheimer’s patients.


4.7.7. Recycle of the extractor chip

To reduce the cost of protein analysis, the extractor chip was recycled by CV to

electrochemically reduce the Au-S bonds and desorb the inactive antibody fragments

from the gold surface of the chip [340]. The electrochemical recycling process was

monitored by SERS (Figure S4.5 a-c). As indicated by the figure, the Raman bands

of the antibody fragments gradually disappeared with the progress of the CV cycles.

This observation was supported by the gradual decrease in the magnitude of the

reduction current of the fragments at -0.8 V (Figure S4.6 a, b).

After the electrochemical desorption, the recycled substrate was re-functionalized

with new antibody fragments and screened by the handheld Raman spectrometer.

The SERS spectra of the extractor chip before recycle, after 130 CV and after re-

functionalization with new antibody fragments are depicted in Figure 4.5 i, ii, iii,

respectively. The reappearance of the antibody Raman spectrum on the extractor chip

after re-functionalization confirms its successful recycle.


Figure 4.5: Raman spectra of (i) CST-C antibody fragments on the extractor chip,

(ii) extractor chip after electrochemical desorption of antibody fragments (after 130

CV cycles), and (iii) fresh CST-C antibody fragments on recycled extractor chip after

the electrochemical treatment (4.5 ii).


4.8. Conclusion

In this work we presented a dual SERS/DPV biosensor for the rapid detection

of protein biomarkers in human blood plasma. To isolate CST-C from blood plasma,

we develop target-specific and recyclable extractor chip using the thiol chemistry of

antibodies where thiol-ended antibody fragments were synthesised and assembled

onto gold coated silicon chip. To recycle of the extractor chip after use, CV was used

to electrochemically desorb the inactive antibody fragments then re-functionalize

with fresh fragments. For reproducible SERS measurements, the disulfide bond

structure of the isolated protein was chemically reduced and assembled, in a highly

oriented monolayer, onto a gold coted silicon nanopillar substrate. This led to the

sensitive quantification of CST-C in blood plasma down to 1 pM and a low RSD of

3.8% which is useful for the early diagnosis of Alzheimer’s disease. The Au-S bonds

between the assembled reduced protein and the gold surface of the substrate were

electrochemically reduced by DPV. This led to the electrochemical quantification of

CST-C down to 62.5 nM which is useful for its quantification in kidney failure

disease. Due to the small size of the used nanostructures substrates and the speed of

the new dual nanosensing methodology, it has strong potential for miniaturization in

a-lab-on a-chip device for protein analysis at pathology labs and POC.



4.9.1. Instrumentation

All the SERS measurements were carried out using a handheld Raman

spectrometer (spectral resolution = 12 cm-1, Ocean Optics, USA), in the raster orbital

scanning (ROS) mode over the wavelength range 400-1800 cm-1. 785 nm laser

source at 5 mW laser power was used for excitation. 10 accumulations with a total

acquisition time of 1 second were used for the SERS measurements. The software

algorithm of the instrument (OceanView Spectroscopy 1.5.07) was operated to

automatically average and correct the acquired spectra for background noise and

florescence [295]. A Raman spectrum of CST-C was also acquired by the Renishaw

InVia Raman microscope (Renishaw, UK) using 785 nm laser excitation and 0.5%

laser power of 450 mW (Figure S4.1).

The electrochemical measurements were carried out using Autolab

PGSTAT204 potentiostat (Metrohm Autolab, NL) that is equipped with NOVA

1.10.5 as operating software. A standard three-electrode cell setup was established

where the extractor chip or the nanopillar substrate were utilized as a working

electrodes, Ag/AgCl/saturated KCl as a reference electrode and platinum wire as the

counter electrode. For the ELISA measurements, VERSAmax Tunable microplate

reader (Molecular Devices, USA) was used to read the optical density at 450 nm.

Software “SoftMax Pro 4.3” was used to process the acquired signal.

4.9.2. Chemicals and reagents

Recombinant human cystatin c (CST-C) standard, phosphate buffered saline

(PBS), 1-butanethiol, glycine, L-cysteine, insulin, L-phenylalanine, hydrochloric

acid, nitric acid, potassium hydroxide and bovine serum albumin were purchased

from Sigma Aldrich (USA). A monoclonal anti-cystatin-C antibody (Cyst-13,

ab24327) and human cystatin C ELISA kit (ab119589) were purchased from Abcam

(UK). Pierce Mouse IgG1 F(ab')2 preparation Kit (number 44980) and neutral tris(2-

carboxyethyl)phosphine (TCEP) solution were purchased from Thermo Fischer

Scientific (USA). All chemicals were of analytical grade and used without any

further purification. Gravity flow size exclusion columns (illustra NAP-5) were

purchased from GE Healthcare Life Science (Au). Eppendorf DNA LoBind vial 1.5

mL (Eppendorf AG, Germany). Human blood plasma was donated by the forensic

biology and analytical toxicology unit, Queensland University of Technology (QUT,


Au) under their ethical clearance. The samples were used under the Human Research

Ethics exemption by QUT (Number 1700000356). Deionized water (18.2 MΩ cm @

25°C) was used in all preparations. The glassware was cleaned by aqua regia and

thoroughly rinsed with deionized water prior to usage. Gold-coated silicon

nanopillars substrates were purchased from Silmeco company (DK). An SEM image

of the purchased substrate is depicted in Figure S4.2.

4.9.3. Preparation of CST-C standard solution

10 µg of recombinant human cystatin-C standard were dissolved in 100 µL of

1x PBS to prepare a 7.5x10-6 M stock solution of the protein. Working standards in

the concentration range of 2x10-6 M to 2x10-12 M were prepared by serial dilutions of

the stock solution using appropriate volumes of 1x PBS.

4.9.4. SERS spectra of authentic and reduced CST-C protein

A reference Raman spectrum for CST-C was not available in the literature.

Therefore, 20 µL of 2x10-7 M CST-C standard solution was loaded onto gold coated

silicon nanopillar substrate and allowed to dry. The protein was then screened by the

Renishaw InVia Raman microscope and handheld Raman spectrometer.

To acquire a SERS spectrum of reduced CST-C, 20 µL of 2x10-7 M of the

protein standard were mixed with 20 µL of 20 mM neutral TCEP reducing agent and

loaded onto nanopillar substrate. The reduced protein was allowed to stand for 10

min to bind to the gold nanostructures via Au-S bonds. The substrate was then rinsed

with 100 µL 1x PBS to remove the TCEP and any unbound protein molecules. After

drying, the substrate was scanned by handheld Raman spectrometer.

4.9.5. SERS quantification of reduced CST-C

For the quantification of the protein biomarker by SERS, 20 µL of CST-C

standard solutions in the concentration range 2x10-7 M to 2x10-12 M were mixed with

20 µL of 20 mM neutral TCEP solution and the above procedure carried out to

acquire SERS spectra of the reduced protein at different concentrations. To develop a

SERS calibration curve, the relationship between the log concentration of reduced

CST-C and the average intensity of the SERS signal at 1363 cm-1 was plotted.

4.9.6. Electrochemical quantification of reduced CST-C by DPV

For the electrochemical quantification of CST-C, 20 µL of CST-C in the

concentration range 2x10-6 M to 1.25x10-7 M were mixed with 20 µL of 20 mM


neutral TCEP, loaded onto gold coated silicon nanopillar substrates and allowed to

stand for 10 min. The substrates were then rinsed with 1x PBS buffer and utilized as

working electrodes in DPV measurements. The DPV was carried out in 0.1 M KOH

as an electrolyte and the potential of the working electrode was swept from -0.2 V to

-1.4 V using -0.005V steps and scan rate of 0.01 VS-1. The pulse amplitude and

width were set to 50 mV and 50 ms, respectively. The magnitude of the DPV current

at ~ -0.9 V, was plotted against the concentration of the CST-C standard solution.

4.9.7. Cross-validation by ELISA

To cross-validated the SERS determination of CST-C in blood, an aliquot of

the blood plasma sample was re-screened by ELISA. The ELISA calibration curve

was constructed by plotting the optical density of CST-C at 450 nm against the

protein concentration in the range 0.312–10 ng mL-1. The relationship between the

optical density at 450 nm and the protein concentration was found to follow the

regression equation y = 0.0663x + 0.0541 (R2 = 0.9985).


Table S4.1: Band assignment of the Raman spectrum of the reduced and un-reduced

forms of CST-C on gold coated silicon nanopillar substrate

Raman Shift (cm-1

) Band assignment Ref.

Un-reduced Reduced

1590 1590 C=C stretching in Tryptophan, tyrosine and

phenylalanine

68, 225, 323

1579 C=C stretching in Tryptophan, tyrosine and

phenylalanine

68, 225, 323

1559 1559 Tryptophan residues 68, 323 1541 1541 Amide II vibration modes 225, 254, 295

1448 1439 CH2 bending/ scissoring and CH deformation 68, 225, 259,

323 1355 1363 Tryptophan residues 72, 225, 324

1295 1295 Amide III vibration modes 72, 262

1274 1280 Amide III vibrations of peptide backbone 68, 72, 262,

323 1242 1242 Amide III vibrations of peptide backbone 72, 262

1203 1207 C-C6H5 stretching mode in phenylalanine/

tryptophan

68, 72, 323,

325 1179 C-N vibration mode of arginine residues 260

1126 1135 NH3+ deformation vibrations of glutamic

acid/ leucine residues, C-N stretching mode

259, 323

1088 C-N stretching mode 323

1066 C-C stretching mode 325

1021 1021 C-H in-plane bending mode of phenylalanine 68, 259, 323 997 997 Symmetric ring breathing mode of

phenylalanine

68, 225, 323,

325

924 931 C-C stretching mode backbone (a-helix

conformation), C-COO- vibrations mode

68, 295, 323,

326 886 880 CH2 rocking of arginine residues, indole ring

H-scissoring in tryptophan

68, 260, 323

848 843 Ring breathing mode of tyrosine 68, 323 763

Phenyl ring CH out-of-plane bending,

symmetric ring breathing of tryptophan

residues

68, 323

787 CH2 rocking of cysteine residues 327, 328

719 716 Methionine residues. COO- deformation and

C-C twisting of alanine and methyl aspartic

acid residues

68, 259

676 Ring breathing vibration mode 329

645 648 C-C twisting of tyrosine residues. COO-

deformation, C-C twisting of alanine and methyl aspartic acid residues

68, 259, 323,

325

618 C-C twisting mode of phenylalanine 68, 323

560 568 Tryptophan residues 68

514 516 S-S stretching mode 68 467 C-S and C-N stretching vibrations 330


Table S4.2: Comparison of different methods used for the detection of CST-C

Method LOQ Ref. Remarks

Chromatography 0.68 nM 338 Requires extensive sample preparation procedures and expensive instrumentation of large footprint.

ELISA 7.52 pM 309 Time consuming procedures (~3 hours).

PETIA 30 nM 310 Fast analysis time (7 min).

Not suitable for ultra-trace analysis.

Uses non-recyclable immunopaticles.

PENIA 17.3 nM 312 Fast analysis time (6 min).

Not suitable for ultra-trace analysis. Uses non-recyclable immunopaticles.

Radioimmunoassay 75 pM 313 Requires long incubation time (16-20 hr).

Fluorescence 1.88 pM 314 Requires long incubation time (3hr).

Provides limited structural information.

NMR 1.5 nM 315 Not suitable for service labs.

Requires instrumentation of large footprint.

NIR fluorescence 3.76 nM 316 Requires instrumentation of large footprint.

Long incubation time (2.5 hr).

Photo-electrochemical

0.72 pM 318 Uses indirect detection. Requires 40 min for binding the target protein.

Uses non-recyclable immunosensor.

Electrochemical

immunoassay

75 fM 339 Requires 8 complementary recognition molecules

and complex procedures for the protein detection.

Indirect and expensive assay.

SERS 1 pM

This

work

Wide detection range for dual sensing of high and low blood concentrations within 50 min.

Provides rich structural information of the analyte.

Uses recyclable nano probes.

Electrochemistry

(DPV)

62.5 nM


Figure S4.1: Raman spectrum of CST-C by the InVia Raman microscope.

Figure S4.2: SEM image of commercial silicon nanopillar SERS substrate.


Figure S4.3: Chemisorption of reduced CST-C onto gold coated silicon nanopillar

substrate via Au-S bond.

Figure S4.4: ELISA calibration curve of CST-C.


Figure S4.5: Raman spectra of CST-C antibody fragments on extractor chip (a) after

30 CV cycles, (b) after 60 CV cycles, and (c) after 90 CV cycles.

Figure S4.6: (a) Electrochemical desorption of the CST-C antibody fragments from

the surface of the extractor chip by cyclic voltammetry (the black arrow depicts the

desorption potential of the antibody fragments at -0.8 V), and (b) voltammogram of

un-functionalized gold-coated silicon nanopillar chip.

Electrochemical pathway for rapid and label-free detection of protein biomarkers in blood 117

Chapter 5: Electrochemical pathway for

rapid and label-free detection of

protein biomarkers in blood

This chapter is made up of the following journal article published in Talanta

An electrochemical biosensor for the rapid detection of erythropoietin in blood

Waleed A. Hassanain, Arumugam Sivanesan, Emad L. Izake, Godwin A. Ayoko

doi.org/10.1016/j.talanta.2018.07.045

https://www.sciencedirect.com/science/article/pii/S0039914018307537?via%3Dihub

118 Electrochemical pathway for rapid and label-free detection of protein biomarkers in blood






6. They meet the criteria for authorship in that they have participated in the conception,


expertise;

7. They take public responsibility for their part of the publication, except for the


8. There are no other authors of the publication according to these criteria;

9. Potential conflicts of interest have been disclosed to (a) granting bodies, (b) the



10. They agree to the use of the publication in the student’s thesis and its

publication on the QUT’s ePrints site consistent with any limitations set by

publisher requirements.


W.A. Hassanain, A. Sivanesan, E.L. Izake, G.A. Ayoko, An electrochemical biosensor for

the rapid detection of erythropoietin in blood, Talanta, 2018, 189, 636-640.



authorship.


Name Signature Date


Waleed A. Hassanain



Arumugam Sivanesan Conducted experiments, data analysis, and primary

manuscript authorship.







5.1. Preface

In the previous chapter, we presented a dual nanosensing approach for protein

analysis. In this chapter, we demonstrate the electrochemical detection of protein

biomarkers in blood plasma by a recyclable nanostructured gold electrode.

Recombinant human erythropoietin was extracted from blood plasma by the same

methodology described in chapter 3, using selective anti-rhuEPO antibody-

conjugated magnetic beads. For sensitive electrochemical detection, the extracted

protein was loaded onto a recyclable gold nanostructured electrode and

electrochemically reduced by chronoamperometry. This was followed by the DPV

desorption of the protein molecule by reducing the Au-S bonds between the protein

and the gold electrode. This electrochemical approach enhanced the quantification

sensitivity of the proteins without signal amplification and allowed for the detection

of the target protein down to 1 pM.


5.2. Abstract

A label-free electrochemical detection method for the rapid detection of

recombinant human erythropoietin (rhuEPO) has been developed. In this method, we

modified the rhuEPO structure for its direct sensing without using a complex signal

amplification strategy. The protein was selectively extracted from blood plasma

sample using target-specific magnetic beads. After releasing rhuEPO from the

magnetic beads, its disulfide bonds were electrochemically reduced and the protein

was spontaneously assembled onto a nanostructured gold electrode via Au-S bonds

formation. For electrochemical quantification, the reduced protein was desorbed

from the electrode surface using differential pulse voltammetry (DPV). The

desorption current was proportional to the concentration of rhuEPO in the range 1 –

1000 pM. By cross-validating against ELISA, we found a 104.85 ± 3.35 %

agreement between the results obtained using the electrochemical biosensor and

ELISA. Therefore, the developed method has a strong potential for the sensitive

detection of rhuEPO doping in sports as well as its rapid screening in pathology labs.

5.3. Keywords

Recombinant human erythropoietin, Disulfide bond reduction, Electrochemical

biosensing, Nanostructured gold electrode, Chronoamperometry, DPV.


5.5. Introduction

Erythropoietin (EPO) is a naturally occurring hormone that is used for the

regulation of erythropoiesis and as a signalling protein for the production of red

blood cells in bone marrow [341]. Recombinant human EPO (rhuEPO) is used to

treat cancer patients suffering from associated anaemia [342]. In the sports industry,

rhuEPO is doped by athletes to increase their oxygen capacity and enhance their

aerobic performance [343]. The widespread doping of rhuEPO has prompted the

World Anti-Doping Agency (WADA) to ban its use in international sports and

sponsor significant research on new sensitive detection methods of the protein in

blood and urine. Similar to other protein biomarkers, the screening of rhuEPO in

biological fluids requires accurate, sensitive and selective detection methods. The

current analysis methods for rhuEPO detection include isoelectric focusing, double

blotting, HPLC-MS and MALDI-TOF techniques [344]. These techniques require

lengthy analysis time, expensive equipment and skilled operators to perform complex

screening procedures.

Electrochemical methods are among the simple and non-expensive techniques

for protein analysis [345]. Electrochemical sensors are characterized by their rapid

response, small sample volume requirement, cost-effectiveness and potential for

miniaturization within portable analytical platforms [22, 346, 347]. The direct

electrochemical detection of proteins suffers from inadequate sensitivity and

selectivity towards the target biomolecule [348-349]. Therefore, the electrochemical

biosensing of proteins is usually limited to those having non-protein redox centres

allowing for the reversible electron transfer between the electrode and the

biomolecule [345]. To improve the sensitivity of the electrochemical biosensors,

derivatization reagents have been employed to lower the detection limit of analytes

to the nanomolar range [350]. In addition, nanostructured electrodes have been

utilized to enhance the sensitivity of the electrochemical detection of analytes [351-

352].

In this work, we present a proof of concept for the rapid and sensitive label-

free electrochemical determination of rhuEPO in blood plasma down to pico molar

concentrations. To selectively extract rhuEPO from blood plasma, an anti-rhuEPO

antibody was conjugated to amine derivatized and silanized iron oxide magnetic

beads. After binding the target protein, the pH of the beads was modified to release


the extracted protein within 5 minutes. For direct detection, the protein disulfide

bond structure was reduced by chronoamperometry on a nanostructured gold

electrode to assemble the reduced protein onto the electrode surface via Au-S bond

formation. The protein was then electrochemically desorbed by differential pulse

voltammetry (DPV) in 1 minute, and the developing current is utilized for the

quantification of the protein (Scheme 5.1). The new direct electrochemical

biosensing of rhuEPO was cross-validated against enzyme linked immunosorbent

assay (ELISA).

Scheme 5.1: A schematic representation of the novel electrochemical biosensing of

rhuEPO.



5.6.1. Chemicals and materials

Amine derivatized and silanized magnetic beads with a particle size of 1-4 µm

were purchased from Thermo Fisher Scientific (Au). Recombinant human EPO

international standard (rhuEPO, 3rd international reference preparation) was sourced

from the national institute for biological standards and control (UK). Anti-

erythropoietin antibody (7D3) was purchased from MAIIA Diagnostics (SWE).

Human EPO ELISA kits were purchased from Abcam (Au). Horse plasma samples

were donated by Dr. Rohan Steel, Project Leader, Biological Research Unit, Racing

Analytical Services Ltd, Melbourne (Au). The samples were collected and shipped to

Queensland University of Technology (QUT) under the Melbourne lab protocols,

ethical clearances and arrangements. 1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide (EDC), N-hydroxysuccinimide (NHS), phosphate buffered saline

(PBS), glycine, potassium hydroxide, gold (III) chloride trihydrate (HAuCl4 .3 H2O)

perchloric acid (HClO4) and protein low bind eppendorf tubes were purchased from

Sigma Aldrich (Au). Gravity flow size-exclusion columns (illustra NAP-5 (17-0853-

01)) were obtained from GE Healthcare Life Sciences (Au). Deionized water

(ultrapure Millipore, 18.2 MΩ cm@25 °C) was used in all preparations. The

glassware and gold discs were cleaned with piranha solution (3:1, 98% H2SO4: 30%

H2O2) and thoroughly rinsed with deionized water prior to use.

5.6.2. Instruments

The electrochemical measurements were carried out using Autolab

PGSTAT204 potentiostat (Metrohm Autolab, NL), equipped with NOVA 1.10.5 as

operating software and a standard three electrode cell. Polycrystalline gold disc

(geometric area = 0.502 cm2) and platinum wire were used as working and counter

electrodes, respectively. Dry leakless Ag/AgCl/saturated KCl (world precision

instruments, USA) was used as a reference electrode. For ELISA screening, the

optical density was measured at 450 nm using a multimode reader GloMax explorer

(Promega, USA).


5.6.3. Preparation of rhuEPO standard solutions

A series of standard solutions of rhuEPO in the concentration range of 1 – 1000

pM were prepared by serial dilution in 1x phosphate buffered saline buffer (PBS)

(pH 7.4).

5.6.4. Preparation of gold nanostructured electrode for DPV detection

Nanostructured gold electrodes were prepared as described by Sivanesan et al.

[353]. Briefly, flat gold disc electrodes were mirror-polished with alumina slurries of

decreasing particle sizes (0.5 µm, 0.05 µm and 0.02 µm), immersed in deionized

water and subsequently sonicated in aqueous ultrasonic bath for 15 minutes. The

electrodes were then immersed in piranha solution [H2SO4 (98%): H2O2 (30%), 3:1

v/v] for 10 minutes and rinsed with deionized water. The gold electrode was utilized

as a working electrode in a three electrode cell that is filled with 4 mM HAuCl4 in

0.1 M HClO4 and purged with argon gas for 30 minutes to remove oxygen gas from

the solution. A negative potential of -0.08 V was applied for 15 minutes to

electrochemically deposit gold nanostructures onto the gold disc. The gold

nanostructured electrode was then removed from the cell, washed with deionized

water, dried under nitrogen stream and stored for future use.

5.6.5. Synthesis of antibody-conjugated magnetic beads

In our work, 100 µL of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

(EDC) solution was mixed with 100 µL of 0.1 M N-hydroxysuccinimide (NHS)

solution in a low bind eppendorf tube and gently vortexed for 2 minutes. This was

followed by the addition of 100 µL of the amine derivatized and silanized iron oxide

magnetic beads. Finally, 10 µL of anti rhuEPO antibody (0.1 mg/mL) were added to

the mixture. The resulting mixture was stored at 4 ºC for 4 hours to allow for the

complete conjugation of the antibody to the magnetic beads. The conjugated beads

were magnetically separated from the solution, re-suspended in 100 µL PBS (pH 7.4)

and stored at 4 ºC for future use.

5.6.6. Selective extraction of rhuEPO

The antibody-conjugated beads were used to selectively extract rhuEPO from

blood plasma samples. Horse blood plasma was spiked with 250 pM of rhuEPO. This

was followed by mixing 100 µL aliquot of the spiked plasma sample with 100 µL of

the antibody-conjugated magnetic beads and left to stand for 15 minutes at room


temperature with occasional shaking. The magnetic beads were collected from the

plasma matrix using an external magnet and washed 5 times, to remove any weakly

adsorbed interfering molecules (each time with 100 µL of 1x PBS buffer, pH 7.4).

To release the captured rhuEPO from the extractor beads, they were re-constituted

into 100 µL of 0.1 M glycine.KOH buffer (pH 11) for 5 minutes at room

temperature. The released rhuEPO solution was loaded onto a size exclusion column

to remove the glycine buffer. The purified protein was eluted using 500 µL of 0.1 M

KOH.

5.6.7. Electrochemical reduction and quantification of rhuEPO

For the electrochemical quantification of the rhuEPO, 50 µL aliquots of

standard rhuEPO solutions in the concentration range 1 - 1000 pM were loaded onto

nanostructured gold electrodes, reduced and assembled by chronoamperometry using

a potential of -1 V for 30 minutes. The reduced protein was electrochemically

desorbed from the electrode surface by DPV where the potential was swept between

-1 V to -1.4 V at step of -0.005 V and a scan rate of 0.01 V/s. The pulse amplitude

and width were set at 50 mV and 50 ms, respectively. The magnitude of the resulting

desorption current at -1.19 V was plotted against the protein concentration to

construct a calibration plot.

For the quantification of rhuEPO in blood plasma, 50 µL aliquot of the eluted

rhuEPO, in section 5.6.6, was loaded onto the working nanostructured gold electrode

of the electrochemical cell and dried under gentle stream of nitrogen gas (Figure

S5.1, supplementary material). KOH was added as an electrolyte solution, and

chronoamperometry followed by DPV measurements were carried out.

5.6.8. ELISA cross-validation

To cross-validate the DPV quantification of rhuEPO, an aliquot of the eluted

protein, in section 5.6.6, was screened using a human EPO ELISA kit. An ELISA

calibration plot was constructed in the concentration range 10.7 – 333.2 pg/mL,

where the average optical density (OD) at 450 nm was plotted against the

concentration of the protein standards (Figure S4.2, supplementary material).



5.7.1. Selective extraction of rhuEPO from blood plasma

Direct electrochemical biosensing of biomolecules requires their selective

isolation from the complex biological matrix prior to measurements [354-355]. For

this purpose, we developed rhuEPO extractor beads by conjugating the C terminal of

an anti-rhuEPO antibody to the amine-terminals of silanized magnetic beads using

the EDC coupling reaction [225, 356]. The EDC/NHS coupling was added to the

amine derivatized and silanized iron oxide magnetic beads to activate the NH2

groups on the surface of the beads. The antibody was then added to the mixture,

where the EDC/NHS reagents activated the COOH groups at the C terminal of the

antibody. The activated NH2 and COOH groups of the magnetic beads and the

antibody, respectively, react to form amide bonds that attach the antibody to the bead

[225].

The antibody-functionalized beads were used to selectively bind and extract

rhuEPO from blood plasma. By changing the pH environment of the beads, the weak

coulombic and van der Waals bonds between the antibody and the bound protein

molecules were easily broken and the isolated rhuEPO molecules retrieved [295,

357, 358, 359].

5.7.2. Electrochemical reduction and assembly of rhuEPO on nanostructured

gold electrode

The average concentration range of EPO in the blood of healthy humans is 0.92

– 3.7 pM [360]. Therefore, an electrochemical biosensor of rhuEPO should have a

detection limit within the pico molar concentration range. We first attempted the

direct electrochemical detection of 10 pM rhuEPO by DPV using a flat gold

electrode. As indicated by Figure 5.1 (blue line), no characteristic desorption current

was observed. Therefore, we repeated the DPV measurement using a nanostructured

gold electrode [103, 237]. However, no characteristic desorption current was

observed for rhuEPO (Figure 5.1, red line).


Figure 5.1: DPV of rhuEPO on a polished gold electrode (blue line) and on a

nanostructured gold electrode (red line).

Another approach to enhance the electrochemical detection of analytes is to

form covalent bonds between the target analyte and the nanostructured electrode

[361]. Therefore, we used chronoamperometry to electrochemically reduce and

assemble the protein onto the nanostructured gold electrode surface. The

electrochemical reduction cleaves the disulfide bond between the cysteine residues of

the protein and generates free sulfhydryl (SH) terminal groups that spontaneously

form Au-S bonds with the electrode surface [362]. The assembled protein molecules

were then desorbed from the electrode by DPV, where a characteristic desorption

current was observed at -1.19 V as depicted by Figure 5.2 (blue line).


Figure 5.2: DPV of reduced EPO on a nanostructured gold electrode, the red dotted

line denoted the blank nanostructured electrode.

To investigate the effect of reduction time on the DPV measurement was

investigated where chronoamperometry was carried at 10, 20, 25, 30, 35 minutes,

respectively, and the DPV current measured after the reduction. As indicated by

Figure S5.3 (supplementary material), the DPV current was maximum after 30

minutes of chronoamperometric reduction. Therefore, to allow for a sensitive

detection of the protein, the chronoamperometry was lasted for 30 minutes before the

DPV measurement.

The electrochemical desorption current of reduced rhuEPO was found to

proportionally increase with the protein concentration in the range 1–1000 pM and

follow a polynomial relationship (Figure 5.3a, b). The inset in Figure 5.3b depicts the

linear relationship between the DPV current and the concentration of rhuEPO in the

concentration range of 1 - 250 pM. The regression equation for the linear relationship

was found to be y = 0.002x + 0.0979 (R2 =0.983). Using this regression equation, the

95% confidence intervals for the slope and the intercept were found to be 0.0008 and

0.109, respectively. The limit of detection was found to be 1 pM.


Figure 5.3: (a) DPV of reduced rhuEPO on nanostructured gold electrode in the

concentration range 1 – 1000 pM, (b) corresponding calibration curve in the same

concentration range. The inset depicts the linear relationship in the concentration

range 1-250 pM.

5.7.3. DPV quantification of rhuEPO in blood plasma and cross-validation by

ELISA

To utilize the developed electrochemical methodology for the biosensing of

rhuEPO in blood plasma, the protein was extracted using the functionalized beads,

reduced by chronoamperometry on a nanostructured gold electrode and quantified by

DPV. The average concentration of rhuEPO in the plasma sample was found to be

221 ± 0.44 pM (n=3) by DPV. The standard deviation within the three measurements

was 1.05.

For a cross-validation purpose, another aliquot of the same extract was

screened by ELISA. The ELISA screening test confirmed the presence of rhuEPO in

the plasma sample at an average concentration of 211 ± 7.17 pM (n=3). Therefore,

the average agreement between the DPV and the ELISA measurements was 104.85 ±

3.35 %.

Zhang et al. recently demonstrated a method for the electrochemical detection

of rhuEPO down to 0.016 fM [363]. The authors used a signal amplification

approach where a glassy carbon electrode was modified with a sandwich-like nano-

Au/ZnO sol-gel/nano-Au compound membrane and used for the detection of the

protein by monitoring the change in the electrode response. The dynamic range of

this method was 0.016 fM – 0.016 nM and the limit of detection was 0.003 fM.

Considering the complex procedures and the long preparation time of the electrode


(~12 hours), our developed direct electrochemical biosensing method is simple, rapid

and can be used for monitoring rhuEPO doping and therapeutic use [364]. In

addition, the nanostructured gold electrode used in this work can be easily recycled

by removing the contaminated gold nanostructures using a simple mechanical

etching procedure, followed by an electrochemical deposition of new gold

nanostructures within 15 minutes.


5.8. Conclusion

In this work, we presented a simple electrochemical method for the rapid and

sensitive detection of proteins, as demonstrated by rhuEPO, in blood plasma. We

developed target-specific antibody-conjugated magnetic beads for the selective

extraction of the protein from blood plasma within 15 minutes. For its sensitive

electrochemical biosensing, the extracted protein was electrochemically reduced and

assembled by chronoamperometry on a nanostructured gold electrode. The

assembled protein was then desorbed from the electrode surface by DPV to give an

electrochemical desorption current that is proportional to the concentration of

rhuEPO within the range of 1-1000 pM which is useful for monitoring rhuEPO

doping and therapeutic use. The DPV determination of rhuEPO was cross-validated

against ELISA where the agreement between the two measurements was 104.85 ±

3.35 %. The developed electrochemical method has a strong potential for the rapid,

sensitive and cost-effective determination of other proteins in molecular diagnosis

applications.



Figure S5.1: The electrochemical cell assembly.

Figure S5.2: ELISA calibration curve of rhuEPO


Figure S5.3: DPV current of reduced rhuEPO at -1.19 V after 10, 20, 25, 30 and 35

minutes of chronoamperometry reduction.

136 Conclusion and future work

Chapter 6: Conclusion and future work

Conclusion

Re-statement of the research aims:

(1) Develop and utilize novel cost-effective nanostructured substrates for the SERS

detection of bioactive molecules such as drug metabolites, environmental toxins and

protein biomarkers in biological fluids.

(2) Develop a novel approach for the fabrication of recyclable target-specific

extractor nanomaterial, using antibodies and/or their fragments as selective

recognition molecules for the selective extraction of proteins and environmental

toxins from blood plasma.

(3) Address the problem of signal irreproducibility in SERS by developing a novel

approach for the oriented immobilization of biomolecules through the chemical

modification of their thiol chemistry.

(4) Develop and cross-validate a label-free approach for the dual SERS and

electrochemical nanosensing of bioactive molecules in biological fluids.

In this research, a proof – of – concept on novel label-free SERS and DPV

nanosensing approaches for the detection of bioactive molecules have been

developed. These methods utilized analyte – selective nanomaterials and cost-

effective SERS substrate for the direct extraction, detection and quantification of the

analytes from complex matrices. A simple and cost-effective paper SERS substrate

has been developed for the detection of pharmaceuticals (sofosbuvir metabolite) in

blood plasma (Aim 1). The compound was chromatographically separated, validated

and detected using paper SERS and a handheld Raman spectrometer. The paper

substrate showed significant sensitivity down to picomolar level. The high sensitivity

and low cost qualified the paper SERS substrate to be used in the detection of the

environmental toxin MC-LR in biological and environmental samples (Aim 1).

To selectively extract the toxin from the biological and environmental samples,

a recyclable target-specific extractor nanomaterial was developed (Aim 2). The

extractor nanomaterial was developed using selective anti-MC-LR – antibody

Conclusion and future work 137

fragments and it offered an excellent selectivity towards its target analyte (Aim 2).

The identity of the extracted sample was confirmed by ELISA. The extractor

nanomaterial was recycled for another extraction cycle of the toxin from the blood

plasma. The extracted toxin was purified and quantified using a commercial gold

coated nanopillar substrate.

The plasmonic and conductive properties of the gold coated nanopillar were

used for the dual nanosensing of CST-C by label-free SERS and electrochemical

methods (Aims 3, 4). For the isolation of the protein biomarker from the human

blood plasma, a selective 2D extractor chip was designed (Aim 2). The extractor chip

design used the same concept as the extractor nanomaterial that was used for the

extraction of MC-LR. However, the flat conductive surface of the extractor chip

optimized the extraction and purification time of the protein. For reproducible SERS

and DPV measurements, the extracted protein was subjected to chemical reduction

using TCEP and immobilized onto the substrate in a unified orientation. This led to

the sensitive quantification of CST-C down to 1 pM and 62.5 nM by SERS and DPV,

respectively (Aim 3). In addition, the extractor chip was recycled by

electrochemically desorbing the used antibody fragments from its surface and re-

functionalized with fresh fragments. The nanosensing approach has strong potential

for the rapid detection / quantification of protein biomarkers at points of care.

A combined electrochemical reduction and detection of rhuEPO in biological

fluids has also been demonstrated (Aim 4). After selective extraction from blood

plasma, the disulphide bonds structure of the isolated protein was modified by

chronoamperometry. The reduced protein was then electrochemically quantified by

DPV. This approach lowered the quantification limits of the screened proteins down

to the picomolar level (Aim 4). The gold nanostructured electrode was also

regenerated for repeated measurements (Aim 1).

Overall, the aims of this research were achieved with the development of

sensitive nanoscale platforms for the SERS and electrochemical detection of

bioactive molecules. The presented techniques have been cross-validated against LC-

MS and ELISA methods with good % agreements. The LOD, LOQ, uncertainty,

confidence intervals for the slope and intercept, % error between measurements, day

to day and batch to batch RSD were investigated. However, a comprehensive large

scale cross-validation study is required for the complete validation of the developed


nanosensing methods and would be the scope of future research. The versatility of

the designed nanosensors provided effective detection with reproducibility, high

selectivity and high sensitivity that are needed in routine trace analysis.

Conclusion and future work 139

Future work

The development of rapid, selective, sensitive and cost-effective nanosensors

for the multimodal determination of bioactive molecules in biological matrices will

remain a focal point of future endeavours in this field. For example, the manufacture

of novel nanostructured carbon fibre cloth will significantly reduce the cost of

protein analysis by SERS and DVP to become commercially viable.

The developed selective capture and SERS sensing methodologies can be

utilized in proteomics to study the conformational changes that occur within

antibodies and proteins upon their interactions. By using isoform-specific antibodies

/ aptamers / affibodies / lectins, the developed methodologies can also be utilized as a

novel and rapid analytical tool for the discrimination of protein isoforms in

foundation biology studies.

When fully optimized and validated, the developed nanosensing methods can

be adopted for commercialization and can be used for the screening of numerous

biomarkers (e.g. proteins, peptides, enzymes and DNA) at pathology labs and points

of care. Some challenges within the developed nanosensing techniques still require

further future optimization, such as:

(1) Improve the manufacture of the paper SERS substrate by using automated

printing instead of hand drawing.

(2) Enhance the sensitivity of the developed substrates by increasing the

nanostructures loading and controlling their surface morphology.

(3) Investigate other Raman studies to study the keto-enol tautomers of bioactive

molecule after selective isolation of the two purified forms.

(4) Identify new proper recognition molecules that have the discrimination power to

differentiate between different isoforms of the same biomolecule.

(5) Enhance the capture efficiency of the developed extractor nanomaterials and

investigate novel bio-conjugation methods for the oriented immobilization of the

selective recognition molecules.

(6) Investigate other sensitive electrochemical methods for the direct and label-free

detection of bioactive molecules at sub-picomolar concentration levels.


(7) Reduce the analytical cycle to make the developed nanosensing techniques more

cost-effective. This can be achieved by avoiding the use of – time consuming – size

exclusion columns to remove the releasing buffer. This will require selecting other

releasing buffers which does not have Raman signatures within the analyte scanned

range.

(8) Apply additional validation parameters and cross-validation methods for the

quantitative measurements against non-ELISA techniques as HPLC-MS-MS. This

will require the use of expensive commercial substrates. Therefore, we aimed to

design a novel, highly reproducible and cost-effective substrate for the dual sensing

of purified biomolecules by SERS and electrochemical methods. For example, work

is currently underway within the research group to develop nanostructured gold

coated ITO as a dual spectro-electro sensor.

Appendix 141

Appendix

Figure A.1: SERS spectra of different concentrations of PSI-6206 on paper SERS


142 References

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