paper-based sensors for contaminant detection using ...(a) fe-sem image of chromatography paper...

Paper-Based Sensors for Contaminant Detection Using Surface Enhanced Raman Spectroscopy

Ishan Jain

Thesis submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of

Master of Science

In

Environmental Engineering

Peter J. Vikesland, Chair

Amy Pruden

Richey M. Davis

April 23, 2015

Blacksburg, Virginia

Keywords:

surface enhanced Raman spectroscopy, gold nanoparticles, paper-based sensors, SERS substrates, pathogen detection, µPads, microfluidic device

Paper-Based Sensors for Contaminant Detection Using Surface Enhanced Raman Spectroscopy

Ishan Jain

ABSTRACT

Surface enhanced Raman spectroscopy (SERS) is highly promising analytical technique

for trace detection of analytes. It is particularly well suited for environmental analyses due to its

high sensitivity, specificity, ease of operation and rapidity. The detection and characterization of

environmental contaminants, using SERS is highly related to the uniformity, activity and

reproducibility of the SERS substrate.

In this thesis, SERS substrates were produced by gold nanoparticle formation on wax

patterned chromatography paper. In situ reduction of hydrogen tetrachloroaurate (gold precursor)

by trisodium citrate dihydrate (reducing agent) was used to produce gold nanoparticles within a

paper matrix. These gold nanoparticle based SERS substrates were analyzed by FE-SEM, UV-

Vis and Raman spectroscopy. This work discusses the SERS signal enhancements for Raman

active MGITC dye for a series of substrates prepared by in situ reduction of gold salt and pre-

produced gold nanoparticles. UV-Vis analysis was performed to understand the effect of

different molar ratio (reducing agent to gold precursor) and reaction time on the size and shape

of the localized surface plasmon resonance (LSPR) band that dictates the SERS enhancements. It

was concluded that lower molar ratio (1:1 and 2:1) of citrate-to gold produced better SERS

signal enhancements and broader LSPR band. Therefore, use of lower molar ratio (MR) was

recommended for paper-based substrates using in situ-based reduction approach.

iii

For my father:

Shri Ishwar Jain

“The beginning of knowledge is the discovery of something we don’t understand.”

-Frank Herbert

iv

Acknowledgments

My MS program experience was the most challenging, yet, great learning experience of

my life. I had the opportunity of working with Dr. Peter J. Vikesland, who was instrumental in

helping me to complete my thesis. I sincerely thank him for his support, guidance, and patience

throughout the period of my MS.

I am also grateful to my committee members Dr. Amy Pruden, and Dr. Richey Davis for

their encouragement throughout the time of my graduate studies.

I am also very thankful to my colleagues, particularly in Dr. Vikesland’s group, for

helping me with data analysis, experiments and lab training. They include Dr. Rebecca

Halvorson Lahr, Dr. Weinan Leng, Xinzhe Zhou, Ron Kent and Marjorie Willner.

This research was supported by the National Science Foundation (NSF), Bill and Melinda

Gates Foundation, and the Virginia Tech Institute for Critical Technology and Applied Science

(ICTAS).

Ishan Jain

Blacksburg, VA, USA

v

TABLE OF CONTENTS

List of Figures .............................................................................................................................. vii

List of Tables ................................................................................................................................ xi

Chapter 1. Introduction ............................................................................................................... 1

1.1 Significance and motivation .................................................................................... 1

1.2 Specific objectives .................................................................................................... 5

1.3 Overview of chapters ............................................................................................... 5

Chapter 2. Literature Review ...................................................................................................... 7

2.1 Raman spectroscopy and its application ............................................................... 7

2.1.1 Origin of Raman ............................................................................................................. 7

2.1.2 Raman scattering ........................................................................................................... 7

2.1.3 Comparison between Raman spectroscopy and other technologies ............................ 8

2.1.4 Limitation of Raman spectroscopy .............................................................................. 10

2.2 Surface enhanced Raman spectroscopy (SERS) ................................................. 11

2.2.1 Introduction ................................................................................................................. 11

2.2.2 SERS mechanism .......................................................................................................... 11

2.2.3 SERS substrates ............................................................................................................ 12

2.3 Gold nanoparticles: synthesis and properties ..................................................... 16

2.3.1 Synthesis of gold nanoparticles ................................................................................... 16

2.3.2 Properties of gold nanoparticles .................................................................................. 19

vi

2.3.3 In situ reduction of metal salts .................................................................................... 19

2.4 Microfluidic paper-based devices (µPADs) ......................................................... 20

Chapter 3. Experimental Methods and Instruments ............................................................... 24

3.1 Material .................................................................................................................................. 24

3.2 Preparation of wax printed microfluidic wells/channels .................................... 24

3.3 Preparation of paper-based SERS substrates ..................................................... 25

3.4 Instrumentation ..................................................................................................... 27

3.4.1 UV-‐Vis .......................................................................................................................... 27

3.4.2 Surface enhanced Raman spectroscopy (SERS) ........................................................... 27

3.4.3 FE-‐SEM ......................................................................................................................... 28

Chapter 4. Results and Discussion ............................................................................................ 29

4.1 Synthesis of gold nanoparticles by in situ reduction within paper .................... 32

4.2 UV-Vis analysis to study effect of the variation in gold precursor and reducing

agent concentration with increase in reaction time .............................................................. 44

4.3 SERS signal optimization strategy for MGITC using paper based substrates 50

Chapter 5. Conclusions and Future Work ............................................................................... 56

5.1 Conclusions ............................................................................................................. 56

5.2 Future work ............................................................................................................ 57

References .................................................................................................................................... 60

vii

List of Figures

Figure 1. Schematic representation of Raman (Stokes & anti-Stokes) and Rayleigh scattering. .. 8

Figure 2. Example of lightning rod effect for Ag Trigonal prism (side length 100 nm and

thickness is 16 nm); Sharp angles and edges produces extra enhancement. Electric field scale is

from 0 to 20 units. ......................................................................................................................... 15

Figure 3. Illustration of gold nanoparticle formation by nucleation and growth mechanism.. ... 18

Figure 4. (A) Paper-based SERS substrates prepared by gold nanorods (AuNRs) solution before

and after exposure showing the strong color change; (B) UV-Vis extinction spectrum of the

AuNRs solution and the AuNRs loaded paper. ............................................................................. 22

Figure 5. (A) Wax printed circular paper-based sensors (2 mm circular wells) (B) Wax printed

microfluidic paper-based channels; dark red color is due to formed gold nanoparticles. .......... 25

Figure 6. In situ reduction of gold precursor (hydrogen tetrachloroaurate solution) by reducing

agent (trisodium citrate); HAuCl4 stands for gold precursor ....................................................... 26

Figure 7. The wax spreads during heating, creating narrower channels in the tested µPADs; (A)

Unheated wax channel and heated wax channel; (B) Unheated and heated circular microfluidic

well. The µPAD dimension describes the printed measurements. ................................................ 30

Figure 8. Characterization of wax printed-paper before and after heating; (A) SEM images of

microfluidic channel before (at magnification of 500x); (B) FESEM images of microfluidic

channel after heating (at magnification of 500x); (C) One of the waxed coated cellulosic fiber is

zoomed in (at magnification of 1000x). ........................................................................................ 31

Figure 9. Example of color change in chromatography paper in 10 mm circular well (left) and 2

mm circular wells (right); HAuCl4 and Na3Ctr stand for gold chloride and sodium citrate

solution respectively. ..................................................................................................................... 33

viii

Figure 10. UV-Vis spectrum of various substrates prepared by different citrate-to- gold ratio

(refer Table 4); Different shape and size of LSPR is observed. (A). 25 mM gold chloride, 23 mM

Citrate Solution (MR= 0.95) (B). 20 mM gold chloride, 27 mM Citrate Solution (MR= 1.35) (C).

15 mM gold chloride, 30 mM Citrate Solution (MR= 2) (D) 10 mM gold chloride, 30 mM Citrate

Solution (MR= 3) .......................................................................................................................... 35

Figure 11. SERS of MGITC dye in MGITC-methanol solution. MGITC assignments are at 1200,

1380 and 1600 cm-1. ...................................................................................................................... 36

Figure 12. Comparative analyses of paper-based substrates prepared by various different molar

ratio of citrate to gold chloride; 2 mm wax printed wells are loaded with gold chloride solution

followed by citrate solution (reducing agent); Reaction time: 7 days with addition of 1 µL of

MGITC dye; (A) the difference in the region of 100 ~ 350 cm-1 may be caused by the size or

shape of Gold NPs in the paper; (B) SERS spectrum of MGITC dye with various molar ratios

(MR). Raman parameters: Laser wavelength 633 nm, and micrometer reading 1.2 mm (0.261

mW for 10x objective lens). ........................................................................................................... 37

Figure 13. (A) FE-SEM image of chromatography paper (blank) at magnification ~10 kx; (B)

Different FE-SEM image of chromatography paper (blank) at magnification ~10 kx; (C) EDX

image showing strong C, O elemental peaks and negligible gold peaks. Gold peak is a result of

gold-palladium coating. ................................................................................................................ 40

Figure 14. (A) SEM image of chromatography paper loaded with 50 nm sized gold nanoparticles

prepared by citrate reduction method at magnification of 30.00 kx; (B) SEM image of

chromatography paper loaded with 50 nm sized gold nanoparticles prepared by citrate reduction

method at magnification of 100.00 kx; (C) EDX image confirming presence of gold

ix

nanoparticles. Images produced at working distance (WD) ~8.5 mm and Voltage (EHT) ~ 20kV.

....................................................................................................................................................... 41

Figure 15. SEM analysis of paper-based SERS substrates prepared by in situ reduction based

gold nanoparticle synthesis (5 mM gold chloride, molar ratio of citrate to gold chloride~ 4, and

allowed reaction time ~ 7 days) (A) Clusters (circles) of formed AuNPs in sample image with

magnification ~ 101.4 KX; (B) Clusters of formed AuNPs in sample image with magnification ~

30.4 KX; (C) Clusters of formed AuNPs in sample image with magnification ~ 100.36 KX; (D)

Clusters of formed AuNPs in sample image with magnification ~ 100.72 K X (used WD~ 5.7 mm,

EHT~ 2 kV). .................................................................................................................................. 42

Figure 16. SEM analysis of paper-based SERS substrates prepared by in situ reduction based

gold nanoparticle synthesis (5 mM gold chloride, molar ratio of citrate to gold chloride~ 4 and

allowed reaction time ~ 7 days); (A) Sample image with magnification ~40 K X, WD~ 5.7 mm,

EHT~ 2 kV; (B) EDX image showing elemental analysis of square box (pink color) in image (A).

....................................................................................................................................................... 43

Figure 17. Temporal variation of maximum absorption of different molar ratios of citrate

solution to gold chloride solution for a given initial concentration of gold chloride solution; (A)

Molar ratio (MR) = 1 (B) Molar Ratio = 3 (C) Molar Ratio= 5. ................................................ 46

Figure 18. Effect of initial gold precursor solution on absorption values for a given molar ratio

of citrate solution to gold chloride solution after reaction time of 35 days; (A) Molar ratio of 1

after 35 days (B) Molar ratio of 3 after 35 days (C) Molar ratio of 6 after 35 days .................... 47

Figure 19. LSPR Band and shape changes with the increase in molar ratio (MR) from 1 to 6.

[HAuCl4]= 20 mM, reaction time 35 days, and diameter of microfluidic wells are 10 mm. ....... 48

x

Figure 20. LSPR Band and shape changes with the increase in molar ratio (MR) from 1 to 6.

[HAuCl4]= 100 mM, reaction time 35 days, and diameter of microfluidic wells = 10 mm. ....... 49

Figure 21. Average SERS spectra (Large area scan) for MGITC dye using paper based

substrates. [HAuCl4]= 20 mM, reaction time= 28 days, Raman Laser 785 nm wavelength with

micrometer reading of 1.2 mm (18.3 mW of laser power for 10x objective lens). MR stands for

Molar Ratio. .................................................................................................................................. 51

Figure 22. Optimization of MGITC assignments with respect to peak at 1800 cm-1 (strategy -1);

Lower molar ratios produce less variability in peak ratios than high molar ratios (circled).

Sample 1 and sample 2 are replicate of same substrates (for initial gold chloride conc. = 20

mM) ............................................................................................................................................... 53

Figure 23. Optimization of MGITC assignments with respect to the peak at 105 cm-1 (strategy-

2); Sample 1 and sample 2 are replicate of same substrates (for initial gold chloride conc. = 20

mM). .............................................................................................................................................. 54

xi

List of Tables

Table 1. Properties and advantages of SERS. Springer Science and Business Media/

Microchimica Acta, 181, 2014, 23-43, Recent Progress in Surface Enhanced Raman

Spectroscopy for the Detection of Environmental Pollutants. Li, D.-W., et al., excerpts,

Copyright © 2013, Springer Science+Business Media [1]. Used with kind permission from

Springer Science and Business Media, 2015. ________________________________________ 2

Table 2. SERS application in organic pollutant detection. Springer Science and Business

Media/ Microchimica Acta, 181, 2014, 23-43, Recent Progress in Surface Enhanced Raman

Spectroscopy for the Detection of Environmental Pollutants, Li, D. -W., et al., excerpts,


Springer Science and Business Media, 2015. ________________________________________ 4

Table 3. Comparison between Raman spectroscopy and other techniques. Springer Science

and Business Media/ Cancer and Metastasis Reviews, 33 (2-3), 2014, 673-693, Emerging

Technology: Applications of Raman Spectroscopy for Prostate Cancer, Kast, R.E., et al.,

excerpts, Copyright © 2014, Springer Science+Business Media [71]. Used with kind permission

from Springer Science and Business Media, 2015. __________________________________ 10

Table 4. Concentration of gold chloride and sodium citrate solution for UV-Vis analysis;

data used in Figure 10. ________________________________________________________ 34

Table 5. Concentration of Gold Chloride and Sodium Citrate (data used in Figure 12) ___ 37

1

Chapter 1. Introduction

1.1 Significance and motivation

Environmental pollutants or toxins include a varied set of natural and synthetic

constituents present in soil, air and water that can cause adverse effects on ecosystems [1, 2].

Exposure to pollutants such as halogenated compounds, dioxins, heavy metals (for instance, lead

and arsenic), pesticides (organophosphates and other insecticides), and various forms of

pathogens (such as cyanobacteria and polio virus) can cause hazardous effects and acute diseases

in humans and animals [1, 3].

Environmental pollutants that pose harmful effects and threats to ecological species

should be regularly monitored and detected to evaluate their risks towards the environment [4-8].

Several analytical techniques including solvent extraction, chromatography, spectrometry, and

electrochemical methods are extensively used for environmental analyses [9-14]. However,

widespread use of many of these techniques is limited as they can be time consuming, complex,

costly or lack the capacity to recognize different contaminant distributions, thus resulting in

incomplete assessment of analytes [1, 15]. Therefore, researchers have been exploring

microfluidics, biosensors, and surface enhanced Raman spectroscopy (SERS) [1, 16, 17]. Among

these techniques, SERS is well suited for environmental analyses because it provides unique

fingerprints for specific analytes with high selectivity, sensitivity, and rapidity [1, 15, 18-23].

Relative to many other analytical techniques, SERS has outstanding advantages. These

advantages are briefly summarized in Table 1.

2

Table 1. Properties and advantages of SERS. Springer Science and Business Media/

Microchimica Acta, 181, 2014, 23-43, Recent Progress in Surface Enhanced Raman

Spectroscopy for the Detection of Environmental Pollutants. Li, D.-W., et al., excerpts,


Springer Science and Business Media, 2015.

Properties Advantages of SERS [1]

Sensitivity [24] SERS is highly sensitive to very low concentration of analytes

(even at the molecular level)

Unique fingerprint of

analytes [19, 20, 24]

It provides information about the inherent chemical structure of

analytes

Rapid detection [20, 25] It requires less than one minute for each measurement (it may

vary with analytes)

Detection in water [8, 15] As “water is a poor Raman scatter” [26], SERS can be applied

to water samples directly

Cost effective detection Good laboratory and field detection

Variable detection [19,

22, 27-30]

It detects chemical and biological variability in analytes

It is advantageous to use SERS for trace-level detection of aqueous and airborne

contaminants, cyanobacteria and other pathogens [15, 18, 19, 21, 31]. The capacity for SERS to

achieve these applications is highly dependent on the activity, plasmon absorption, and

reproducibility of the SERS substrates [18, 24, 32].

Lin XM et al. discussed various methods to prepare SERS substrates, which are briefly

covered in Chapter 2 [32]. SERS substrates made of roughened noble metal (Au, Ag) surfaces

3

are used to get signal enhancement factors ranging from 104 to 1010 [15, 33]. A summary of

different examples of SERS substrates and their applications are described in Table 2.

An SERS substrate (an ideal substrates) should have high SERS activity, decent

reproducibility (less than 20%), good stability and uniformity (deviation less than 20%). It

should provide high sensitivity, which can be controlled by changing the nanoparticle size and

inter-particle spacing (less than 10 nm) of nanoparticles [32, 34]. Uniform and reproducible

SERS substrates minimize variations in signal enhancement of the analytes [34, 35]. However, it

is difficult to design ideal SERS substrates to satisfy all of the above requirements [32, 34].

Therefore, researchers have recently begun fabricating paper based microfluidic

substrates [36-40]. Depositing metal (usually Au, Ag, Cu) nanoparticles on the patterned paper

produces paper-based SERS substrates [21, 37, 41-46]. These SERS substrates may be prepared

by using pre-formed metal nanoparticles and then subsequent addition of these nanoparticles to

paper [21, 22, 27, 38, 47, 48] or synthesis of metal nanoparticles within paper by the addition of

metal precursor and the subsequent addition of reducing agent [49-53]. This latter approach can

be considered as in situ reduction based synthesis of metal nanoparticles, which is further

discussed in Chapter 3.

Lee C. H. et al. fabricated SERS substrates by directly depositing pre-formed gold

nanorods (AuNRs) on the filter paper [21]. However, direct deposition of metal nanoparticles on

cellulose filter paper usually causes aggregation and heterogeneous distribution of nanoparticles

at the edge (boundary), producing a “coffee ring effect” due to drying process [13, 21, 37, 54]. In

this thesis, we hypothesized that we could reduce the potential “coffee ring effect” [54] by

4

producing more uniform substrates by using in situ reduction based synthesis of gold

nanoparticles on chromatography (or filter) paper.

Table 2. SERS application in organic pollutant detection. Springer Science and Business

Media/ Microchimica Acta, 181, 2014, 23-43, Recent Progress in Surface Enhanced Raman

Spectroscopy for the Detection of Environmental Pollutants, Li, D. -W., et al., excerpts,


Springer Science and Business Media, 2015.

Pollutant Category

Analytes SERS Substrates Sensitivity (LOD)

Pesticides Thiram Gold Nanorods ~34 nM [48] Chlortoluron Ag-quantum dots " sponge"

nanocomposite ~0.94 µM [55]

PAHs & PCBs Fluoranthene 1-Hexanethiol functioned Ag NPs ~24.7 nM [29]

Pyrene Viologen functionalized Ag NP 1 nM [56] Anthracene CD-SH functionalized Au NPs 100 nM [25] Pyrene Au NPs-modified alginate gel

network 10 nM [57]

Explosives 3,3’, 4,4’-tetrachlorobiphenyl

Ag nanosheet-assembled hemispheres modified with CD-SH

0.1 µM [58]

2,4-dinitroanisole Ag NPs modified by L-cysteine methyl ester hydrochloride

~0.1 µM [59]

Other Organic Pollutants

Phosphorus triphenyl Metal nanostructured film/graphene oxide

1 nM [60]

Rhodamine 6G Ag NPs-decorated reduced graphene oxide

10 nM [61]

Herein, we fabricated simple paper-based microfluidic SERS substrates via in situ

reduction of hydrogen tetrachloroaurate (gold precursor) to form gold nanoparticles. We

illustrated that in situ reduction of gold precursor produced gold nanoparticles within the paper

5

matrix to develop SERS substrates. These SERS substrates were used to measure the SERS

signal enhancement of MGITC dye. An attempt to understand the localized surface plasmon

resonance (LSPR) band, uniformity, and optimization of these substrates (with different molar

ratio of reducing agent to gold precursor) was made.

1.2 Specific objectives

The primary objective of this research was to develop paper based SERS substrates

(sensors) using in situ -based reduction of gold precursor. This goal was achieved by focusing on

the following specific objectives:

1) Synthesis of gold nanoparticles (AuNPs) using in situ reduction of hydrogen

tetrachloroaurate (gold precursor) within paper, and

2) Effect of various parameters such as gold precursor, and reducing agent concentration

and reaction time on SERS signal enhancement for MGITC dye.

1.3 Overview of chapters

Chapter 2. Literature Review

This chapter provides an overview of relevant topics including the review of surface

enhanced Raman spectroscopy and its applications. The three primary topics covered in this

review are Raman and SERS principles, microfluidic paper-based devices for SERS applications,

and the preparation of gold nanoparticles (AuNPs).

Chapter 3. Experimental Methods and Instruments

This chapter summarizes various experimental methods and processes used for synthesis

of paper based sensors. It also provides a detailed discussion about the instruments (UV-Vis,

Raman, SEM, and TEM) used to characterize the paper-based SERS substrates.

6

Chapter 4. Results and Discussion

This chapter summarizes the experimental results that describe the synthesis of gold

nanoparticles using in situ based reduction of gold salt within paper. It also discusses the effect

of variations in gold salt and reducing agent (citrate solution) concentrations on the SERS signal

enhancements of Raman active MGITC dye. Also, detailed analysis of collected UV-Vis data is

performed to understand shape and size of LSPR band.

Chapter 5. Conclusions and Future Work

This chapter summarizes the conclusions drawn from the previous chapters and the future

direction for this research.

7

Chapter 2. Literature Review

2.1 Raman spectroscopy and its application

2.1.1 Origin of Raman

Dr. Chandrasekhara Venkata Raman, an Indian professor of physics, described the

important phenomena of light scattering in a paper on “molecular diffraction of light” in 1928

[62]. This observed phenomena indicated that “when a light is scattered by any material some of

the scattered light will have different frequency than the frequency of incident light” [62]. This

phenomenon was named as “Raman effect”.

2.1.2 Raman scattering

When a monochromatic light is scattered by a material, two types of scattering i.e., elastic

or inelastic scattering occur [40, 62-64]. The first type of scattering is known as Rayleigh

scattering, which doesn’t have any energy transfer between molecules and the incident light

photons [62]. Therefore, it is also called elastic scattering [62, 65].

The latter type of scattering involves the exchange of energy between incident photons of

monochromatic light and molecules of analytes, therefore, the scattered light photon energy will

be different from the incident photon energy [62]. This second type of scattering is known as

inelastic, or Raman scattering [62]. Raman scattering can be further classified as either “Stokes”

or “anti-Stokes” [64]. Stokes scattering reflects the lower energy of scattered photons than the

incident light, whereas, anti-Stokes scattering has higher energy of scattered light photons than

the incident light photon [62]. Inelastic scattering provides information about various transitions

(such as vibrational, and rotational) in the samples [66]. However, Raman scattering is a very

weak phenomena as Rayleigh scattering is six order of magnitude higher than the Raman

8

scattering [62, 64]. The schematic representation of Raman and Rayleigh scattering is shown in

Figure 1.

A Raman spectrometer consists of a laser, illumination system (collection of lens and

optical systems), filter or spectrophotometer, and a detector [26, 62, 67, 68].

Figure 1. Schematic representation of Raman (Stokes & anti-Stokes) and Rayleigh

scattering. Modified from: (http://www.doitpoms.ac.uk/tlplib/raman/raman_scattering.php.)

Used under fair use, 2015.

The sample of interest (analyte) is normally illuminated with a monochromatic laser

beam and the laser beam is then reflected by mirrors [67, 69, 70]. Afterwards, scattered light is

filtered and detected to obtain the Raman spectrum of the desired sample [67, 69, 70].

2.1.3 Comparison between Raman spectroscopy and other technologies

Raman spectroscopy is very useful for obtaining molecular and chemical information

about the desired molecules in the sample as it enables non-destructive, rapid and real time

9

spatial resolution of micron size [71]. Detailed comparison between Raman spectroscopy and the

other technologies are in Table 3.

X-ray diffraction (XRD) is commonly used to analyze solid structures, but not liquid or

gas samples [71, 72]. Whereas, Raman is fast and it can produce unique structure/spectrum for

target analytes [71, 72]. Raman Spectroscopy is based on the changes in the polarizability of

vibrating molecules whereas, infrared (IR) spectroscopy is based on the changes in dipole

moment [71]. Raman can be used for aqueous samples, and IR inactive samples can often be

analyzed by Raman spectroscopy [73].

10

Table 3. Comparison between Raman spectroscopy and other techniques. Springer Science

and Business Media/ Cancer and Metastasis Reviews, 33 (2-3), 2014, 673-693, Emerging

Technology: Applications of Raman Spectroscopy for Prostate Cancer, Kast, R.E., et al.,

excerpts, Copyright © 2014, Springer Science+Business Media [71]. Used with kind permission

from Springer Science and Business Media, 2015.

Properties Raman Fourier Transform Infrared Spectroscopy

X-Ray Diffraction

Gas chromatography/ Mass Spectroscopy

Spatial Resolution <1 µm <1 µm Generally for crystal analysis

Generally Bulk analysis

Destructive No No Yes Yes Extensive sample preparation

No No Yes No

Influenced by water

Limited, Depends

Yes Yes No

Surface Near surface penetration

Near surface penetration

Crystal structure

Bulk

Phases Solid, Liquid, Gas

Solid, Liquid, Gas

Solid

2.1.4 Limitation of Raman spectroscopy

Raman scattering is a very weak phenomena as Rayleigh scattering is six order of

magnitude higher than the Raman scattering [62]. Rayleigh scattering doesn’t provide any useful

information for molecular characterization [62]. Only the inelastic scattering (Raman scattering)

is practically useful for the purpose of molecular detection [62]. However, Raman scattering is

difficult to detect as the Raman signals are often overshadowed by Rayleigh scattering,

competitive fluorescence (another type of inelastic scattering) and thermal emission [68, 74].

11

For these reasons, researchers are trying to develop various techniques to improve Raman

signal intensity for detection of desired analytes. One of the techniques used for Raman signal

enhancement is ‘surface enhanced Raman spectroscopy (SERS)’ [18, 71, 75-77]. In this thesis,

SERS is used for signal enhancements of Raman active MGITC dye.

2.2 Surface enhanced Raman spectroscopy (SERS)

2.2.1 Introduction

SERS is a reliable, economical, highly surface selective and ultrasensitive method to

qualitative identify and quantitative analyze aqueous and airborne contaminants, cyanobacteria

and other pathogens [15].

2.2.2 SERS mechanism

SERS signal enhancement depends on roughened (nanoparticles based) metal surface

[78-80]. Usually, silver or gold metal are used to produce rough surface as they produce high

signal enhancements. Compared to normal Raman, SERS may produce signal enhancements of

up to ~1014, in general, enhancements of 106 or 108 are most readily observed [15, 37]. The

SERS signal enhancement can be attributed to two main mechanisms: a) electromagnetic

enhancement by electromagnetic field produced at or near nanostructured surfaces, and b)

chemical enhancement by chemical/ physical adsorption of the analyte to the metal surface [37].

When an electromagnetic wave from an excitation laser interacts with the roughened

metal surface, it leads to enhancements of the e-field by surface plasmon resonance (with

incident laser wave) and hence enhancement in Raman signals [32, 81-84]. Detailed discussion

on surface plasmon resonance is performed later in this chapter.

12

Chemical enhancement involves covalent bonds formation (i.e. adsorption) between the

analytes molecules and the roughened metal surface [84, 85]. These chemical bonds lead to

charge transfer from the metal surface to adsorbate thus resulting in an increased polarizability of

the analyte molecules (adsorbate) [84]. It is generally believed that enhancement due to charge

transfer mechanism is lesser with respect to electromagnetic enhancements [84].

Roughness of the metal surface and wavelength of the Raman laser are important

component for a good SERS enhancement [31, 78, 80]. Smooth surface will not produce

effective surface plasmons [31]. Usually, size of nanoparticles is commonly in the range of 20 to

100 nanometers for Raman laser wavelengths in the range of 532 to 780 nanometers [31].

2.2.3 SERS substrates

The choice of SERS substrate/support material (such as silicon, paper, polymers or glass

plate) and noble metals (Au, Ag, or Cu) are very important for SERS [18, 24, 31, 86]. In general,

SERS substrates are prepared by depositing molecules of noble metals on the support material.

One of the main reason to select noble metals (such as Ag, Au or Cu) is that they are easy to

handle in acidic/basic, ambient, electrochemical or vacuum conditions and they also produce

significant Raman signal enhancements for a wide variety of analytes [87].

2.2.3.1 Methods to prepare SERS substrates

The applications of SERS mainly depend on activity and reproducibility of the SERS

substrates [38, 48, 58, 88]. According to Lin X.M. et al., electrochemical oxidation and reduction

cycle (EC-ORC), wet chemical synthesis methods, or lithography methods are often used to

produce SERS substrates [32].

13

Besides above-mentioned methods, other methods such as chemical assembly method,

aluminum oxide template method, and lithography are also used to fabricate SERS substrates

[32, 89, 90]. In this research, paper-based substrates are synthesized by chemical reduction

method; therefore, it is reviewed in detail to discuss synthesis of various types of nanoparticles

(especially gold nanoparticles) and their effect on LSPR band and Raman signal enhancements.

Gold nanoparticles are mainly produced by reduction of metal precursor in aqueous or

non-aqueous solution [32, 51, 52, 91-96]. Various reducing agents such as sodium citrate

(Na3Ctr), and Sodium borohydride (NaBH4) are widely used for this purpose [32]. However,

these formed nanoparticles tend to aggregate or oxidized back to metal atoms [34, 94, 97].

Therefore, various capping agents, usually surfactants, are also added to avoid aggregation, and

oxidation of formed nanoparticles [32, 34, 53]. These surfactants (capping agents) also help to

regulate growth rate of various features/sides to produce controlled size or shaped nanoparticles

[32, 53]. Different methods to synthesize gold nanoparticle are also discussed later in this

chapter.

Various factors such as type of metal salt, reducing agent, or capping agent, and their

concentration in solution, and reaction conditions (such as temperature, and pH) affects the size,

and shape of nanoparticles [32, 33, 50, 94, 98, 99]. In this review, metal salts of silver (Ag) and

gold (Au) are discussed in detailed as they are widely used to prepare SERS substrates. It is

estimated that Raman signal enhancements for a single AuNP and a single AgNP is about 103 -

104 and 106-107 respectively [18, 30, 32, 38, 47, 100, 101].

In a study by Emory SR et al., Ag colloidal nanoparticles were used to study SERS of

rhodamine 6G (R6G) [32, 102]. Single Ag nanoparticle exhibited maximum SERS enhancement

14

(as large as 1014 - 1015) compared to colloidal nanoparticles aggregates or bulk with 647-nm

excitation [32, 102]. Similarly, AuNPs (~60 nm) with 647 nm laser excitation showed maximum

enhancement of SERS signals [32, 103].

Kelly et al., studied the electrostatic field generated by spherical, non-spherical and

truncated tetrahedrons shaped nanoparticles, and it was observed that metal nanoparticles with

the sharp edge and angles would produce extra enhancement (Figure 2) due larger electric field

values at sharp edge and angles [32, 104]. This is also called ‘Lightning rod effect’. Due to

lightning rod effect non-spherical SERS enhancements were higher [32, 104].

15

Figure 2. Example of lightning rod effect for Ag Trigonal prism (side length 100 nm and

thickness is 16 nm); Sharp angles and edges produces extra enhancement. Electric field

scale is from 0 to 20 units. Kelly, K.L., et al., The optical properties of metal nanoparticles: The

influence of size, shape, and dielectric environment. Journal of Physical Chemistry, 2003.

107(3): p. 668-677. Copyright © (2003) American Chemical Society. Used with kind permission

from Rightslink, 2015.

16

Orendorff et al. studied the effect of shape and architecture of gold nanoparticle on SERS

spectrum of 4-mercaptobenzoic acid (4-MBA) [32, 88]. It was concluded that larger signal

enhancements were produced due to increase in localized electromagnetic field enhancement at

the interface of 4-MBA and nanoparticles on the surface of substrates [32, 88]. It was also

established that shape of AuNPs may vary enhancement factors by a factor of 102 [88].

Gold nanorods produced transverse and longitudinal modes in absorption spectrum in

UV-Vis analysis [105, 106]. According to the study by Huang et al., Au nanorods (aspect ratio =

4) produced surface plasmon band at about 800 nm; therefore, these gold nanorods based

substrates were suitable for 785-nm (Raman laser) excitation [105].

As mentioned before, ideal SERS substrates should have high SERS activity and good

uniformity; also should provide sensitive detection of analytes [32, 37]. Substrates should be

uniform and reproducible to minimize the deviation/variation in signal enhancement [36, 37,

107]. It is challenging to prepare ideal SERS substrates that can be simultaneously be uniform,

reproducible, and produce good signal enhancements [21, 32]. Therefore, researchers are

fabricating microfluidic paper-based SERS substrates [21, 37, 108]. The detailed discussion of

microfluidic paper-based devices (sensors) is performed later in this chapter.

2.3 Gold nanoparticles: synthesis and properties

2.3.1 Synthesis of gold nanoparticles

In this research, gold nanoparticles are used to produce paper-based sensors (refer

Chapter 3). Therefore, in this thesis, gold nanoparticle synthesis is reviewed in detail. Various

approaches have been developed to produce gold nanoparticles of different dimensions and

functionality [34, 53, 109]. In “top down” approach, bulk state gold is broken into smaller

17

dimensions of required size and shape. However, “bottom up” approach involves chemical or

biological reduction of individual molecules (gold precursor) to form gold nanoparticles [53].

Chemical reduction of gold precursor involves nucleation or growth mechanisms of gold

nanoparticles (bottom up approach) [53]. In “in situ synthesis” of gold nanoparticles both

nucleation and growth steps are performed in the same process whereas, in “seed-growth

method” nucleation and successive growth happens in two different processes [53].

The schematic illustration of gold nanoparticle formation through nucleation or growth

mechanism is shown in Figure 3. Reduction of gold chloride by various reducing agents

converts Au+3 ions into neutral gold atoms in the solution [53]. As more and more gold atoms are

formed, nucleation of gold atoms occur and gold atoms will start precipitating in nanometer

particles [110]. These nucleated gold atoms combine and grow to form nanoparticles (Figure 3).

In this thesis, a modified Turkevich method approach is used for in situ based reduction

of hydrogen tetrachloroaurate [53]. Turkevich et al. produced spherical gold nanoparticles in

1951 by simply adding and stirring hydrogen tetrachloroaurate (HAuCl4) with sodium citric acid

in aqueous solution [34, 95, 111]. In this approach, various parameters such as reaction

temperature, the ratio of citrate to gold, and gold salt (HAuCl4) concentration affect the size

distribution of gold NPs [53, 95].

18

Figure 3. Illustration of gold nanoparticle formation by nucleation and growth mechanism.

Polte, J., et al., Nucleation and Growth of Gold Nanoparticles Studied via in situ Small Angle X-

ray Scattering at Millisecond Time Resolution. ACS Nano, 2010. 4(2): p. 1076-1082. Copyright

© 2010, American Chemical Society. Used with kind permission from Rightslink, 2015.

Turkevich method was further improved by changing the citrate-to-gold ratio for

production of relatively small sized spherical gold nanoparticles (diameter = 10 to 20 nm) [53,

99]. Use of surfactant during this reaction can prevent the aggregation of citrate reduced AuNPs

[21, 53, 96].

Seeding techniques are also used to produce large spherical and non-spherical particles

[53, 112]. It is also a popular method to make gold nanorods. In this method, gold precursor is

reduced to seed particles for controlled formation of nanorods [92, 97].

19

2.3.2 Properties of gold nanoparticles

AuNPs possess important property to produce surface plasmon resonance (SPR), and to

reduce fluorescence produced during SERS analysis [35, 99]. Because of these properties,

AuNPs has applications in imaging, sensing and sensor fabrications [35].

Spherical AuNPs exhibit red or purple color in aqueous solution depending on the

diameter and UV-Vis extinction (absorption and scattering) peak [33, 34]. Toma et al. defined

surface plasmon band as “the result of the collective oscillations of the conduction band

electrons” [113]. It is effected by dimension of NPs, type of solvent, reducing agents, reaction

conditions (i.e. temperature), closeness and aggregation of nanoparticles [35, 113].

Accumulation or aggregation of nanoparticles allows electronic coupling of gold nanoparticles,

which causes broadening and shifting of spectrum peaks [113]. A SERS spectrum is a function

of various factors such as shape, size, proximity and extent of plasmon coupling and the nature

of molecules adsorbed [30, 35, 99].

Typically extinction spectrum (UV-Vis) of spherical AuNPs have the plasmon band

range from 520 nm to 560 nm [113]. Non-spherical gold NPs shows two distinct longitudinal and

transverse dipole polarization modes (transverse and longitudinal) [113]. Transverse modes and

longitudinal modes show resonance coinciding with the plasmon band of gold NPs at blue shift

(520- 560 nm) and red shift (650-750 nm) respectively [94, 99, 111, 112].

2.3.3 In situ reduction of metal salts

To deposit nanoparticles on cellulose based polymer/paper various methodology such as

blending of components, in situ reduction of metal salt, electrostatic assembly, and UV reduction

are used [35, 114]. Blending of metal NPs and cellulose i.e. direct deposition of metal

20

nanoparticles on cellulose filter paper usually causes aggregates and heterogeneous distribution

of nanoparticles at the edge producing a “coffee ring effect” due to drying process [13, 21, 37,

54].

In situ reduction of metal precursors by external reducing agents is a simple process [35,

51-53, 114-118]. It entraps metal salt in the fibers, and then, entrapped metal salt are reduced by

an external reducing agent [33, 34, 96, 114]. In situ reduction avoids aggregation of

nanoparticles (NPs) and improves homogenous distribution of NPs [50, 52, 53]. Sodium

borohydride, and tri-sodium citrate are effective and widely used external reducing agents for

gold precursors [114].

2.4 Microfluidic paper-based devices (µPADs)

Paper, a cellulose fiber web, is hydrophilic in nature and has high surface area [41, 119,

120]. Structure and porosity of paper can be modified easily to achieve desired properties [13,

42, 100]. It is portable, disposable, degradable, cheap and easy to use [119]. Therefore, paper is

widely used to fabricate microfluidic devices for variety of applications in healthcare and

environmental monitoring [108, 121, 122].

According to Thuo et al., paper microfluidic is defined as: “a methodology in which fluid

flow in paper is driven by capillary wetting and directed by channel boundaries of hydrophobic

wax or polymer patterned in the paper by printing, photolithography, or other methods” [123].

Wax printing and photolithography are widely used fabricating and patterning technology

for preparations of paper-based microfluidic devices [42, 46, 120]. Wax printing is a rapid,

inexpensive and easier than photolithography and other mentioned techniques [42]. In this

research, wax printing is used to prepare SERS substrates therefore; only wax printing method of

21

patterning is discussed. Wax printer deposits wax on the paper; wax is melted by a heat source;

and spreads in lateral and vertical direction creating a 3-D hydrophobic barrier across the

thickness [42, 124]. Molten Wax spreads and hence hydrophobic walls are wider than original

printed pattern (Figure 7).

Wax printed microfluidic devices are suited for aqueous solutions of various pHs as it

(aqueous solution) doesn’t cross the hydrophobic barrier of wax on cellulose paper but flow with

in the hydrophobic barriers (channels). Analytes transport can also be controlled by changing the

channel shape, size and by adding soluble or insoluble barriers [68].

Microfluidic devices (µPads) are used for colorimetric detections of glucose, protein, and

copper using silver nanoparticles [41, 125]. Low cost paper-based sensors present a great

platform for detection of cancer biomarkers, heavy metal, identification of blood type and

various bacteria [15, 17, 23, 41, 105, 126, 127]. They are also used with SERS for detection of

various environmental contaminates and pathogens [23, 100, 101].

Depositing metal (usually Au, Ag, Cu) nanoparticles on the patterned paper forms paper-

based SERS substrates [21, 37, 41-46]. These SERS substrates may be prepared by using pre-

formed metal nanoparticles and then subsequent addition of these nanoparticles to paper [21, 22,

27, 38, 47, 48] or synthesis of metal nanoparticles within paper by the addition of metal

precursor and the subsequent addition of reducing agent [49-53].

As mentioned in Chapter 1, Lee C.H. et al. fabricated SERS substrates by directly

depositing gold nanorods (AuNRs) solution in filter paper, which can be used for detection of

desired analytes using SERS (Figure 4) [21]. Also, LSPR band in the UV-Vis analysis was

observed to be in between from 520 nm - 700 nm.

22

Figure 4. (A) Paper-based SERS substrates prepared by gold nanorods (AuNRs) solution

before and after exposure showing the strong color change; (B) UV-Vis extinction spectrum

of the AuNRs solution and the AuNRs loaded paper. Lee, C.H., L. Tian, and S. Singamaneni,

Paper-Based SERS Swab for Rapid Trace Detection on Real World Surfaces. Applied Materials

& Interfaces, 2010. 2(12): p. 3429-3435. Copyright (2010) American Chemical Society. Used

with kind permission from Rightslink, 2015.

23

However, very little is known and reported for SERS substrates prepared by in situ

reduction of gold salt to produce gold nanoparticles (AuNPs) in the paper [114]. This method can

reduce effect of “coffee ring” due to nanoparticle transport and it can be expected to get more

uniform substrates. Therefore, in this thesis, an effort is made to fabricate simple paper-based

SERS substrates using in situ reduction based synthesis of gold nanoparticles.

24

Chapter 3. Experimental Methods and Instruments

3.1 Material

Tri-sodium citrate dihydrate (F.W. 294.1) was purchased from Fisher Chemical. ACS

reagent grade hydrogen tetrachloroaurate (F.W 393.8) was purchased from Sigma Aldrich. Stock

solutions of 200 mM hydrogen tetrachloroaurate and 600 mM trisodium citrate dihydrate were

prepared at ambient temperature in nanopure water. The stock solution was diluted to achieve

various concentrations from 5 to 200 mM of hydrogen tetrachloroaurate and 5 to 600 mM

trisodium citrate dihydrate. In this thesis, tri-sodium citrate dehydrate solution is simply referred

as citrate solution and similarly, hydrogen tetrachloroaurate is called gold chloride solution or

gold precursor.

3.2 Preparation of wax printed microfluidic wells/channels

Wax printed Microfluidic channels/wells were designed using Adobe Photoshop CS5

software. Various designs such as a) circular wells of 2 mm diameter (Figure 5), b) circular

wells of 10 mm diameter, and c) channels of 3 mm diameter with 1.2 mm channel length each

with wax wall thickness of 300 µm (Figure 5) were produced on chromatography paper using a

wax printer (Xerox Colorqube 8570).

The wax printed microfluidic channels or wells were heated to spread the wax in the

paper to create a 3D hydrophobic barrier. After printing these designs on chromatography paper,

the channels/wells were heated at 150 ℃ for 2 minutes in the following manner: a) the back side

of the wax printed channels/spots was heated for 1 min; b) the front side of the sample was

heated for 30 sec; and c) the back side of the samples was heated again for 30 sec. In this way,

the wax melted into the paper creating a three-dimensional microfluidic channel or well with

25

hydrophobic boundaries. These wax boundaries provide the barrier for aqueous solutions of

hydrogen tetrachloroaurate or trisodium citrate (Figure 7).

Figure 5. (A) Wax printed circular paper-based sensors (2 mm circular wells) (B) Wax

printed microfluidic paper-based channels; dark red color is due to formed gold

nanoparticles.

3.3 Preparation of paper-based SERS substrates

Paper-based SERS substrates (Figure 5) were prepared by in situ reduction (Figure 6) of

hydrogen tetrachloroaurate with trisodium citrate in microfluidic channels/wells under room

temperature conditions (Temperature ~25oC & RH ~65%). The citrate reduction method

(Turkevich method [95, 111]) was used with the following modifications (Figure 6). In the

Turkevich method, gold nanoparticles are synthesized by citrate reduction in aqueous solution

whereas, in this case in situ reduction involved reaction of hydrogen tetrachloroaurate with

sodium citrate within the microfluidic channels/wells. First, addition of hydrogen

tetrachloroaurate was done in microfluidic channels/spots under room temperature conditions

A B

26

(Temperature ~25oC & RH ~65%). Afterwards, trisodium citrate (reducing agent) was added to

reduce hydrogen tetrachloroaurate (gold precursor). The time gap between the addition of gold

salt precursor and reducing agent was approximately 1-2 minutes. Hydrogen tetrachloroaurate

and trisodium citrate were adsorbed and transported within the prepared 3D microfluidic

channel. The gold precursor was then reduced by citrate and gold nanoparticles were formed

within the paper after a given reaction time Figure 6. The molar ratio of reducing agent

(trisodium citrate solution) to gold precursor (gold chloride) was systematically varied from 1 to

6 and the substrates were analyzed over a fixed time interval (immediately after preparation, on

day 1, and week-1 to week-5)

Figure 6. In situ reduction of gold precursor (hydrogen tetrachloroaurate solution) by

reducing agent (trisodium citrate); HAuCl4 stands for gold precursor

The prepared substrates were kept under ambient laboratory conditions to dry or for the

reaction to proceed (reaction time varies). The samples were then used as SERS substrates for

analysis of the Raman active MGITC dye. Parameters such as the concentration of reducing

agent, the room temperature and type of reducing agent, humidity, pH of aqueous solution and

the molar ratio of reducing agent to gold salt precursor affect the rate of in situ reduction of gold

chloride solution.

HAuCl4

27

3.4 Instrumentation

3.4.1 UV-Vis

The UV-Vis extinction (scattering and absorption) spectra between 400-800 nm of 10

mm circular microfluidic substrates (µPads) were collected using a Varian Cary 5000

spectrophotometer with a solid sample analyzer with reduced slit height. Samples were analyzed

at regular time intervals (immediately after preparation, on day 1, and week-1 to week-5) for

different molar ratios (MRs) of reducing agent to gold precursor. It was found that transmittance

of the light beam through these µPads was negligible (less than 1%) with respect to adsorption

and reflection of light. Therefore, the UV-Vis solid sample analyzer measured the percentage of

light beam reflected by each sample substrate. On the basis of the percentage reflected data,

extinction coefficient (varies from 0 to 1) vs. wavelength (400 nm - 800 nm) graphs were plotted

and analyzed.

3.4.2 Surface enhanced Raman spectroscopy (SERS)

SERS spectra were collected using a WITec Alpha500R AFM Raman spectrometer. It

consists of a UHTS300 spectrometer, DU 401 BR-DD CCD camera, 10× and 100× microscope

objectives, and 633 nm and 785 nm He-Ne lasers. SERS substrates were prepared primarily

using 2 mm wax printed microfluidic circular wells. Various substrates of different molar ratio

(MR) of reducing agent to gold precursor were prepared according to the above-mentioned

method (Figure 6). MGITC dye (1 µL, 1 µM) was added to each SERS substrate to enable

collection of the surface-enhanced Raman spectrum. After addition of MGITC dye, these

substrates were taped to glass microscope slides.

Before collecting the SERS of substrates, the laser alignment was adjusted, the sample

position was calibrated, and the Raman spectrum of a reference silica wafer was collected. For

28

sampling, the laser needed to be focused at the center of the sample by using a 100× microscope

objective lens. Optical images, single Raman spectra and large area scans were collected using a

particular set of parameters such as 10× microscope objective lens, 785 nm or 633 nm laser,

spectral center of 1800 cm-1 and laser power of 1.0 or 1.2 on the micrometer. For 785 nm laser

1.0 and 1.2 reading on micrometer corresponds to 5 mW and 18.3 mW of power respectively for

10x microscopic objective. After obtaining optical images of a sample of interest, large area

scans (i.e., image scans) of 50 × 50 µm2 with 50 points per line, 50 lines per image and an

integration time of 0.11 sec/sample were collected.

3.4.3 FE-SEM

A Leo ZEISS Field Emission Scanning Electron Microscope (FE-SEM) was used to

observe the formation of gold nanoparticles in the microfluidic channels/wells by in situ

reduction of gold precursor. To prepare samples for FE-SEM analysis, wax was removed from

the SERS substrates to minimize charging and they were attached to the SEM stubs using double

stick tape. A sputter-coater was used to coat samples with gold and palladium (60% Au and 40%

Pd). SEM images of interest were collected by setting the voltage source (EHT) around 5 kV and

using a working distance of 10 mm. Energy Dispersive X-Ray Spectroscopy (EDS/EDX) was

used to analyze the chemical composition of selected SEM images of substrates by adjusting the

voltage to 10 kV and using a working distance of approximately 8.5 mm.

29

Chapter 4. Results and Discussion

Paper based SERS substrates (µPADs) were prepared using a wax printer to create 3D

hydrophobic wax boundaries within chromatography paper (refer Section 3.2 & 3.3). It was

observed that after heating, the width of the channels or the diameter of the circular wells was

reduced due to the spreading of the wax (Figure 7). In general, for a wax printed channel

(µPAD), the available area for transport of analyte will be reduced; however, the as formed wax

boundaries constrain the transport of analyte and solution as illustrated in Figure 7.

After heating, the cellulosic fibers of the filter paper were covered by the hydrophobic

layer of wax. Figure 8 illustrates wax-coated paper prior to heating and after heating. Because of

the wax coating, the aqueous analytes were forced/constrained to flow within the wax printed

boundaries. These wax-printed paper based microfluidic channels or wells (µPADs) were used to

further develop SERS substrates using in situ reduction of gold precursor.

30

Figure 7. The wax spreads during heating, creating narrower channels in the tested

μPADs; (A) Unheated wax channel and heated wax channel; (B) Unheated and heated

circular microfluidic well. The μPAD dimension describes the printed measurements.

31

Figure 8. Characterization of wax printed-paper before and after heating; (A) SEM images

of microfluidic channel before (at magnification of 500x); (B) FESEM images of

microfluidic channel after heating (at magnification of 500x); (C) One of the waxed coated

cellulosic fiber is zoomed in (at magnification of 1000x).

Unheated wax Fresh paper, no wax

100 μm

100 μm 2 μm

B C

A

After Heating

32

4.1 Synthesis of gold nanoparticles by in situ reduction within paper

The synthesis of gold nanoparticles by citrate reduction is a commonly used method that

involves hydrogen tetrachloroaurate, sodium citrate, and water [33, 111]. In this research, gold

nanoparticles were synthesized within the microfluidic paper based substrates by ‘in situ

reduction’. As described in Section 3.3, ‘in situ reduction’ occurs when nucleation and growth of

nanoparticles occur in the same process/step within the paper matrix [50-52, 110, 115, 116, 128].

Here, paper-based SERS substrates were prepared by loading gold precursor on chromatography

paper or microfluidic paper based devices (Figure 6), followed by addition of sodium citrate as a

reducing agent.

Hydrogen tetrachloroaurate (also referred as gold chloride or HAuCl4) and trisodium

citrate (referred to as citrate or Na3Ctr) are adsorbed and transported on microfluidic paper based

devices. Due to redox reaction, gold ions (Au+3) are reduced to gold atoms that nucleate to form

gold nanoparticles (Figure 3).

As shown in Figure 9, the observed color of the microfluidic paper based sensors

(µPADs) changed to dark pink (red/violet) as a result of in situ nanoparticle formation. In situ

nanoparticles synthesis was affected by the change in the concentration of gold salt, reducing

agent, the ambient conditions, and the reaction time. Figure 12 shows the UV-Vis spectra for

samples with variable gold salt and citrate solution concentrations (Table 4). The molar ratio of

citrate to gold chloride has been shown to be the primary parameter that controls nucleation and

growth of AuNPs in suspension and therefore during preparation of paper based samples the

molar ratio (MR) was varied from 1 to 6 by changing the gold chloride and sodium citrate

concentrations.

33

Figure 9. Example of color change in chromatography paper in 10 mm circular well (left)

and 2 mm circular wells (right); HAuCl4 and Na3Ctr stand for gold chloride and sodium

citrate solution respectively.

The UV-Vis spectrum of spherical gold nanoparticle suspensions typically consists of a

maximum absorption peak with size dependent wavelength of 520-580 nm [53, 113]. However,

the UV-Vis spectra of non-spherical gold nanoparticles typically consist of two maximum

absorption peaks that correlate to two different nanoparticle dimensions (longitudinal and

transverse). These two different peaks are blue shifted (500 nm - 580 nm) and red shifted (600

nm - 700 nm) relative to the band location for spherical particles [97, 100, 113].

In the UV-Vis spectra (Figure 10) of the paper-based substrates, two absorption peaks

were observed for the samples where a lower molar ratio of reducing agent to gold precursor was

used. For higher molar ratio of citrate-to-gold precursor only one peak was observed. The

observed LSPR band provides an indication of the formation of gold nanoparticles within the

paper matrix. However, as the reaction time increases, the maximum absorbance intensity

increases with time.

34

Table 4. Concentration of gold chloride and sodium citrate solution for UV-Vis analysis;

data used in Figure 10.

Gold Chloride Conc. (Volume used: 10 µL)

Sodium Citrate Conc. (Volume used: 10 µL)

Molar Ratio (Citrate/ Gold Chloride)

25 mM 23 mM 0.95 25 mM 27 mM 1.08 20 mM 27 mM 1.35 15 mM 27 mM 1.8 15 mM 30 mM 2 10 mM 30 mM 3

For the lowest concentration of citrate (when MR is from 0.95 to 1.35) two plasmon

bands (blue shifted and red shifted) are observed. It is likely that this is because of the non-

spherical nature of the as formed gold nanoparticles or is the result of the coupling of the surface

plasmons of multiple particles. For excess citrate (molar ratio of citrate to gold chloride 2 to 3)

only one maximum absorbance peak (blue shifted) was observed. The effect of gold precursor

concentration, reducing agent and reaction time are discussed later in this chapter in Section 4.2

and Section 4.3.

35

Figure 10. UV-Vis spectrum of various substrates prepared by different citrate-to- gold

ratio (refer Table 4); Different shape and size of LSPR is observed. (A). 25 mM gold

chloride, 23 mM Citrate Solution (MR= 0.95) (B). 20 mM gold chloride, 27 mM Citrate

Solution (MR= 1.35) (C). 15 mM gold chloride, 30 mM Citrate Solution (MR= 2) (D) 10

mM gold chloride, 30 mM Citrate Solution (MR= 3)

A B

C D

Extin

ction coeff.

Extin

ction coeff.

Extin

ction coeff.

Wavelength (nm) Extin

ction coeff.

36

SERS spectrums of MGITC dye were collected from a concentrated MGITC-methanol

solution (Figure 11). In MGITC-methanol solution, MGITC has primary bands at 1200, 1380

and 1600 cm-1 and therefore the spectrum from 1100 cm-1 to 1800 cm-1 was primarily considered

for further analysis in Section 4.3.

Figure 11. SERS of MGITC dye in MGITC-methanol solution. MGITC assignments are at

1200, 1380 and 1600 cm-1. Leng, W. and P.J. Vikesland, MGITC Facilitated Formation of

AuNP Multimers. Langmuir, 2014. 30(28): p. 8342-8349. Copyright (2014) American Chemical

Society. Used with kind permission from Rightslink, 2015.

Different molar ratio (MR) of reducing salt and gold precursor also affect the SERS

signals intensity/enhancements. Large area scans (SERS spectrum) of different MR were plotted

in Figure 12 to understand the effect of MR, and the effects of changes in the initial

concentration of gold precursor. The general method to obtain SERS spectrum is reported in

Section 3.4.2. However, to obtain Figure 12, Raman laser of 633 nm wavelengths was used

instead of 785 nm Raman Laser. The Raman laser of 785 nm produced less fluorescence and

background noise for these SERS substrates.

37

Table 5. Concentration of Gold Chloride and Sodium Citrate (data used in Figure 12)

Gold Chloride Conc. (Volume used= 1 µL)

Sodium Citrate Conc. (Volume used= 1 µL)

Molar Ratio (Citrate/ Gold Chloride)

25 mM 17 mM 0.68 15 mM 20 mM 1.33 10 mM 27 mM 2.7 5 mM 20 mM 4.0 5 mM 27 mM 5.4 4 mM 27 mM 6.75 4 mM 30 mM 7.5

Figure 12. Comparative analyses of paper-based substrates prepared by various different

molar ratio of citrate to gold chloride; 2 mm wax printed wells are loaded with gold

chloride solution followed by citrate solution (reducing agent); Reaction time: 7 days with

addition of 1 µL of MGITC dye; (A) the difference in the region of 100 ~ 350 cm-1 may be

caused by the size or shape of Gold NPs in the paper; (B) SERS spectrum of MGITC dye

with various molar ratios (MR). Raman parameters: Laser wavelength 633 nm, and

micrometer reading 1.2 mm (0.261 mW for 10x objective lens).

38

The SERS spectra for substrates with MR of 0.68 and 1.33 have higher initial gold

precursor than other samples and they (MR= 0.68 and 1.33) have clear and uniquely

distinguishable MGITC spectrum. Higher gold chloride concentrations produce more gold

nanoparticles (for same conditions and same reducing salt concentration). Also, if MR of

reducing agent (citrate solution) to gold chloride solution is less than the 1:2 (reaction

stoichiometry) then it may produce larger and irregular dimensions of gold nanoparticles in the

paper based substrates that will result into higher peak heights in the SERS spectrum of MGITC

dye [104]. This analysis shows that paper based substrates prepared by in situ based reduction

based method can be used for detection and monitoring of analytes. However, ideal paper-based

substrates should be uniform and reproducible so that variation in signal enhancement may be

reduced. For this purpose, an additional set of experiments and optimization strategies were

conducted and are further discussed in Section 4.3.

A Leo ZEISS Field Emission Scanning Electron Microscope (FE-SEM) was used to

observe the formed gold nanoparticles in microfluidic channels/wells by in situ reduction of gold

salt. Figure 13 shows chromatography paper SEM image and EDX analysis. A very small

elemental peak for gold (Au) element was observed, which can be attributed to the gold-

palladium sputter coating. Figure 14 shows SEM images and EDX analysis of chromatography

paper loaded with pre-formed gold nanoparticles (size 50 nm) prepared by Turkevich method.

Due to the presence of 50 nm size AuNPs a significant elemental peak for Au element was

observed. Analysis of paper-based substrates prepared by in situ reduction of gold salt was also

performed. Figure 15 shows clusters of AuNPs formed via in situ reduction method. Figure 16

illustrates SEM and EDX images for paper based SERS substrates prepared by loading 5 mM

39

auric acid (3 µL) followed by 20 mM citrate solution (3 µL) with allowed reaction time of 7 days

(with a desiccator time ~ 1 day). Detailed discussion on FE-SEM method is discussed in Section

3.4.3. It is observed that the elemental peak of gold in paper-based substrates prepared by in situ

reduction of gold salt is significant.

40

Figure 13. (A) FE-SEM image of chromatography paper (blank) at magnification ~10 kx;

(B) Different FE-SEM image of chromatography paper (blank) at magnification ~10 kx;

(C) EDX image showing strong C, O elemental peaks and negligible gold peaks. Gold peak

is a result of gold-palladium coating.

B A

C

41

Figure 14. (A) SEM image of chromatography paper loaded with 50 nm sized gold

nanoparticles prepared by citrate reduction method at magnification of 30.00 kx; (B) SEM

image of chromatography paper loaded with 50 nm sized gold nanoparticles prepared by

citrate reduction method at magnification of 100.00 kx; (C) EDX image confirming

presence of gold nanoparticles. Images produced at working distance (WD) ~8.5 mm and

Voltage (EHT) ~ 20kV.

A B

C

42

Figure 15. SEM analysis of paper-based SERS substrates prepared by in situ reduction

based gold nanoparticle synthesis (5 mM gold chloride, molar ratio of citrate to gold

chloride~ 4, and allowed reaction time ~ 7 days) (A) Clusters (circles) of formed AuNPs in

sample image with magnification ~ 101.4 KX; (B) Clusters of formed AuNPs in sample

image with magnification ~ 30.4 KX; (C) Clusters of formed AuNPs in sample image with

magnification ~ 100.36 KX; (D) Clusters of formed AuNPs in sample image with

magnification ~ 100.72 K X (used WD~ 5.7 mm, EHT~ 2 kV).

A B

C D

43

Figure 16. SEM analysis of paper-based SERS substrates prepared by in situ reduction

based gold nanoparticle synthesis (5 mM gold chloride, molar ratio of citrate to gold

chloride~ 4 and allowed reaction time ~ 7 days); (A) Sample image with magnification ~40

K X, WD~ 5.7 mm, EHT~ 2 kV; (B) EDX image showing elemental analysis of square box

(pink color) in image (A).

44

On the basis of scanning electron microscope (SEM), UV-Vis absorption analysis and

signal enhancement of surface Raman enhancement spectroscopy, formation of AuNPs within

the microfluidic paper-based device (µPADs) was confirmed. However, to study effect of

variation in gold precursor i.e. gold chloride and reducing agent concentration on LSPR band and

shape, detailed UV-Vis and SERS analysis of different samples were performed.

4.2 UV-Vis analysis to study effect of the variation in gold precursor and reducing agent

concentration with increase in reaction time

UV-Vis spectra of paper-based sensors were collected using a solid UV-Vis analyzer as

discussed in Section 3.2. It is expected that different gold precursor and reducing agent

concentrations will produce different UV-Vis spectrum. In this analysis, the effect of variation in

gold precursor and reducing agent concentration with an increase in reaction time was studied.

For this purpose four different gold salt concentrations (10, 20, 50, and 100 mM) were

considered. The molar ratio of citrate to gold chloride was varied from ~ 1 to ~ 6. Reaction time

for the reaction and analysis were day 1, week 1, week 3 and week 5. Detailed experimental

method for UV-Vis is described in Section 3.4.

Temporal changes in the maximum extinction peak of various molar ratios (MR) are

reported in Figure 17. It is observed that the initial rate of change in the maximum extinction

peak intensity is fast, and finally an equilibrium value of the maximum extinction peak was

achieved. As the gold precursor concentration increased from 10 to 100 mM, the maximum

extinction value also increased for a given reaction time. The use of 100 mM gold chloride

resulted in the highest equilibrium value of maximum extinction coefficient (Figure 17). This

may be because of the high gold nanoparticle density for the given molar ratio of citrate to gold

chloride. This trend was also observed for other MRs of citrate to gold chloride. After 35 days,

45

maximum extinction peak value associated to 50 mM initial gold precursor approaches to

maximum absorption value 100 mM gold precursor (red circle in Figure 17). It is observed that

as the initial gold precursor concentration is increased from 10 mM to 100 mM the spectrum

became broader (Figure 18). It can be concluded that for [HAuCl4] greater than 20 mM, a

broader LSPR is observed. In this analysis, initial concentration of 20 mM for gold precursor

was used for further SERS analysis (refer Section 4.3).

46

Figure 17. Temporal variation of maximum absorption of different molar ratios of

citrate solution to gold chloride solution for a given initial concentration of gold chloride

solution; (A) Molar ratio (MR) = 1 (B) Molar Ratio = 3 (C) Molar Ratio= 5.

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20 25 30 35 40

Max. A

bsorpW

on

Time (days)

Temporal change in Max. AbsorpWon (MR: 1)

10 mM gold precursor

20 mM gold Precursor



A

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

0 5 10 15 20 25 30 35 40

Max. A

bsorpW

on

Time (days)






B

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20 25 30 35 40

Max. A

bsorpW

on

Time (days)






C

47

Figure 18. Effect of initial gold precursor solution on absorption values for a given molar

ratio of citrate solution to gold chloride solution after reaction time of 35 days; (A) Molar

ratio of 1 after 35 days (B) Molar ratio of 3 after 35 days (C) Molar ratio of 6 after 35 days

0

0.2

0.4

0.6

0.8

400 500 600 700 800

Abs

Wavelength

Molar RaWo 1 (Days 35)

Paper

10 mM

20 mM

50 mM

100 mM

A

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

400 500 600 700 800

Abs

Wavelength


Paper

10 mM

20 mM

50 mM

100 mM

B

0

0.2

0.4

0.6

0.8

1

400 500 600 700 800

Abs

Wavelength


Paper

10 mM

20 mM

50 mM

100 mM

C

As [HAuCl4] increases



48

The LSPR band and shape of the curve changes as MR is increased (Figure 19). In

Figure 19, the gold chloride concentration was kept constant (20 mM) and the concentration of

citrate solution was varied. Because of different concentration of reducing agent, different

dimensions (size or shapes) of AuNPs were produced in the paper-based substrates (circular

wells of size = 10 mm).

Figure 19. LSPR Band and shape changes with the increase in molar ratio (MR) from 1 to

6. [HAuCl4]= 20 mM, reaction time 35 days, and diameter of microfluidic wells are 10 mm.

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

400 450 500 550 600 650 700 750 800 850

Normalize

d exWn

cWon

Wavelenght (nm)

[HAuCl4]= 20 mM and ReacWon Time= 35 days

MR 1

MR 2

MR 3

MR 4

MR 5

MR 6

Increase in Molar Ratio (MR)

49

Figure 20. LSPR Band and shape changes with the increase in molar ratio (MR) from 1 to

6. [HAuCl4]= 100 mM, reaction time 35 days, and diameter of microfluidic wells = 10 mm.

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

400 450 500 550 600 650 700 750 800 850

Normalize

d exWn

cWon

Wavelength (nm)

[HAuCl4]= 100 mM and reacWon Time= 35 days

MR 1

MR 2

MR 3

MR 4

MR 5

MR 6

Increase in Molar RaWo

50

Similarly, for [HAuCl4] = 100 mM concentration, by changing molar ratio (MR) change

in LSPR shape and band is observed (Figure 20). For molar ratio (MR = 1), both blue shift and

red shift are observed.

It can be concluded that for a given gold chloride (gold precursor) solution concentration

and given reaction time, the highest molar ratio produces the most intense extinction peak in the

UV-Vis spectrum. This may be because of excess citrate reduction of gold salt into AuNPs is

faster than lower citrate to gold salt molar ratio. As reaction time increased, different molar ratios

produced overlapping or similar maximum absorption peaks with different LSPR band shapes

and sizes.

For a given molar ratio (MR) and reaction time, the highest molar concentration of gold

chloride solution showed highest absorption peak. It may be because lower citrate conc.

(reducing agent) is available to gold salt, which produces larger nanoparticle in the paper. Larger

particle will scatter and absorb more UV-Vis light.

4.3 SERS signal optimization strategy for MGITC using paper based substrates

As discussed in Section 4.1, various molar ratios (MR) of citrate solution (reducing

agent) to gold chloride solution (gold precursor) produce different SERS signal enhancements,

and therefore, optimization of SERS signal enhancements for detection of MGITC dye or other

contaminants was performed in this section. An example of SERS spectrum for MGITC dye on

paper-based substrates is plotted in Figure 21. Using the procedure listed in Section 3.4.2, these

SERS spectra (large area scan, laser 785 nm and micrometer reading ~1.2 mm i.e. 18.3 mW of

laser power) were collected

51

Figure 21. Average SERS spectra (Large area scan) for MGITC dye using paper based

substrates. [HAuCl4]= 20 mM, reaction time= 28 days, Raman Laser 785 nm wavelength

with micrometer reading of 1.2 mm (18.3 mW of laser power for 10x objective lens). MR

stands for Molar Ratio.

As shown in Figure 21, higher molar ratio (such as MR = 5) produces lower signal

enhancements (lower peak heights) than lower molar ratio (i.e., MR= 1 or MR= 3). By

subtracting background signals (noise) from the spectrum, normalized SERS spectrum of various

initial gold precursor concentration and different molar ratios were collected. It can be concluded

that lower molar ratios such as MR= 1, 2 or 3 produce distinguishable signals/assignments for

MGITC dye.

660

680

700

720

740

760

780

800

-‐500 0 500 1000 1500 2000 2500 3000

Raman Intensity

(CCD

cou

nts)

Wavenumber (1/cm)

SERS spectrum of MGITC dye on paper based substrates

MR 1

MR 3

MR 5

1400 cm-‐1

1180 cm-‐1

230 cm-‐1

105 cm-‐1

[HAuCl4]= 20 mM and Reaction time= 28 days

1800 cm-‐1

52

It is desirable that variability of peak intensities (peak heights) are minimized for uniform

and reproducible SERS signals by using paper based SERS substrates. To understand peak

intensity variability of SERS signals, the MGITC assignments were compared to the peak at

1800 cm-1 wavenumber, which is one of the MGITC assignments (Figure 22). As discussed in

Figure 11, major MGITC assignments are at 1200 cm-1, 1400 cm-1, and 1600 cm-1. Therefore, in

these optimization strategies major MGITC assignments were normalized with respect to

smallest peak at 1800 cm-1 and at 105 cm-1 (peak associated to the gold nanoparticles).

53

Figure 22. Optimization of MGITC assignments with respect to peak at 1800 cm-1 (strategy

-1); Lower molar ratios produce less variability in peak ratios than high molar ratios

(circled). Sample 1 and sample 2 are replicate of same substrates (for initial gold chloride

conc. = 20 mM)

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6

Normalize

d pe

ak intensity

/ height

Molar RaWo of reducing agent to gold chloride sol

Peak raWo (1180/1800) vs. molar raWo

Sample 1

Sample 2

Avg Value

A

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

0 1 2 3 4 5 6 7

Normalize

d pe

ak intensity

/heights

Molar RaWo of reducing agent to gold chloride sol

Peak raWo (1400/1800) vs. molar raWo

Sample 1

Sample 2

Avg Value

B

54

Figure 23. Optimization of MGITC assignments with respect to the peak at 105 cm-1

(strategy-2); Sample 1 and sample 2 are replicate of same substrates (for initial gold

chloride conc. = 20 mM).

0 10 20 30 40 50 60 70

0 1 2 3 4 5 6 7

100* ra

Wo of p

eaks 1180/105

RaWo

RaWo of ~1180/105 vs. Molar RaWo

Sample 1

Sample 2

Avg value

0 5

10 15 20 25 30 35

0 1 2 3 4 5 6 7

100* ra

Wo of p

eaks 1400/105

RaWo


Sample 1

Sample 2

Avg

0 1 2 3 4 5 6 7

0 1 2 3 4 5 6 7

100* ra

Wo of p

eaks 1800/105

RaWo


Sample 1

Sample 2

Avg

55

For lower MRs (such as of MR = 1, 2 or 3), the variability in peak intensities is

minimized (circled in Figure 22) and for higher ratios, the variability in peak intensities is

relatively high. Similar results were obtained if normalization was performed with respect to the

initial gold nanoparticle peaks i.e., 105 or 230 cm-1 (Figure 23). Also, SERS spectra (large area

scan) of various initial gold precursor concentrations and different MR were collected and it was

observed that lower molar ratio such as MR= 1, 2 or 3 produces lower variability in peak ratios

(peak intensities).

Similar analysis was performed for different initial gold precursor concentration

([HAuCl4]= 10 mM), and it was observed that for lower ratios (such as MR= 1, 2 or 3) the

variability in normalized peaks are lower than higher molar ratios (such as MR= 5 or 6). Also,

Raman laser of 785 nm or 633 nm can be used with citrate-to-gold molar ratio of 1:1 or 2:1 to

produce high signal enhancements of analyte.

As paper-based SERS substrates produce less variation in peak intensities for lower ratio

in comparison of higher molar ratios (MR), therefore, use of lower molar ratio is recommended

for further analysis of desired Raman active compound or environmental contaminants using

paper-based substrates.

56

Chapter 5. Conclusions and Future Work

5.1 Conclusions

In this thesis, in situ reduction based paper-based microfluidic SERS substrates were

discussed. These substrates were prepared for detection of environmental contaminants using

AuNPs, which were synthesized by reduction of gold chloride (gold precursor) within the wax

printed chromatography paper. These prepared substrates were mainly analyzed and

characterized by UV-Vis spectroscopy, scanning electron microscope (SEM), and SERS.

It was concluded that in situ reduction produced AuNPs in the microfluidic paper based

devices (Figure 14 and Figure 16) that can be further used for contaminant detection using

SERS. From EDX analysis (Figure 16), formed gold NP clusters were confirmed. From UV-Vis

analysis, it was concluded that for a given gold salt conc. and a given reaction time, the highest

molar ratio produced the highest peak of extinction coefficient in the UV-Vis spectrum (Figure

19 and Figure 20). Similarly, the highest molar concentration of gold salt showed the highest

extinction coefficient (maximum peak) for a given molar ratio and the reaction time (Figure 18),

which may be because of available higher gold density. UV-Vis analysis also showed that molar

ratio of citrate-to-gold around 1:1 or 2:1 produced broader LSPR band including red shift (~650

nm). Therefore, it is recommended to use lower molar ratio (MR) for SERS analysis using laser

source of 633 nm or 785 nm wavelength so that optimum signal enhancement can be achieved to

detect contaminants.

By optimizing conditions (such as gold precursor conc., reducing agent conc., and

temperature) paper based substrates (prepared by in situ reduction based method) may be

produced to identify analyte below limit of detection (LOD). Also, these paper-based SERS

57

substrates produced lesser variation in peak intensities for lower citrate-to-gold ratio in

comparison of higher molar ratio (Figure 22 and Figure 23).

In summary, lower molar ratio of reducing agent to gold precursor produced broader

LSPR band and Raman laser of 785 nm or 633 nm can be used with these substrates to produce

good signal enhancements (peak intensity) of MGITC dye. Therefore, use of lower molar ratio in

paper-based substrates (prepared by in situ method) is recommended for further analysis of the

desired Raman active compound or environmental contaminants.

5.2 Future work

SERS has a great potential for detection of pathogens, cyanobacteria and environmental

contaminants [15, 127, 129]. With SERS, detection of these pollutants may be handy, and

accurate. However, it requires proper SERS substrates that can be effectively used for this

purpose [18, 21, 24, 31].

Paper-based SERS substrates may hold a great hope for the future of detection and

characterization of environmental contaminants. However, there are few barriers associated with

uniformity and reproducibility of substrates [8, 38, 88]. It is still difficult to achieve ideal

substrates having uniformity and reproducibility, which are necessary to produce ideal detection

of environmental analyses [32, 34]. In situ reduction of gold precursor based approach may hold

the key to production of ideal SERS substrates. However, following steps may be taken to

further this research:

1. The effect of temperature on reaction yield

In situ reduction of metal salt in the paper is a complex process, which also depends on

ambient conditions such as humidity and temperature. Effect of these ambient conditions on

58

SERS signals enhancements should be studied further, which will help in better decision-making.

In this thesis, it was observed that at room temperature, reaction yield or conversion of gold

chloride was low. Therefore, use of higher temperature may increase the reaction yield. Higher

temperature may be maintained in a hot bath or a heating plate.

2. The effect of change in reducing agent

By increasing the reducing agent concentration, variation in peak enhancement/intensity

of MGITC dye was observed. However, effect of change in concentration of reducing agent on

the size and shape of formed nanoparticles should be further studied.

3. Microfluidic paper based devices for multiple analyte detection

In this thesis, an attempt is made to understand various parameters that may affect the

uniformity of paper-based substrates. However, extensive efforts are needed to develop specific

substrates for various contaminates. For example, specific substrates can be produced for

detection of various microcystins and particular viruses such as poliovirus [15, 127, 129].

Specific detection of various contaminants may also be achieved by developing proper

antibodies that can bind desired contaminants [43]. Also, paper surface may be modified to

achieve ideal detection of specific analytes.

4. Characterization and optimization of SERS signals of Raman active compounds

Currently, we used MGITC dye for characterization of paper-based substrates and for

optimization of Raman signals. However, other Raman active compounds may be used for

characterization of these substrates.

59

5. Quantitative studies for reaction conversion and AuNPs formation

Further quantitative study of the reaction conversion (in situ based reduction of gold

chloride) and density calculation of formed AuNPs may help in optimization of SERS signals.

Various methods such as IPC-MS may be used for this purpose.

60

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