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BIOFUNCTIONALIZED NANOPOROUS
MEMBRANE/NANOPARTICLES-BASED RAPID
AND ULTRASENSITIVE SENSING PLATFORM
FOR BIOMOLECULE DETECTION
YE WEIWEI
Ph.D
THE HONG KONG POLYTECHNIC UNIVERSITY
2014
lbsysText BoxThis thesis in electronic version is provided to the Library by the author. In the case where its contents is different from the printed version, the printed version shall prevail.
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The Hong Kong Polytechnic University
Interdisciplinary Division of Biomedical Engineering
Biofunctionalized Nanoporous
Membrane/Nanoparticles-Based Rapid and
Ultrasensitive Sensing Platform for Biomolecule
Detection
YE Weiwei
A thesis submitted in partial fulfillment of the requirements for
the degree of Doctor of Philosophy
Jul 2014
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CERTIFICATE OF ORIGINALITY
I hereby declare that this thesis is my own work and that, to the best of my
knowledge and belief, it reproduces no material previously published or written, nor
material that has been accepted for the award of any other degree or diploma, except
where due acknowledgement has been made in the text.
_________________
YE Weiwei
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Abstract
Biomolecule detection plays important roles in various applications including food
safety detection, biomedical diagnosis, and environmental detection. Traditional
biomolecule detection methods include polymerase chain reaction (PCR), enzyme-
linked immunosorbent assay (ELISA) and fluorescent dye labeled detection methods.
However, they are time-consuming, labor-intensive, and expensive and require
sophisticated instrumentation. A simple, rapid and ultrasensitive sensing platform
based on biofunctionalized nanomaterials is required to be developed.
This study investigates biofunctionalized nanoporous membrane/nanoparticles based
sensing platforms via electrochemical and optical detection mechanisms for nucleic
acid hybridization and bacterial toxin protein detection. The whole study includes
three parts.
The first part was the development of nanoporous alumina membrane based
electrochemical biosensor with gold nanoparticles (AuNPs) amplification and silver
enhancement for deoxyribonucleic acid (DNA) hybridization detection. Nanoporous
alumina membranes have the advantageous properties of high surface reaction area,
which allowed huge numbers of probe DNA segments to be adsorbed on the
nanopore walls by covalent bonding for target DNA hybridization detection and
significantly increased detection sensitivity. Probe DNA was immobilized in
nanopores by covalent bonding of chemical linkers. Target DNA hybridization in
nanopores led to nanopore blockage and ion current decrease, which could be
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detected by electrochemical impedance spectroscopy (EIS). AuNP conjugation with
silver enhancement in nanochannels could further increase the pore-blocking
efficiency and consequently the detection sensitivity. The results demonstrated that
AuNPs labelling and silver enhancement could significantly increase the sensitivity
for DNA hybridization detection for both two complementary strands hybridization
and sandwich structure assay detection. Compared with two complementary strands
hybridization detection, sandwich structure assay detection was more suitable for real
applications. The nanopore size effect on detection sensitivity was also explored. We
found 100 nm nanopore size was optimal for DNA hybridization detection with
AuNP labelling and silver enhancement. The limit of detection (LOD) was as low as
single digit of pM.
The second part was the development of nanoporous alumina membrane based
electrochemical biosensor for monitoring botulinum neurotoxin type A (BoNT/A)
light chain protease activity. In this part, green fluorescent protein (GFP) modified
SNAP-25 peptides were first immobilized in nanopores by covalent bonding causing
blockage of electrolyte ions passing through the nanopores. On chip cleavage of
immobilized SNAP-25-GFP was analyzed via impedance spectroscopy with
nanoporous substrate exposure to bacterial toxin BoNT/A light chain. The limit of
detection was around 500 pM.
The third part was the development of a nanoporous alumina membrane based
Luminescence Resonance Energy Transfer (LRET) biosensor using upconversion
nanoparticles (UCNPs) and AuNPs pairs for rapid and ultrasensitive detection of
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avian influenza virus H7 subtype. Both LRET processes in solution and on solid
phase nanoporous alumina membrane were explored. Lanthanide-based UCNPs
could absorb multiple low-energy near-infrared (NIR) photons and convert them into
visible emission. They acted as donors with the advantages of biocompatibility, low
toxicity, and photostability. AuNPs could act as good acceptors with the strong
surface plasma absorption in the NIR-to-IR region. In this part, poly(ethylenimine)
(PEI) modified BaGdF5:Yb/Er UCNPs were conjugated with the amino modified H7
capture oligonucleotide probes by glutaraldehyde linker. AuNPs were conjugated
with thiol modified H7 hemagglutinin gene oligonucleotides. The UCNPs and
AuNPs were brought near with a distance of 10 nm by the hybridization process
between two complementary oligonucleotides. With 980 nm laser excitation, the
emission energy of UCNPs was transferred to AuNPs and quenched. This solution
based system could reach a low limit of detection 7 pM. Nanoporous alumina
membranes based LRET biosensor was also constructed as a solid phase platform for
carrying UCNPs for detection. Large surface area to volume ratio of nanoporous
alumina membranes made it possible to immobilize large amount of UCNPs on
nanpores by covalent bonding. An ultralow limit of detection of 300 fM was
achieved for solid phase nanoporous alumina membrane based LRET platform.
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List of Publications
Journal papers
[1] Weiwei Ye, Jingyu Shi, Chunyu Chan Mo Yang, A nanoporous membrane
based electrochemical sensor for E. coli O 157:H7 DNA detection via
sandwich assay. (In preparation)
[2] Weiwei Ye, Mingkiu Tsang, Xuan Liu, Mo Yang, Jianhua Hao. Upconversion
luminescence resonance energy transfer (LRET) based biosensor for rapid and
ultrasensitive detection of avian influenza virus H7 subtype, Small, 2014, 12:
2390-2397.
[3] Weiwei Ye, Jingyu Shi, Chunyu Chan, Yu Zhang, Mo Yang. A nanoporous
membrane based impedance sensing platform for DNA sensing with gold
nanoparticle amplification, Sensors and Actuators B: Chemical, 2014, 193:
877-882.
[4] Weiwei Ye, Jiubiao Guo, Sheng Chen, Mo Yang. Nanoporous membrane
based impedance sensors to detect the enzymatic activity of botulinum
neurotoxin A, Journal of Materials Chemistry B, 2013, 1: 6544-6550.
[5] Baojian Xu, Weiwei Ye, Yu Zhang, Jingyu Shi, Chunyu Chan, Xiaoqiang
Yao,Mo Yang. A hydrophilic polymer based microfluidic system with planar
patch clamp electrode array for electrophysiological measurement from cells,
Biosensors and Bioelectronics, 2014, 53: 187-192.
[6] Kayiu Chan, Weiwei Ye, Yu Zhang, Lidan Xiao, Polly P.H.M. Leung, Yi Li,
Mo Yang. Ultrasensitive detection of E coli O157:H7 with biofunctional
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magnetic bead concentration via nanoporous membrane based electrochemical
immunosensor, Biosensors and Bioelectronics, 2013, 41: 532-537.
Conference papers
[7] Weiwei Ye, Jingyu Shi, Chunyu Chan, Lidan Xiao, Mo Yang. Nanoporous
alumina membrane and nanoparticle based microfluidic sensing platform for
direct DNA detection, Transducers 2013, Barcelona, Spain, 16-20 June 2013.
[8] Weiwei Ye, Mo Yang. Optimal surface functionalization of nanoporous
alumina membrane for DNA detection, Advanced Materials Research, 2013,
631: 572-575.
[9] Weiwei Ye, Mo Yang. A functionalized nanoporous alumina membrane
electrochemical sensor for DNA detection with gold nanoparticle
amplification. 10th Pacific Rim Conference on Ceramic and Glass Technology,
San Diego, USA, June 2-7, 2013.
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Acknowledgements
I wish to give my greatest thanks to many people who gave their generous help to me
on finishing this dissertation.
First, I would like to express my sincere and deepest gratitude to my supervisor,
Associate Professor Mo Yang, for his continuous professional guidance,
encouragement, inspiration, and unreserved support throughout my PhD study. His
wide scope of knowledge and insightful comments are fundamental and significant to
my thesis. His enthusiasm and intelligence on scientific research have deep impact
on my career and future work.
I wish to owe my great thanks to Associate Professor Jianhua Hao in Department of
Applied Physics in the Hong Kong Polytechnic University for upconversion
nanoparticles preparation and providing detection instrument. I also want to thank Dr.
Sheng Chen in Department of Applied Biology & Chemical Technology in the Hong
Kong Polytechnic University for protein preparation.
I warmly thank Professor Renaud Philippe for kindly giving me the chance of being
a visiting PhD student for three months in Microsystems laboratory 4 (LMIS 4) in
Ecole Polytechnique Federale de Lausanne, Switzerland.
I also thank Dr. Wei Lu from MRC for TEM experiments and Dr. Hardy Lui from
MRC for SEM experiments. I give my great thanks to professors and all staff in
Interdisciplinary Division of Biomedical Engineering for their continuous support
and encouragement.
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I am deeply grateful to collaborators, Ming-Kiu Tsang from Department of Applied
Physics, Jiubiao Guo from Applied Biology & Chemical Technology and Xuan Liu
from Institute of Textiles & Clothing in the the Hong Kong Polytechnic University
for their hard work and experiences in their research area. Special thanks to the
labmates in the group, Mr. Chan Chun Yu, Miss. Jinyu Shi, Mr. Feng Tian, Dr Lidan
Xiao, Dr. Jinjiang Yu, Mr. Fei Tan, Dr. Zongbin Liu, Dr. Baojian Xu and other
colleagues and friends, Mr. Cheng Liu, Dr. Jacky Kwun Fung Wong, Miss. Qijin He,
Miss. Jing Sun, Mr Yaoheng Yang and so on in Interdisciplinary Division of
Biomedical Engineering. Without their help and encouragements, this work could
not have been completed so smoothly.
The financial support from the Hong Kong PhD Fellowship Scheme is greatly
acknowledges. Acknowledges are also extended to the Hong Kong Polytechnic
University for the fellowship award and the award of Research Students Attachment
Programme Out-going Polyu Students, 2014.
Finally I want to express my great thanks to my parents and brother for their support
and continuous encouragement which have enriched me confidence and strength.
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Table of Contents
Certificate of Originality…………………………………………………………….i
Abstract…………………………..…………………….……………………………ii
List of Publications…………………….…………..……………………………..v
Acknowledgements………………………………..……………………………..vii
Table of Contents……………………………………..….…………………………ix
List of Figures……………………………………..….…………………………xv
List of Abbreviations………………………………………………….…………xxvi
Chapter 1 Introduction……………………………………………………………1
1.1 Nanoporous membranes………………………………………..........……………1
1.2 Nanoporous alumina membrane………………………………………………….6
1.2.1 Fabrication of nanoporous alumina membrane………………..……………9
1.2.2 Pore parameters of nanoporous alumina membrane…………...………….11
1.3 Nanoporous alumina membrane for biosensing application……………...……..17
1.3.1 Requirements of biosensors……………………………...………………..17
1.3.2 Surface functionalization of nanoporous membranes for biosensors.…….18
1.3.3 Anti-biofouling properties of nanoporous membranes for biosensing ….. 21
1.3.4 Recent development of nanoporous membrane based biosensing………...21
1.3.4.1 Glucose detection…………………………………....……………...22
1.3.4.2 Cholesterol detection……………………………....……………….23
1.3.4.3 Biomolecule analysis………………………….……………………23
1.3.4.4 Cancer biomarker detection………………………..……………….25
1.3.4.5 Bacteria and cell detection………………………….………………26
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1.4 Gold nanoparticles (AuNPs)……………………………………….……………29
1.4.1 Synthesis of AuNPs………………………………………………………31
1.4.2 Physical properties of AuNPs……………..……………………………32
1.4.3 Sensing application of AuNPs……………………………………………32
1.5 Upconversion nanoparticles (UCNP)…………………………………………..35
1.5.1 Mechanisms of upconversion luminescence……………………………37
1.5.2 Synthesis of UCNPs………………………………………………………38
1.5.3 Properties and applications……………………………….………………40
1.6 Significance and objectives of the study.…………………….…………………44
Chapter 2 Materials and Experiments …………………………………….……50
2.1 Nanoporous alumina membrane based biosensor with AuNPs tags amplification
for DNA hybridization detection …………………………………….....…50
2.1.1 Materials and instrumentation.……………..…………..………………50
2.1.2 Surface modification of nanoporous alumina membrane………………52
2.1.3 Oligonucleotide immobilization inside nanopores………………………54
2.1.4 Fabrication of microfluidic chip integrated with nanoporous alumina
membranes ………………………………………………………………56
2.1.5 EIS analysis by nanoporous alumina membrane integrated with PDMS
chamber……………………………………………………………..…58
2.1.6 AuNPs synthesis………………………………………………………....59
2.1.7 AuNPs-oligonucleotide conjugation preparation………………………60
2.1.8 Silver enhancement for signal amplification…………………………….61
2.1.9 Two complementary strands for DNA hybridization detection …………62
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2.1.10 Sandwich structure assay for DNA hybridization detection………….64
2.2 Nanoporous alumina membrane based biosensor for botulinum neurotoxin
detection………………………………………………………………..……66
2.2.1 Plasmid construction and protein expression …………………...………66
2.2.2 Establishment of nanoporous alumina membrane based biosensing
platform for BoNT LcA detection ………………….………………67
2.2.2.1 Nanoporous alumina membrane surface modification……………67
2.2.2.2 Immobilization of SNAP-25-GFP on nanoporous alumina
membrane……………………………………………………....68
2.2.2.3 Toxin enzymatic activity detection mechanism…………...………68
2.2.3 Protease activity detection by impedance spectroscopy measurement …70
2.3 Nanoporous alumina membrane/UCNPs based LRET biosensor for Avian
Influenza Virus (AIV) H7 subtype detection ………………………………71
2.3.1 Materials and instrumentation………………..………….………………71
2.3.2 One-pot hydrothermal synthesis of PEI-modified BaGdF5:Yb/Er
UCNPs …………………………………………………….…………72
2.3.3 Conjugation of the probe DNA to BaGdF5: Yb/Er UCNPs ……………73
2.3.4 Conjugation of AIV H7 gene oligonucleotide with AuNPs………….....74
2.3.5 Upconversion quenching measurement for solution based detection.…75
2.3.6 Upconversion quenching measurement for solid phase nanoporous
alumina membrane based detection………………………………..….75
Chapter 3 Results………………………………………….…………………....77
3.1 Nanoporous alumina membrane based biosensor with AuNPs tags amplification
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for DNA hybridization detection ………………………………………....77
3.1.1 Nanoporous alumina membrane surface modification for oligonucleotide
immobilization………………………………………………….………77
3.1.2 Oligonucleotide immobilization on nanoporous alumina membrane…81
3.1.3 AuNPs-oligonucleotide conjugation ……………..…………..…………84
3.1.4 DNA hybridization with AuNPs tags on nanoporous alumina
membrane………………………………………………………………86
3.1.5 Two complementary strands of DNA hybridization detection………..90
3.1.5.1 Impedance spectroscopy monitoring of DNA immobilization and
hybridization process with AuNPs tags…………………..90
3.1.5.2 AuNPs concentration effect on signal amplification……………..93
3.1.5.3 Silver enhancement for signal amplification……………………..95
3.1.6 Sandwich structure assay for DNA hybridization detection…………..99
3.1.6.1 Probe DNA immobilization on nanoporous alumina membrane with
different nanopore sizes……………………………………...100
3.1.6.2 DNA hybridization detection based on nanoporous alumina
membrane with different nanopore sizes………………….104
3.1.6.3 Impedance sensing with various target DNA concentrations based
on nanoporous alumina membrane with different nanopore sizes.111
3.2 Nanoporous alumina membrane based biosensor for botulinum neurotoxin
detection…………………………………………………………………...124
3.2.1 Immobilization of SNAP-25-GFP on the nanoporous alumina
membrane …………………………………..…………………..125
3.2.2 Impedance spectrum monitoring of SNAP-25-GFP immobilization on the
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nanoporous alumina membrane ………………………………..……127
3.2.3 Protease activity detection …………………………………………....129
3.3 Nanoporous alumina membrane/UCNPs based LRET biosensor for Avian
Influenza Virus (AIV) H7 subtype detection …….........................134
3.3.1 Upconversion luminescence of BaGdF5:Yb/Er UCNPs………...….….136
3.3.2 Design of LRET sensor ……………………………………………...138
3.3.3 Structural and phase characterizations of BaGdF5:Yb/Er UCNPs and
AuNPs..………………..………………………………………..…..….140
3.3.4 Characterization of UCNPs-oligo and AuNPs-oligo …………….…145
3.3.5 H7 hemagglutinin gene detection using solution based LRET
system………………………………………………………….…..147
3.3.6 H7 hemagglutinin gene detection using nanoporous alumina membrane
based LRET system………………………………………………… 152
Chapter 4 Discussions…………….…………………………………………….157
4.1 Nanoporous alumina membrane biosensor with AuNPs tags amplification for
DNA hybridization detection…….…………………………………………157
4 .2 Botu l i sm neurotox in detec t ion based on nanoporous a lumina
membrane………………………………….………….…………………….163
4.3 Upconversion LRET based detection of AIV H7 subtype……………….…165
Chapter 5 Conclusions………………………………………………………….170
Chapter 6 Suggestions for Future Work………………………………………..174
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References…………………………………………………………………..……..176
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List of Figures
Figure 1.1.1 Illustration of ordered mesoporous silica film formation processes by
spontaneous growth procedure (Adapted from [18])…………………………..…..5
Figure 1.1.2 Cross-sectional SEM image of the carbon nanotube. Scale bar, 20 µm
(a); Top view SEM image of MCE supported carbon nanotube membrane with an
average pore size of 220 nm. Scale bar, 1 µm (b); Photo of a MCE supported carbon
nanotube membrane. Scale bar, 1 cm (c). (Adapted from [20])……………………..6
Figure 1.2.1 SEM of nanoporous alumina membrane (a) top view; (b) cross-section
(Adapted from [29])………………………………………………………………….7
Figure 1.2.2 Fabrication scheme of nanoporous alumina membrane by two-step
anodization process (Adapted from [40]) ……………………………………..……10
Figure 1.2.3 Anodic alumina porous structures (a) and a cross-sectional view of the
anodized layer (b) (Adapted from [41]) ………………………………………..11
Figure 1.2.4 Factors influence on the nanopore diameter during anodization process
(Adapted from [41])… …………………………………………………………..12
Figure 1.2.5 ZnO nanowires obtained from nanoporous alumina membrane
templates with different pore diameters. The average diameter of membrane template
was 130 nm in (a); the average diameter of nanowires was 85 nm; the average
diameter of membrane template was 60 nm in (c); the average diameter of nanowires
was 50 nm. Scale bar was 500 nm (Adapted from [46]) ………………………….14
Figure 1.2.6 (a) SEM image of a gold nanotube membrane (inset: enlarged top view).
(b) Inclined view of the gold nanotube membrane (Adapted from [46])…………..16
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Figure 1.3.1 Scheme of nanoporous alumina membrane surface modification by
APTES (Adapted from [50])…………………..…………………………………..19
Figure 1.3.2 Scheme of nanoporous alumina membrane surface modification by
isocyanatopropyl triethoxysilane and immobilization of amino modified DNA
(Adapted from [50])……………………………………………………………….20
Figure 1.3.3 Scheme of nanoporous alumina membrane surface modification by n-
alkanoic acid (Adapted from [51])….……………………………………………..20
Figure 1.3.4 Schematic illustration of nanoporous alumina membrane in glucose
affinity sensor (Adapted from [59])… ……………………………………………22
Figure 1.3.5 Scheme of nanoporous alumina membrane based impedimetric
biosensing for DNA detection (Adapted from [63])… …………………………24
Figure 1.3.6 Nanoporous alumina membrane based platform for cancer biomarker
detection in blood sample. Left: SEM images of a nanoporous alumina membrane
with a top view and cross-section view and confocal microscopy image; Center:
Protein sensing scheme based on nanoporous alumina membrane; Right: Sensing
principle for protein detection (Adapted from [65])… …………………………..26
Figure 1.3.7 Scheme illustration of nanoporous alumina membrane based biosensor
for bacteria sensing (Adapted from [67]) ……………………………………..……27
Figure 1.3.8 Scheme of microfluidic device for cell impedance spectroscopy with
integrated mesoporous membrane and embedded electrodes (Adapted from
[70]) …………………………………………………………………………………28
Figure 1.4.1 Physical properties of AuNPs and schematic illustration of an AuNP-
based detection system (Adapted from [72])… ………………………………..30
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Figure 1.4.2 Scheme of FRET-based system for DNA detection (Adapted from
[72])… …………………………………………………………………………….34
Figure 1.4.3 Scheme of electronic DNA detection methods based on ‘sandwich’
structure of DNA functionalized with AuNPs and followed by silver enhancement (a);
Sequences of capture, probe and target DNA segments (b) (Adapted from [67])…..35
Figure 1.5.1 Schematic diagram of ESA (w’>w1, w0). E0, E1 and E2 represent
ground state, intermediate, and excited state, respectively……………………..37
Figure 1.5.2 Energy diagram of the Er3+
/ Yb3+
codoped materials excited with NIR
to blue, green and red emission (Adapted from [99])… ………………………..38
Figure 1.5.3 Schematic representation of the binding of biotinylated AuNPs to
avidinylated UCNPs (A); colorless suspension of UCNP under visible light (a);
UCNPs with green luminescence under 980-nm laser excitation (b); adding red Au-
NPs under visible light (c) (Adapted from [108])… ……………………………….41
Figure 1.5.4 Luminescence of the UCNPs (photo-excited at 980 nm) after addition
of varying concentrations of biotinylated AuNPs (B) (Adapted from [108])……….42
Figure 1.5.5 Schematic illustration of the upconversion FRET process between
ssDNA-UCNPs and GO for adenosine triphosphate (ATP) sensing (Adapted from
[109])… ………………………………………………………………………….43
Figure 1.5.6 PL spectra of the UCNPs-GO FRET aptasensor with varying
concentrations of ATP (Adapted from [109])… …………………………………..44
Figure 2.1.1 Processes of nanoporous alumina membrane surface modification by
GPMS……………………………………………………………………………….52
Figure 2.1.2 Ramé-Hart contact angle goniometer………………………………..54
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Figure 2.1.3 Scheme of oligonucleotide immobilization on GPMS modified
nanoporous alumina membrane surface (Adapted from [116])… ………………..55
Figure 2.1.4 Fabrication of microfluidic chip integrated with nanoporous
membranes………………………………………………………………………57
Figure 2.1.5 Schematic image of a PDMS chip integrated with nanoporous alumina
membrane for DNA hybridization detection with EIS analysis (Adapted from
[116]). ……………………………………………………………………………….59
Figure 2.1.6 Sensing principle of nanoporous membrane impedance sensor for two-
strand DNA hybridization detection. (a) Relative large electrolyte current through
nanoporous membranes immobilized with single strand probe DNA_a; (b) DNA
hybridization caused partial blockage; (c) AuNP tags increased blocking degree; (d)
silver enhancement on AuNPs further increased blocking degree (Adapted from
[116])… ……………………………………………………………………………..63
Figure 2.1.7 Sensing principle of nanoporous membrane impedance sensor for
sandwich structure assay of DNA hybridization detection. (a) Relative large
electrolyte current through nanoporous membranes immobilized with single strand
probe DNA_c; (b) Hybridization of probe DNA_c with long target DNA_c’d’ caused
partial blockage; (c) Hybridization of AuNPs-reporter DNA_d with target DNA_c’d’
increased blocking degree; (d) silver enhancement on AuNPs further increased
blocking degree…………………………………………………………………….65
Figure 2.2.1 Scheme of protein SNAP-25-GFP immobilization on nanoporous
alumina membrane………………………………………………………………..68
Figure 2.2.2 Scheme of the nanoporous membrane based electrochemical biosensor
for protease activity detection of the BoNT-LcA. (a) Segment of SNAP-25-GFP
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immobilized in nanopores increased the blocking degree and decreased the current
signals; (b) cleavage of BoNT-LcA on specific site of SNAP-25-GFP; (c) the current
increased and impedance signals decreased by washing away the cleaved part of the
protein (Adapted from [121]).……………………………………………………..70
Figure 3.1.1 Water contact angle change of nanoporous alumina membrane surface
before treatment (a), after GPMS treatment (b)… ………………………………..78
Figure 3.1.2 Average water contact angle before treatment (A), after GPMS
treatment (B)… ………………………………………………………………….79
Figure 3.1.3 Scanning electron microscopy (SEM) images of top view (a) and cross-
sectional view of unmodified nanoporous alumina membranes (b), cross-sectional
view of modified nanoporous alumina membranes (c) and element analysis by EDX
(d).………………………………………………………………………………….81
Figure 3.1.4 Fluorescence images of fluorescein labeled oligonucleotide with
different concentrations immobilized on GPMS modified nanoporous alumina
membrane. Images of a, b and c correspond to oligonucleotide concentrations of 1
μM, 2 μM and 3 μM, respectively. Moreover, fluorescence intensity increase analysis
of fluorescence images a, b and c is shown in the histogram……………………….83
Figure 3.1.5 (a) TEM image of AuNPs dispersed in DI water; (b) HRTEM image of
a single AuNP and SAED pattern (inset); (c) and (d) comparison images of AuNPs
and AuNPs-oligonucleotide conjugation dispersed in DI water………………….....85
Figure 3.1.6 UV-Vis absorption spectra of AuNPs and AuNPs-oligonucleotide
conjugation….……………………………………………………………………….86
Figure 3.1.7 Fluorescence images of different concentrations of fluorescein labeled
target DNA hybridized with probes on nanoporous alumina membrane. a, b, c, and d
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represent the fluorescence results of 0.05 μM , 0.5 μM, 1 μM, and 2 μM target DNA
hybridized with probe oligonucleotide, respectively…………………………….87
Figure 3.1.8 Fluorescence intensity change of fluorescein labeled complementary
target DNA hybridized with probes on nanoporous alumina membrane……….88
Figure 3.1.9 Scanning electron microscopy (SEM) images of cross-sectional view of
bare nanoporous alumina membrane (a) and after DNA hybridization with AuNPs
and silver enhancement (b)… …………………………………………………….89
Figure 3.1.10 (a) Impedance spectra of functionalized nanoporous membranes
before and after probe DNA_a immobilization (ssDNA), target DNA_b hybridization
(dsDNA), and target DNA_b hybridization with AuNP tags (AuNP-dsDNA); (b)
relative impedance amplitude change for various cases compared with silane
modified bare nanoporous membrane (Adapted from [116])… …………………....92
Figure 3.1.11 (a) Impedance spectra of various AuNP concentrations for signal
amplification; (b) relative signal amplification for various AuNPs conjugation
compared with silane modified bare nanoporous membranes (Adapted from
[116])… …………………………………………………………………………….94
Figure 3.1.12 (a) Impedance spectra of probe DNA_a immobilization (ssDNA),
DNA hybridization with AuNP tags (dsDNA-AuNP), AuNP tags with silver
enhancement (silver enhancement), and dehybridization; (b) relative impedance
amplitude change relative to silane modified bare nanoporous membranes for DNA
hybridization with AuNP tags, silver enhancement, and dehybridization (Adapted
from [116])… ……………………………………………………………………..96
Figure 3.1.13 (a) Impedance amplitude change for label free assay and nanoparticle
tags amplification assay for various target DNA_b concentrations. The control value
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was based on probe DNA_a immobilized nanoporous membranes; (b) correlation
curve between impedance amplitude increase and target DNA_b concentrations. The
LOD was 50 pM (Adapted from [116]) …………………….…………………….98
Figure 3.1.14 Impedance change of different probe DNA concentrations in
nanoporous alumina membranes with the diameter of 20 nm…………………….102
Figure 3.1.15 Impedance change of different probe DNA_c concentrations in
nanoporous alumina membranes with the diameter of 50 nm…………………..…102
Figure 3.1.16 Impedance change of different oligonucleotide concentrations on
nanoporous alumina membranes with the diameter of 100 nm………………….103
Figure 3.1.17 (a) Impedance spectra of 0.5 µM probe DNA_c immobilization, 0.5
µM target DNA_c’d’ hybridization based on nanoporous alumina membrane with the
diameter of 20 nm; (b) relative impedance amplitude change relative to silane
modified bare nanoporous membranes for DNA immobil izat ion and
hybridization………………………………………………………………………106
Figure 3.1.18 (a) Impedance spectra of 0.4 µM probe DNA_c immobilization, 0.05
µM target DNA hybridization, AuNPs tags amplification, and silver enhancement
based on nanoporous alumina membrane with the diameter of 50 nm; (b) relative
impedance amplitude change relative to silane modified bare nanoporous membranes
for 0.4 µM probe DNA_c immobilization, 0.05 µM target DNA hybridization,
AuNPs tags amplification, and silver enhancement…………………………….108
Figure 3.1.19 (a) Impedance spectra of 0.2 µM probe DNA_c immobilization, 1 nM
target DNA hybridization, AuNPs tags amplification, and silver enhancement based
on nanoporous alumina membrane with the diameter of 100 nm; (b) relative
impedance amplitude change relative to silane modified bare nanoporous membranes
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for 0.2 µM probe DNA_c immobilization, 1 nM target DNA hybridization, AuNPs
tags amplification, and silver enhancement……………………………………….110
Figure 3.1.20 Impedance amplitude changes with different target concentrations
based on 20 nm nanoporous alumina membranes…………………………….112
Figure 3.1.21 (a) Impedance amplitude changes with different target concentrations
under label free, AuNP label, and silver enhancement based on 50 nm nanoporous
alumina membranes; (b) Impedance amplitude change for label free, AuNP label and
silver enhancement amplification assay for various low target DNA_c’d’
concentrations of 1 nM, 5 nM, 10 nM and 50 nM. The control value was based on
impedance value of probe DNA_c immobilized nanoporous membranes……….115
Figure 3.1.22 (a) Impedance amplitude changes with different target concentrations
under label free, AuNP label, and silver enhancement based on 100 nm nanoporous
alumina membranes; (b) Impedance amplitude change for label free, AuNP label and
silver enhancement amplification assay for various low target E. coli O157:H7
bacterium DNA concentrations of 1 nM, 5 nM, 10 nM and 50 nM. The control value
was based on probe DNA_c immobilized nanoporous alumina membranes……...118
Figure 3.1.23 (a) Impedance spectra without probe DNA, with immobilized 0.4 µM
probe DNA, with 0.2 µM target DNA hybridization detection, 0.2 µM 6 bases
mismatch and totally noncomplementary DNA detection based on 50 nm nanoporous
alumina membrane; (b) Impedance amplitude change for 0.2 µM target DNA
hybridization detection (A), 6 bases mismatch DNA detection (B) and
noncomplementary DNA detection (C). The control value was based on probe
DNA_c immobilized nanoporous alumina membranes………………………….120
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Figure 3.1.24 (a) Impedance spectra of detecting DNA segments without probe
DNA, with immobilized 0.2 µM probe DNA, with 0.1 µM target DNA, 0.1 µM 6
bases mismatch and totally noncomplementary DNA detection based on 100 nm
nanoporous alumina membrane; (b) Impedance amplitude change for 0.1 µM target
DNA hybridization detection (A), 6 bases mismatch DNA detection (B) and
noncomplementary DNA detection (C). The control value was based on probe
DNA_c immobilized nanoporous alumina membranes……………………….…122
Figure 3.2.1 (a) Top view and (b) cross-sectional SEM image of a GPMS modified
nanoporous alumina membrane; (c) cross sectional SEM image of a nanoporous
membrane after SNAP-25-GFP immobilization; (d) fluorescence image of a
nanoporous membrane after SNAP-25-GFP immobilization. The inset shows the
fluorescence image of the nanoporous alumina membrane without SNAP-25-GFP
immobilization (Adapted from [121])… ……………………………………….126
Figure 3.2.2 (a) Impedance spectroscopy of nanoporous membranes with various
SNAP-25-GFP concentrations; (b) impedance amplitude change at 1 Hz with SNAP-
25-GFP concentrations. The saturation concentration of SNAP-25-GFP was around 4
µM (Adapted from [121])… …………………………………………………….129
Figure 3.2.3 (a) Fluorescence images of the nanoporous membrane based activity
assay at a LcA concentration of 5 nM with various cleavage incubation times of 0
minutes, 10 minutes, 20 minutes and 30 minutes; (b) time courses of the relative
impedance amplitude signal change with the cleavage incubation time with a LcA
concentration of 5 nM at 1 Hz; (c) correlation curve between the relative impedance
change and the cleaved SNAP-25-GFP percentage (Adapted from [114])………..131
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Figure 3.2.4 (a) Time courses of the relative impedance amplitude change at 1 Hz
with various LcA concentrations; (b) relative impedance amplitude decrease at 1 Hz
at 25 minutes for various LcA concentrations (Adapted from [121])…….……….133
Figure 3.3.1 Upconversion spectrum of the PEI-modified BaGdF5:Yb/Er UCNPs.
The left inset shows the photograph of DI water and the right inset shows the
photograph of 1 wt% of UCNPs with 980 nm laser excitation..………….137
Figure 3.3.2 (a) Power dependence of UC emission of PEI-modified BaGdF5:Yb/Er
UCNPs.(b) A simplified energy level diagram of Yb/Er system…………………..138
Figure 3.3.3 Schematic diagram of H7 hemagglutinin gene detection by LRET
biosensor based on energy transfer from BaGdF5 :Yb/Er UCNPs to AuNPs (Adapted
from [152])… …………………………………………………………………..….139
Figure 3.3.4 Scheme of nanoporous alumina membrane based UCNPs and AuNPs
LRET system for DNA detection……………………………………………….140
Figure 3.3.5 XRD pattern of the (a) BaGdF5:Yb/Er UCNPs, (b) BaGdF5:Yb/Er
UCNPs-oligo, (c) AuNPs, and (d) BaGdF5:Yb/Er UCNP-oligo-AuNPs (Adapted
from [152])… …………………………………………………………………..142
Figure 3.3.6 (a) TEM image (b) SAED pattern (c) Size distribution of as-prepared
PEI-modified BaGdF5:Yb/Er UCNPs; (d) EDX of oligonucleotide conjugated
BaGdF5:Yb/Er UCNPs; HRTEM of (e) PEI-modified BaGdF5:Yb/Er UCNPs (f)
oligonucleotide conjugated BaGdF5:Yb/Er UCNPs; (g) and (h) oligonucleotide
conjugated BaGdF5:Yb/Er UCNPs assembled with AuNPs (Adapted from
[152])… ………………………………………………………………………….144
Figure 3.3.7 UC emission spectra of BaGdF5:Yb/Er UCNPs and oligonucleotide
coated BaGdF5:Yb/Er UCNPs in water under 980 nm laser excitation, and UV-Vis
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absorption spectra of AuNPs and AuNPs-oligo (Inset: green UC emission of
BaGdF5:Yb/Er UCNPs) (Adapted from [152])… …………………………….145
Figure 3.3.8 FTIR spectra of the as-prepared UCNPs and UCNPs-Oligo (Adapted
from [152]) ……………………………………………………………………….146
Figure 3.3.9 The Zeta potential of UCNPs-Oligo (Adapted from [152])……...….147
Figure 3.3.10 (a) Luminescence spectra of UCNPs-oligo with various concentrations
of H7 gene target oligonucleotide conjugated with AuNPs; (b) quenching efficiency
with concentrations of H7 gene target oligonucleotide (Adapted from [152])……149
Figure 3.3.11 Lifetime of UCNPs-oligo emission at 540 nm before and after
conjugation with AuNPs-oligo (Adapted from [152])……………………………..150
Figure 3.3.12 (a) Fluorescence signal quenching (F0-F) versus H7 gene
oligonucleotide concentration from 1 pM to 10 nM. (b) Linear relationship between
fluorescence signal quenching and H7 gene oligonucleotide in the range between 1
pM to 10 nM as y = 4957.29 ln(x) + 2419.28, with R2=0.9412 (Adapted from
[152]) ………………………………………………………………………………151
Figure 3.3.13 Luminescence intensity of capture with AuNP-H7 hemagglutinin gene
segment, non AuNP-H7 hemagglutinin gene segment and dehybridization (Adapted
from [152]) ………………………………………………………………………..152
Figure 3.3.14 (a) Luminescence spectra of UCNPs-oligo on nanoporous alumina
membrane with various concentrations of H7 gene target oligonucleotide conjugated
with AuNPs; (b) the normalized intensity with different H7 oligonucleotide
concentrations at the wavelength of 540 nm……………………………………….154
Figure 3.3.15 Quenching efficiency with concentrations of H7 gene target
oligonucleotide…………………………………………………………………….155
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List of Abbreviations
2D 2-dimensional
A adenine
AAO aluminum anodic oxide
AIV avian influenza viruses
APTES 3-aminopropyltrimethoxysilane
ATP adenosine triphosphate
AuNPs gold nanoparticles
BoNT LcA botulinum neurontoxin type A light chain
C cytosine
CTAB silicate-cetrimonium bromide
CW continuous wave
DI water deionized water
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
DTT dithiothreitol
E. coli Escherichia coli
EBL electron-beam lithography
EDC (N-(3-dimethylaminopropyl)-N’-ethyl-carbodiimide)
EDX energy dispersive X-ray spectroscopy
EIS electrochemical impedance spectroscopy
ELISA enzyme-linked immunosorbent assay
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ESA excited state absorption
ETU energy transfer upconversion
FRET Förster resonance energy transfer /fluorescence resonance
energy transfer
FTIR fourier transform infrared spectrum
G guanine
GFP green fluorescence protein
GO graphene oxide
GPMS (3-glycidoxypropyl) trimethoxysilane
GQDs graphene quantum dots
HA Hemagglutinin
HRTEM high resolution TEM
IBL ion-beam lithography
LBL layer-by-layer
LOD limit of detection
LRET luminescence resonance energy transfer
NA Neuraminidase
NHS N-hydroxysuccinimide
NIR near-infrared
OA oleic acid
ODE 1-octadecence
OM Omeylamine
PBS phosphate buffer solution
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PC polycarbonate
PC12 cells Pheochromocytoma
PCR polymerase chain reaction
PDMS Polydimethylsiloxane
PEG poly(ethylene glycol)
PEI poly(ethylenimine)
PET polyethylene terephthalate
QDs quantum dots
RA retinoic acid
RE rare earth
RT-PCR reverse transcription-PCR
SAED selected area electron diffraction
SEM scanning electron microscopy
SPR surface plasmon resonance
ssDNA single strand DNA
T Thymine
TEM transmission electron microscopy
TEOS Tetraethylorthosilicate
UC Upconversion
UCNPs upconversion nanoparticles
UV Ultraviolet
XRD X-ray diffraction
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Chapter 1 Introduction
1.1 Nanoporous membranes
In the past ten years, nanomaterials have attracted great interests in the development
of biocompatible and controllable interconnections for biomedical applications [1].
Nanoporous materials as the subset of nanomaterials include nanoporous carbons,
nanoporous silica, colloidal crystals and nanoporous membranes. They have been
fully developed and widely applied for catalysis, separation and purification due to
their large internal surface area [2]. Particularly, nanoporous membranes with ideal
biocompatibility that can be used for selecting molecules in the nanoscale attract lots
of interest. Many biological membranes in nature such as cell membranes are good
filters in regulating the diffusion of small molecules through protein pores [3]. In
order to mimic the cell membrane functions, researchers have paid much attention to
synthetic nanoporous membranes.
Nanoporous membranes consist of numerous nanopores, which are small channels
and their diameters range from 1 to 500 nm [4]. Based on the different sizes and
shapes of nanopores, nanoporous membranes are capable of discriminating and
selectively separating biomolecules. Nanoporous membrane is highly stable with
respect to its function and structure under various conditions of temperature, pH and
chemical agents [3, 5]. Nanopore size, morphology as well as distribution could be
well controlled by the established fabrication techniques [6]. The surface properties
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of nanopores, such as wettability, biocompatibility, and biomolecule adhesion
capability can be changed by chemical or physical surface modification techniques.
Using various surface modification/functionalization techniques, different functional
groups are immobilized on nanopore walls for biomolecule capture and interaction.
With these advantages, nanoporous membranes have become more and more popular
in molecular biology, tissue engineering and cell biology for sensing, sorting and
separation [7].
Based on pore sizes, material constituents, and fabrication methods, nanoporous
materials can be classified into many types. Nanoporous materials are categorized
macroporous materials (50 nm), mesoporous materials (2 to 50 nm) and microporous
materials (less than 2 nm) according to the International Union of Pure and Applied
Chemistry (IUPAC) [8]. Both organic and inorganic materials can be used for
fabricating nanoporous membranes. Polyethylene terephthalate (PET) and
polycarbonate (PC) are typical organic materials [9, 10]. Inorganic membranes
contain metals, elementary carbon or oxides [11]. They have gained a lot of interests
due to the low cost, well-established fabrication process and availability for massive
production. Nanoporous membranes vary a lot in porosity, permeability, strength,
thermal stability, chemical stability, cost and durability [12].
Recently, due to the advances of synthesis and processing techniques, various
nanoporous polymers are developed. The common fabrication techniques include
lithography, track etching pattern-transfer and so on [13]. The lithographic technique
has the primary advantage of being able to produce user-defined patterns.
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3
Photocrosslinkable or photodegradable polymer is required in direct use of
lithographic techniques. Ion-beam lithography (IBL) and electron-beam lithography
(EBL) can be used to pattern polymeric materials using electrons and charged
particles [14]. The EBL and IBL techniques are limited by their low throughput due
to the repeated spatical scanning process.
Template fabrication was applied to produce nanostructured materials for a variety of
applications over the past several decades [15]. Imprint techniques physically
transfer a master pattern to the polymer target. Using this method, nanopores can be
fabricated in polymer. Both of the lithographic and pattern-transfer techniques
fabricate nanopores according to a prepared template. Solvent-based precipitation
techniques make a target polymer have variable solubility based on different
concentrations, solvent and process conditions. Layered structures were formed by
Layer-by-layer (LBL) assembly by sequential deposition of cationic and anionic
polymers by electrostatic force [16]. The electrostatical attraction between polymers
on the surface and in solutions results in a monolayer deposition.
Inorganic membranes consist of oxides or metals. Due to the advantages of high
selectivity and permeability for specific molecules, they are good candidates for
biomolecule separation and drug delivery. Three types of inorganic membranes,
including mesoporous silica film, carbon nanotube membrane and nanoporous
alumina membrane are illustrated as examples.
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4
Mesoporous silica films have unique structures and functions. The nanopore size and
geometry can be controlled by different hydrothermal treatment, pH of precursor
solution and organic surfactants [17]. The ordered mesoporous silica films are
formed according to the detailed formation process shown in Figure 1.1.1[18].
Assisted by ammonia hydrogen bonding and controlled silicate polymerization,
spherical micelles of silicate-cetrimonium bromide (CTAB) form cylindrical
micelles. The substrate including glass or ITO, is firstly processed to be negatively
charged. The cations (CTA+) of surfactant (CTAB) are adsorbed strongly on the
substrate by electrostatic attraction forming spherical CTAB micelle on the substrate.
Then, tetraethylorthosilicate (TEOS) is added and slowly hydrolyzed in the
solution containing ammonia and ethanol to form negatively charged silicate
species. They are attracted on the spherical micelle surface by electrostatic
interaction. Free silicate species with negative charges prefer to be deposited
between the spherical micelles and the junction of spherical micelle and
substrate. At the same time, ethanol diffuses to CTAB micelles causing
reduction of alkyl tail interaction. Therefore, the hydrophobic micelle volume
increases with lowered curvature [19]. Due to the above factors, the morphology
can be changed from spherical to cylindrical micelles. In longitudinal direction,
a continuous and large-domain film grows by continuous diffusing and re-
assembling process. Perpendicular channels formed mesoporous silica films
after solvent extraction of surfactants.
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5
Figure 1.1.1 Illustration of ordered mesoporous silica film formation processes by
spontaneous growth procedure (Adapted from [18]).
Mesoporous silica films have 2-dimensional (2D) architectures with perpendicular
channels aligned in the substrate. These films have varieties of shapes, sizes and
spatial arrangement, which result in a wide range of porosity. Therefore, they can be
applied in separating small molecules, catalysis and other biomedical applications.
Carbon nanotube membranes are films consisting of an array of nanoscale cylinders
oriented perpendicularly to the surface of an impermeable film. They have the
characteristics of high aspect ratio, large surface area, and high mechanical strength.
The morphology and property of carbon nanotube membrane is shown in Figure
1.1.2 [20]. Figure 1.1.2a shows scanning electron microscopy (SEM) image of a
cross-sectional view of the carbon nanotubes, which have a vertically aligned and a
closely packed structure. When a conformal thin layer of carbon nanotubes is
deposited on the porous mixed cellulose ester (MCE) support, an MCE supported
carbon nanotube membrane forms with the top view shown in Figure 1.1.2b. The
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6
resultant MCE supported carbon nanotube membrane has excellent mechanic
properties. It can be bent at an angle of 90º more than 20 times without damage or
loss of structural integrity (Figure 1.1.2c). Carbon nanotube membranes have
remarkable electrical and thermal conductivity. Combined with excellent stiffness
and strength, they have attracted intense study in various applications [21, 22].
Figure 1.1.2 Cross-sectional SEM image of the carbon nanotube. Scale bar, 20 µm
(a); Top view SEM image of MCE supported carbon nanotube membrane with an
average pore size of 220 nm. Scale bar, 1 µm (b); Photo of a MCE supported carbon
nanotube membrane. Scale bar, 1 cm (c). (Adapted from [20]).
Carbon nanotube membranes could be made in extreme magnetic fields [23]. They
can be made of open-ended carbon nanotubes and water can flow through the porous
membrane forced by voltage. Therefore, carbon nanotube membranes have wide
chemical application, such as water desalination [24], water purification, gas
separation, and sensing.
1.2 Nanoporous alumina membrane
Nanoporous alumina membrane has honeycomb like structure, which is fabricated by
anodic etching pure aluminum using chromic aqueous, sulfuric or oxalic solution [25,
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7
26]. The types of electrolyte and the anode voltage used can control the pore
geometry and morphology, which are shown in Figure 1.2.1 [27]. The fabrication
process for nanoporous alumina membrane is well-established. Together with
stability and ease of surface modification, nanoporous alumina membranes have
achieved great attention [28]. Well-established fabrication process makes mass
fabrication possible, which decreases the cost of each piece. The pore sizes of
nanoporous alumina membranes can be controlled and they are chemically and
thermally stable. Nanopores allow ions in electrolyte to pass through.
Figure 1.2.1 SEM of nanoporous alumina membrane (a) top view; (b) cross-section
(Adapted from [29]).
To realize the capture for specific target molecules, biological sensing elements, such
as enzyme, antibody, nucleic acid, microorganism or cell, should first be
immobilized on nanoporous membrane surface by covalent bonding methods or
physical adsorption, which is easy to operate but has weak adhesion force with low
stability, while covalent bonding is strong and highly stable [30]. It requires surface
modification techniques, which can change membrane surface properties, surface
wettability and adhesion properties. For example, poly(ethylene glycol) (PEG) was
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8
applied to produce thin films covered on nanoporous membrane surface to reduce
non-specific adsorption [31].
Compared with imperforate substrate, nanoporous alumina membranes own the
advantages of fast electrolyte transfer rate, larger surface area, which increases area
for molecule affinity and reaction leading to enhanced signals. Nanoporous
membrane based platforms are applied for separation and sorting to isolate and
purify molecules from many kinds of biological feed streams. Although many other
techniques can be used in separation, such as size exclusion chromatography and gel
electrophoresis, nanoporous alumina membranes which have ordered pores have
been investigated in the application of supporting kidney cells and filtering blood to
retain serum proteins and export waste out [32, 33]. Small molecules, including
insulin and oxygen, could get through the nanopores, while the nanopores block
large molecules. In addition, the nanoporous alumina membranes have many
biological applications in lab on a chip, immunoisolation and drug delivery [3]. It is
potential to make capsules, which are capable to control pharmacologic agent release
[34].
Nanoporous alumina membranes are good scaffolds for cell culture and tissue
engineering with excellent biocompatibility, which are suitable for cell and tissue
existing in harmony with nanoporous alumina membranes without deleterious
changes, toxins, or adverse effect [28, 35]. Therefore, they have been extensively
used as substrates for tissue construction [36]. With the advantages of good stability
and biocompatibility, they have been more and more widely used in the area of
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molecular biology, cell detection and tissue engineering for sensing, sorting and
separation, so they attract many researchers’ interest [37].
Nanoporous alumina membranes were applied for neuronal cell cultures to develop
promising sensing devices in many studies [38]. Pheochromocytoma (PC12 cells)
was cultured on nanoporous alumina membranes coated with gold to monitor the
neurite development [39]. By observing cell growth on gold-coated nanoporous
alumina membranes, they assumed PC12 cells spatially sensed the underlying
nanotopography, which could be applied to control neurite outgrowth. Due to
increasing osteoblastic cells on the implant surface and the excellent mechanical
performance, surface modification of nanoporous alumina membranes becomes more
and more important for bone implants [36]. Due to the excellent properties of
nanoporous alumina membranes, they are promising for biomedical applications.
1.2.1 Fabrication of nanoporous alumina membrane
Nanoporous alumina membranes, which have reproducible geometries could be
produced by the well-established two-step anodization process shown in Figure 1.2.2
[40]. Generally, a piece of pure aluminum sheet is first prepared by sonication in the
solution of acetone, washed in deionized water (DI water) and dried by nitrogen gas.
During the first anodization step, the aluminum sheet is immersed in 0.3 M oxalic
acid with the 60 V voltage for 6 h. A platinum electrode can be used as the cathode
and the aluminum piece is the anode. After the first anodization step, a mixture of
chromic acid (4%) and phosphoric acid is added to get rid of the alumina layer with
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10
irregular pores. A second anodization process is then performed at the same
condition resulting in excellent pore size distribution. A drop of NaOH solution (10%)
is poured to remove unnecessary thin alumina layer. The whole piece is then
thoroughly washed by DI water. It is etched in solution of HCl (10%) and 0.1 M
CuCl2 to get rid of aluminum and expose alumina.
First anodization
Second anodization
Barrier thinning
Figure 1.2.2 Fabrication scheme of nanoporous alumina membrane by two-step
anodization process (Adapted from [40]).
The voltage affect nanopore sizes. Generally, larger voltage produces larger pore
sizes. At the first anodization process, an oxide layer is formed leading to uneven
surface. Current dense concentrates at the barrier layer, where pores form and grow
along the perpendicular direction. Pores grow along the horizontal direction under
the balance of alumina formation and dissolution. Both the repulsion between pores
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11
and volume expansion can regulate pore structure. When large voltage is applied,
ions transfer rate increases leading to increased pore size. Moreover, pore depth is
determined by anodizing time duration.
1.2.2 Pore parameters of nanoporous alumina membrane
After anodization, anodized aluminum forms amporphous alumina with hexagonally
ordered pore arrays (Figure 1.2.3a) [41]. Parameters including nanopore diameter,
interpore distance and nanopore thickness are usually applied to describe the
nanostructures of nanoporous alumina membranes (Figure 1.2.3b).
Figure 1.2.3 Anodic alumina porous structures (a) and a cross-sectional view of the
anodized layer (b) (Adapted from [41]).
The different anodizing conditions form various pore diameters. The pore depth can
even reach to 100 μm, which is an important factor to make nanoporous alumina
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12
membrane have high surface to area ratio. The diameter of pores has linear
relationship to the anodizing voltage. The proportionality constant λp is approximate
1.29 nm/V [42].
Dp =λp U (1.1)
Where Dp is the diameter of pores (nm), and U refers to the potential for anodizing
(V). The diameter of pore is determined by electrolyte temperature and
hydrodynamic conditions. High temperature speeds the chemical dissolving of the
outer oxide layer. It also accelerates electrochemical formation of anodic oxide layer.
Moreover, the current density increases which makes pore diameter decrease in the
inner oxide layer.
Figure 1.2.4 Factors influence on the nanopore diameter during anodization process
(Adapted from [41]).
In addition, electrolyte temperature, and acid concentration also affect the pore
diameter (Figure 1.2.4) [41]. When electrolyte concentration increases, and pH of
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13
solution decreases, pore diameter increases. With the increasing anodizing time and
widening time, the pore diameter increases.
The nanoporous alumina membrane porosity is mostly affected by anodizing
potential and pH of the electrolyte. The porosity varies under different experimental
conditions with the estimated porosity ranging from 8% to 30% and even more. In
sulfuric acid and oxalic acid, the porosity exponentially decreases due to the increase
of anodization potential. On the other hand, porosity increases slightly as the increase
of anodizing potential in sulfuric acid [43]. In oxalic acid, when the temperature for
anodizing increases, the porosity decreases, while in sulfuric acid, the increase of
anodizing temperature increases the porosity [44].
Nanoporous alumina membrane has high pore density, so the number of pores is one
of the most important parameters. The density is defined as the total pore number in
the surface area of 1 cm2.
n = 1014
/Ph (1.2)
where Ph is a surface area of a single cell. From Equation 1.2, the number of pores
formed within the membrane decreases as the surface area of a single cell increases.
Nanoporous alumina membranes have hexagonal pattern of nanopores and the pore
length is controllable. So they are promising and inexpensive platforms for
synthesizing other nanostructured materials. A wide variety of nanoscale materials,
such as nanotubes, nanowires, nanorods, and nanodots can be fabricated with the
assistance of nanoporous alumina membranes [45]. Figure 1.2.5 shows ZnO
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14
nanowires obtained from corresponding nanoporous membrane templates [46]. Here,
the nanopore diameters are 130 nm and 60 nm in Figure 1.2.5a and c, respectively.
The corresponding nanowires obtained are shown in Figure 1.2.5b and d. The mean
diameters for nanowires are 85 nm and 50 nm, respectively.
Figure 1.2.5 ZnO nanowires obtained from nanoporous alumina membrane
templates with different pore diameters. The average diameter of membrane template
was 130 nm in (a); the average diameter of nanowires was 85 nm; the average
diameter of membrane template was 60 nm in (c); the average diameter of nanowires
was 50 nm. Scale bar was 500 nm (Adapted from [46]).
The increasing development of nanoimprinting and nanopattern technology has led
to increasing fabrication of nanomaterials assisted with nanoporous alumina
membrane. Figure 1.2.6 shows SEM images of gold nanotubes, which are fabricated
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15
using nanoporous membrane templates. They have perfect hexagonal arrangement
and uniform alignment shown in Figure 1.2.6b, which mirror the initial pore arrays
of nanoporous membrane template in Figure 1.2.6a. These kinds of materials have
promisingly applied in many analyzing areas including disease analysis, and sensors.
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16
Figure 1.2.6 (a) SEM image of a gold nanotube membrane (inset: enlarged top view).
(b) Inclined view of the gold nanotube membrane (Adapted from [46]).
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17
1.3 Nanoporous alumina membrane for biosensing application
Generally, biosensors should consist of biologically sensitive elements and
transducers that can detect biological components such as tissues, cells,
microorganisms, virus, nucleic acid, proteins and so on. The transducers can be
optical, piezoelectric and electrochemical [47]. An ideal biosensor should have low
limit of detection, high sensitivity, and high specificity (low interference). There are
always efforts for researchers to look for new sensing platforms and materials to
improve the performance of biosensors. In recent years, nanoporous alumina
membranes have attracted many interests in biosensing area due to the advantages of
high surface-to-volume ratio, enhanced sensitivity, ease of surface functionalization
and biocompatibility.
1.3.1 Requirements of biosensors
A biosensor typically includes a biological element and a physiochemical signal
detection and transduction element. The biological elements could be biomolecules
including DNA molecules, proteins, cells or enzymes[47]. Therefore, a biosensing
system can be used to detect biological species. The biosensor could be classified
based on various transducer properties, which play an important role in biological
sensing. The most common types are optical, piezoelectric, or electrochemical [48].
Specific receptors are immobilized on transducer surface, and capture bioanalyte
targets. The physiochemical signal is transferred to optical or electrical signals for
detection and analysis. Compared with imperforate substrate, nanoporous alumina
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18
membranes own increased surface area for biomolecule capturing and reaction. The
signals could be enhanced significanty. Owing to the capabilities of integration of
electrochemical or optical detection, nanoporous membranes are widely used in
different biosensing areas. Biosensors based on nanoporous membranes have
advantageous properties of fast response, excellent sensitivity and low LOD.
1.3.2 Surface functionalization of nanoporous membranes for biosensors
Surface modification and functionalization of nanoporous alumina membrane are
expected to expand the scope for nanoporous alumina membrane based biosensing
applications. When nanoporous alumina membranes are boiled in hydrogen peroxide,
rich content of hydroxyl groups is generated on membrane surface, which allows the
membrane to be easily modified with silanes. Generally, the surface modification
techniques include wet chemical modification and gas-phase techniques.
Chemical modification is a relatively effective method to control surface properties
of nanoporous alumina membrane. Wide varieties of silanes, such as 3-
aminopropyltrimethoxysilzne (APTES) and (3-glycidoxypropyl) trimethoxysilane
(GPMS) are commercially available and used as coupling agents or linkers to
immobilize oligonucleotide, proteins and some polymers. The silanes could be self-
assembled on nanoporous alumina membrane surfaces. The modification process of
nanoporous alumina membrane using APTES is shown in Figure 1.3.1 [49, 50].
Nanoporous alumina membranes are boiled in hydrogen peroxide and generate
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19
hydroxyl on the membrane surface. APTES is then employed to aminate hydroxyl
groups and assembled on the nanoporous alumina membrane surface.
NH2
Si(OH5C2)3
H5C2O OC2H5H2O2
OH OH
Si
NH2
Figure 1.3.1 Scheme of nanoporous alumina membrane surface modification by
APTES (Adapted from [50]).
Biomolecules such as DNA and proteins are immonilized on nanoporous alumina
membranes by covalent bonding of functional groups [50]. The biomolecules are
immobilized on APTES functionalized membrane surface through glutaraldehyde.
One aldehyde group of glutaraldehyde covalently binds with the amino group on the
APTES and the other aldehyde group binds with the amino end of molecules. When
nanoporous alumina membranes are silanized by isocyanatopropyl triethoxysilane,
amino group can react with aldehyde group to form a covalent bonding between
DNA molecules and membrane surface with the scheme shown in Figure 1.3.2.
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NCO
Si(OH5C2)3
H5C2O OC2H5 OC2H5H5C2O
NCO
NH
NH
OH
Si
O
CO
Si
O
H2N
Figure 1.3.2 Scheme of nanoporous alumina membrane surface modification by
isocyanatopropyl triethoxysilane and immobilization of amino modified DNA
(Adapted from [50]).
In addition to silane, organic acid can self-assemble on metal oxide surface and has
been used for nanoporous alumina membrane surface modification [50, 51]. The
proposed reaction scheme of n-alkanoic acid on a hydroxylated membrane surface is
shown in Figure 1.3.3.
Figure 1.3.3 Scheme of nanoporous alumina membrane surface modification by n-
alkanoic acid (Adapted from [51]).
The surface properties of nanoporous alumina membranes are expected to be
changed by gas-phase techniques [52]. Various materials including metals, metal
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oxides, and ceramics can be deposited on nanoporous alumina membrane and offer
opportunities to change membrane surface properties for specific applications [53].
1.3.3 Anti-biofouling properties of nanoporous membranes for biosensing
When nanoporous alumina membranes are used to construct biosensors, non-specific
adsorption of biomolecules, such as nucleic acids, proteins and cells may cause some
interference. For example, the adsorbed proteins may block active sensing area,
decrease diffusion efficiency of small molecules, and decrease sensing signals.
Many materials, such as hydrogels, surfactants, and phospholipids are used to modify
nanoporous alumina membrane to avoid biofouling [54]. One important aspect is that
the use of these materials should not decrease detection sensitivity and efficiency of
biosensors. PEG hydrogel is a kind of popular materials, which can be grafted on the
membrane surface to decrease non-specific protein adsorption. PEG is hydrophilic,
and can decrease nonspecific adsorption of protein on the surface. PEG grafted
nanoporous alumina membrane forms hybrid organic-inorganic membrane interface
and provides significantly improved non-biofouling properties [55]. PEG was grafted
on membrane surface forming micropatterns for detecting bacteria [56].
1.3.4 Recent development of nanoporous membrane based biosensing
Nanoporous alumina membranes have been used for various biosensing applications
including glucose, cholesterol, single molecule, cancer biomarker, bacteria and cell,
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protein and virus detection due to the preferred properties such as increased surface
reactive area, ease of fabrication, enhanced sensitivities and biocompatibility.
1.3.4.1 Glucose detection
Nowadays, diabetes mellitus increasingly threatens people’s health. Blood glucose
level is a crucial indicator of diabetes mellitus. Many efforts have been paid to blood
glucose monitoring for early detection. Nafion-based membrane glucose biosensor
was developed by Moussy et al. for in vitro and in vivo evaluation in dogs [57]. Due
to the high adsorption ability and excellent biocompatibility, porous nanocrystalline
TiO2 film was immobilized with glucose oxidase for amperometric detection [58].
Boss et al. developed a glucose sensor integrated with a nanoporous alumina
membrane as size-selective interface for glucose level detection with the schematic
illustration shown in Figure 1.3.4 [59].
Figure 1.3.4 Schematic illustration of nanoporous alumina membrane in glucose
affinity sensor (Adapted from [59]).
This sensor consisted of an actuating diaphragm, a sensing diaphragm and a flow-
resistive microchannel for viscosity detection. The nanoporous alumina membrane
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was semi-permeable and played two important roles in this system. It could not only
confine large molecules passing through but also allow glucose to pass through,
ensuring that the concentrations of liquids were the same in the sensor and in the
liquid. This nanoporous alumina membrane based biosensor used ConcanavalinA in
dextran as the sensing fluid which was suitable for continuous blood glucose
detection.
1.3.4.2 Cholesterol detection
In recent years, cardiovascular diseases increasingly threaten human’s health. High
concentration of cholesterol in blood is the main factor causing cardiovascular
diseases. Therefore, measurement of cholesterol concentration in blood is of great
essence. Li et al. developed a cholesterol biosensor with cholesterol oxidase
immobilized on porous silica sol-gel matrix [60]. This biosensor had a high
specificity and the detection limit could reach as low as 1.2×10-7
mol/L. Another kind
of cholesterol biosensor was based on zinc oxide nanoporous film surface, which was
much easier in preparation with cholesterol physically adsorbed on the membrane
surface without any functionalization.
1.3.4.3 Biomolecule analysis
Cell membranes are composed of a lipid bilayer, composed of phospholipids, which
is important for the permeability property. The hybrid system with lipid bilayer
coupled with inorganic solids has attracted many researchers’ interests in the past
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24
years [61]. Many types of artificial lipid membrane structures were studied to be
attached on a solid surface for transport activity detection [62]. Recently, Deng et al
developed a nanoporous alumina membrane-based biosensor to detect DNA (Figure
1.3.5) [63]. In this application, alumina pores were modified by silane for covalent
linkage of probe DNA. Two sides of the nanoporous alumina membrane were
deposited by platinum layers as a working electrode and a reference electrode,
respectively. The processes of DNA immobilization and hybridization in the
nanopores had effect on the pore size and ionic conductivity. By measuring the
electrode Faradaic current, target DNA capture and hybridization process could be
monitored.
Figure 1.3.5 Scheme of nanoporous alumina membrane based impedimetric
biosensing for DNA detection (Adapted from [63]).
The morphology, diameters and physical or chemical properties of nanoporous
membranes can be modulated. It allows biomolecules to interact with membrane
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surfaces, which provides potential opportunities for biomolecule detection and
analysis [64].
1.3.4.4 Cancer biomarker detection
Cancer is another deadly disease threatening people’s health. It becomes more and
more important to screen rapidly blood samples for cancer early diagnostics. A
nanoporous alumina membrane based system was developed for cancer marker
CA15-3 filtering and detection shown in Figure 1.3.6 [65]. The nanoporous
membrane was functionalized by APTES and immobilized with anti-CA15-3
antibodies. Cancer biomarker could be captured by antibody, which was immobilized
in the nanopores and caused blocking effect of the nanopores. The blocking effect
could be detected by monitoring the impedance signals change, which could be
enhanced by gold nanoparticle conjugation and silver enhancement. With signal
amplification methods, low concentrations of cancer biomarker in blood samples
could be detected. The detection limit reached as low as 52 U/mL. This nanoporous
alumina membrane based immunoassay platform had the potential for cancer
diagnosis.
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Figure 1.3.6 Nanoporous alumina membrane based platform for cancer biomarker
detection in blood sample. Left: SEM images of a nanoporous alumina membrane
with a top view and cross-section view and confocal microscopy image; Center:
Protein sensing scheme based on nanoporous alumina membrane; Right: Sensing
principle for protein detection (Adapted from [65]).
1.3.4.5 Bacteria and cell detection
It is important to detect pathogenic bacteria for ensuring food safety and people’s
health. It is of significant necessity to investigate on effective detection methods for
bacteria control in food and water supply. Single strand DNA (ssDNA) probe-
functionalized nanoporous alumina membrane was developed for detecting E. coli
O157:H7 [66]. A dynamic polymerase-extending DNA hybridization process was
proposed. The hybridization process occurred with the appearance of TaqDNA
polymerase and dNTPs with controllable reaction temperature. DNA hybridization
process caused ionic conductivity change, which could be monitored by
electrochemical biosensors.
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A microfluidic chip integrated with nanoporous alumina membrane immobilized
with antibody was developed for E. coli O157:H7 capture shown in the Figure 1.3.7
[67]. Antibodies were immobilized on the nanoporous membrane surface for bacteria
detection. When the bacteria were captured by antibody, electrolyte ions passing
through the nanopores were blocked causing the increase of impedance, which was
analyzed by exterior electrochemical impedance spectroscopy (EIS). This
nanoporous alumina membrane based microfluidic immunosensor for E. coli
O157:H7 sensing achieved fast and sensitive bacteria detection of 102 CFU/mL and
specificity.
Impedance
analyzer
Electrode
Bacteria
Fluorescence
labeled antibody
Modified alumina
membraneNanopore
Immobilized
antibody
Figure 1.3.7 Scheme illustration of nanoporous alumina membrane based biosensor
for bacteria sensing (Adapted from [67]).
Cell-based biosensors have attracted more and more interests during the past years.
Cell-based biosensors using the whole cells as sensing elements are widely used for
cell physiological effect investigation. They are simple, sensitive and low cost. It is
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important to develop a good substrate for cell culture to investigate cell response to
many different agents. Yu et al. developed nanoporous alumina membrane based
electrochemical biosensors for investigating anticancer drug effect of retinoic acid
(RA) on human esophageal squamous epithelial KYSE30 cancer cells [68].
Nanoporous alumina membranes acted as the ideal interface, which were chemically
and mechanically suitable for interaction with biomolecules for stable and long-time
detection. A microarray fabricated by PEG on nanoporous alumina membrane with
micropatterning was further developed by Liu et al [69]. It was investigated to
monitor drug delivery to cytotoxic effects of cisplatin. A microfluidic device was
developed for cell investigation via impedance spectroscopy with integrated
mesoporous membrane and embedded electrodes (Figure 1.3.8) [70].
Co
un
ter
elec
tro
de
Impedance
analyzer
Cells in culture mediumPDMS
Wo
rkin
g e
lect
rod
e
Inlet
Glass slideMesoporous membrane
Figure 1.3.8 Scheme of microfluidic device for cell impedance spectroscopy with
integrated mesoporous membrane and embedded electrodes (Adapted from [70]).
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1.4 Gold nanoparticles (AuNPs)
Gold nanoparticles (AuNPs) can interact with visible light and generate bright colors.
Recently, researchers have focused on the distinct physical and chemical properties
of AuNPs and utilized them in therapeutic agents, sensing, and drug delivery in
biological and medical applications [71]. Their properties include surface plasmon
resonance phenomena, good conductivity, ease of surface functionalization,
fluorescence quenching effect and redox catalyst properties and the scheme to
illustrate the system for AuNP-based detection is shown in Figure 1.4.1 [72]. These
properties can be controlled by changing the shape, size and the surrounding
chemical environment.
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Figure 1.4.1 Physical properties of AuNPs and schematic illustration of an AuNP-
based detection system (Adapted from [72]).
The interaction of recognition elements with analytes can cause physicochemical
property change of AuNPs, such as plasmon resonance absorption and generate a
response signal for exterior analyzer to detect and analyze. The high surface-to-
volume ratio and wonderful biocompatibility of AuNPs provides an appropriate
platform for wide applications of small molecules and biological targets detection
[73-75]. These attributes allow researchers to develop sensors with excellent
sensitivity, stability and selectivity. Easy synthesis and functionalization, together
with physical and chemical properties make AuNPs suitable candidates for
developing biological and chemical sensors.
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1.4.1 Synthesis of AuNPs
AuNPs can be synthesized in numerous ways including both ‘top down’ and ‘bottom
up’ methods. ‘Top down’ is a physical procedure. The desired AuNP dimensions and
shapes are resulted from breaking down of bulk state Au. This method is limited by
the precise control of size and shape [76]. On the contrast, ‘bottom up’ is an
approach involving chemical reduction transformation.
The earliest reported scientific synthesis of AuNPs was developed by Faraday’s
group in 1857, in which AuNPs were reduced from chloroaurate by phosphorus
dissolved in carbon disulfide [77]. Nearly one century later in 1951, AuNPs were
synthesized by citrate reduction of chloroauric acid (HAuCl4) in water by Turkevitch
[78]. In this approach, citrate acid acted as a reducing agent and stabilized AuNPs in
solution. AuNP size cou