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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.

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

i

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

ii

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

iii

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

iv

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.

v

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

vi

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.

vii

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.

viii

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.

ix

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

x

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

xi

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

xii

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

xiii

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

xiv

References…………………………………………………………………..……..176

xv

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

xvi

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

xvii

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

xviii

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

xix

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

xx

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

xxi

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

xxii

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

xxiii

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

xxiv

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

xxv

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

xxvi

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

xxvii

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

xxviii

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

1

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

2

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.

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.

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.

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

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,

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

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

9

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

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

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

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

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

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

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.

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]).

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

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

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.

20

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

21

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,

22

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

23

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

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

25

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.

26

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

28

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.

30

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