BIOASSAYS FOR DISEASE-RELATED NUCLEIC

ACID DETECTION

SHEN WEI

NATIONAL UNIVERSITY OF SINGAPORE

2015

BIOASSAYS FOR DISEASE-RELATED NUCLEIC

ACID DETECTION

SHEN WEI

(B.Sc., ZHEJIANG UNIVERSITY)

A THESIS SUBMITTED

FOR THE DEGREE OF DOCTOR OF

PHILOSOPHY

DEPARTMENT OF CHEMISTRY

NATIONAL UNIVERSITY OF SINGAPORE

2015

DECLARATION

I hereby declare that the thesis is my original work and it has been written by

me in its entirety, under the supervision of Professor Gao Zhiqiang, Department

of Chemistry, National University of Singapore, between August 2011 and July

2015.

I have duly acknowledged all the sources of information which have been used

in the thesis.

This thesis has also not been submitted for any degree in any university

previously.

The contents of this thesis have been partially published as the following articles:

[1] "A ferrofluid-based homogeneous assay for highly sensitive and

selective detection of single-nucleotide polymorphisms," Chem.

Commun., 2013, 49, 8114-8116.

[2] "Synthesis of polyaniline via DNAzyme-catalyzed polymerization of

aniline," RSC Adv., 2014, 4, 53257-53264.

[3] "A highly sensitive microRNA biosensor based on hybridized

microRNA-guided deposition of polyaniline," Biosens. Bioelectron.,

2014, 60, 195-200.

[4] "A simple and highly sensitive fluorescence assay for microRNAs,"

Analyst, 2015, 140, 1932-1938.

[5] "Genotyping and quantification techniques for single-nucleotide

polymorphisms," TrAC, Trends Anal. Chem., 2015, 69, 1-13.

____________

Shen Wei

22th July 2015

I

Acknowledgements

I would like to express my sincere gratitude to all the people who made my PhD

journey meaningful and unforgettable.

First of all, I would like to express my utmost gratitude to my supervisor,

Professor Gao Zhiqiang, for accepting me in his lab and giving me the

opportunity to embark on my PhD research projects under his supervision. His

invaluable instructions and inspirations supported me throughout this four-year

journey and benefited me greatly in my academic research.

Secondly, I would like to cordially thank honors students, Ms. Lim Cai Le and

Ms. Yeo Kiat Huei, for their remarkable contributions in the ferrofluidic

nanoparticle-based bioassay and miRNA bioassay projects, respectively. Their

contributions promoted the good progress of these two projects.

Thirdly, I would also like to thank all my friendly labmates, especially Ms. Deng

Huimin, Dr. Yang Xinjian, and Mr. Teo Kay Liang Alan, for their kind

assistance offered and valuable experiences imparted in lab.

In addition, I am grateful to National University of Singapore and Ministry of

Education for providing me the scholarship to support my research, and also to

Department of Chemistry for all the facilities, technical services and academic

resources provided that greatly facilitated my research.

Last but not least, I would like to express my deepest gratitude to Mr. Tang

Sheng, my family, and friends for their encouragement and moral support

throughout this journey.

II

Table of Contents

Acknowledgements .......................................................................................... I

Table of Contents ............................................................................................ II

Summary ........................................................................................................ VI

List of Tables ................................................................................................. IX

List of Figures .................................................................................................. X

List of Abbreviations ................................................................................. XIV

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

1.1 Disease-related nucleic acids .............................................................. 1

1.1.1 Single-nucleotide polymorphisms in DNA .......................................... 1

1.1.2 Abnormal expression of miRNA ......................................................... 3

1.2 General principle of nucleic acid detection ...................................... 7

1.3 Detection techniques of disease-related nucleic acids ..................... 7

1.3.1 Colorimetric detection ......................................................................... 8

1.3.2 Fluorescent detection ......................................................................... 10

1.3.3 Impedimetric detection ...................................................................... 11

1.4 Detection of SNPs in DNA ................................................................ 14

1.4.1 Difficulties in the detection of SNPs in DNA .................................... 14

1.4.2 Developed SNP genotyping and quantification techniques ............... 14

1.4.3 Detection techniques developed in this thesis .................................... 25

1.5 Detection of abnormal expression levels of miRNAs ..................... 25

1.5.1 Difficulties in the detection of miRNAs ............................................ 25

1.5.2 Developed techniques for miRNA detection ..................................... 26

1.5.3 Detection techniques developed in this thesis .................................... 32

1.6 Purpose, significance and scope of this thesis ................................ 32

Chapter 2. A Ferrofluid-Based Homogeneous Bioassay for Highly

Sensitive and Selective Detection of Single-Nucleotide Polymorphisms ... 35

III

2.1 Introduction ...................................................................................... 36

2.2 Materials and methods ..................................................................... 38

2.2.1 Reagents and apparatus ...................................................................... 38

2.2.2 Preparation of ferrofluidic MNP-PAA ............................................... 39

2.2.3 Prepare ferrofluidic nanoparticle probes ............................................ 40

2.2.4 Buffer optimization for hybridization ................................................ 41

2.2.5 TMB-based signal amplification ........................................................ 41

2.2.6 Ligation chain reaction ...................................................................... 42

2.3 Results and discussion ...................................................................... 42

2.3.1 Principle of the assay ......................................................................... 42

2.3.2 Optimization of the conditions ........................................................... 48

2.3.3 LOD of the assay ............................................................................... 51

2.3.4 SNP discrimination of the assay ........................................................ 54

2.4 Conclusion ......................................................................................... 56

Chapter 3. A Simple and Highly Sensitive Fluorescence Assay for

MicroRNAs ..................................................................................................... 57

3.1 Introduction ...................................................................................... 58

3.2 Materials and methods ..................................................................... 62

3.2.1 Reagents and apparatus ...................................................................... 62

3.2.2 Preparation and quantification of DNA detection probe-MB

conjugates ................................................................................................... 64

3.2.3 Optimization of experimental conditions and miRNA detection ....... 65

3.2.4 Analysis of miRNA in total RNA extracted from cultured cells ....... 66

3.3 Results and discussion ...................................................................... 67

3.3.1 Assay principle ................................................................................... 67

3.3.2 Optimization of conditions................................................................. 68

3.3.3 Analytical performance ...................................................................... 74

3.3.4 Sample analysis .................................................................................. 79

3.4 Conclusion ......................................................................................... 79

IV

Chapter 4. Application of Catalytic Nucleic Acids in Bioassay for

MicroRNA Part I: Synthesis of Polyaniline via DNAzyme-Catalyzed

Polymerization of Aniline ............................................................................. 81

4.1 Introduction ...................................................................................... 82

4.2 Experimental section ........................................................................ 85

4.2.1 Reagents and apparatus ...................................................................... 85

4.2.2 Preparation of the G-quadruplex DNAzyme...................................... 87

4.2.3 DNAzyme-catalyzed polymerization of aniline................................. 87

4.3 Results and discussion ...................................................................... 88

4.3.1 DNAzyme-catalyzed polymerization of aniline................................. 88

4.3.2 Optimization ...................................................................................... 92

4.3.3 Kinetics of the DNAzyme-catalyzed polymerization of aniline ........ 94

4.3.4 Characterization of the polymerization product ................................. 96

4.3.5 Proton doping ................................................................................... 100

4.4 Conclusion ....................................................................................... 100

Chapter 5. Application of Catalytic Nucleic Acids in Bioassay for

MicroRNA Part II: A Highly Sensitive MicroRNA Biosensor Based on

Hybridized MicroRNA-guided Deposition of Polyaniline........................ 102

5.1 Introduction .................................................................................... 103

5.2 Experimental section ...................................................................... 104

5.2.1 Materials and reagents ..................................................................... 104

5.2.2 Biosensor fabrication ....................................................................... 106

5.2.3 MicroRNA hybridization and EIS detection .................................... 107

5.3 Results and discussion .................................................................... 108

5.3.1 Detection scheme ............................................................................. 108

5.3.2 Feasibility study ............................................................................... 109

5.3.3 Optimization .................................................................................... 113

5.3.4 Analytical performance of the biosensor ......................................... 118

5.4 Conclusion ....................................................................................... 121

V

Chapter 6. Conclusions and Suggestions for Future Research................ 122

References ..................................................................................................... 130

List of Publications ...................................................................................... 154

VI

Summary

Single-nucleotide polymorphisms (SNPs) in DNA and abnormal expression of

microRNAs (miRNAs) are two significant types of abnormalities of nucleic

acids. Recent studies have revealed that numerous diseases, including various

types of cancer and metabolic syndromes, are originated from these two types

of abnormalities. These facts intrigued scientists to find methods to qualitatively

and quantitatively discriminate disease-related DNAs and miRNAs. The works

in this thesis focuses on establishing colorimetric, fluorescent, and

electrochemical bioassays for the detection of disease-related nucleic acids.

Chapter 1 gives an introduction of the two types of disease-related nucleic acids

and the necessity to detect them. Representative detection techniques developed

previously for genotyping and quantifying SNPs in DNA as well as for profiling

expression levels of miRNAs will be reviewed and discussed.

Chapter 2 presents a ferrofluid-based homogeneous bioassay for highly

sensitive and selective detection of SNPs. The assay utilized a ligase as a target

amplifier through ligase chain reaction (LCR) and ferrofluidic nanoparticle

probes (FNPs) as signal generators and amplifiers at the same time by catalyzing

the colorimetric oxidation of 3,3’,5,5’-tetramethylbenzidine. The exponential

amplification ability of the LCR and FNP-catalyzed signal amplification offered

the assay excellent sensitivity and selectivity as well as low limit of detection.

The attractive properties of simple, rapid and low cost colorimetric SNP

VII

genotyping could open a new perspective in the development of SNP

genotyping tools for uses at point-of-care.

Chapter 3 demonstrates a simple and highly sensitive fluorescence assay for the

detection of miRNAs. Briefly, fluorescein-capped DNA detection probes were

first conjugated to magnetic beads (MBs) for capturing target miRNA let-7a. A

duplex-specific nuclease (DSN) was engaged to selectively cleave the DNA

probes in the miRNA-DNA duplexes and release the target miRNA strands back

to solution, thereby establishing a target recycling amplification mechanism and

a cumulative signal amplification process. The remained DNA detection probes

were completely removed when the MBs were separated by permanent magnet,

attaining a negligible background. The high specificity of the DSN to perfectly

matched duplexes, the accumulative signal amplification in association with a

negligible background endowed this assay a good sensitivity and selectivity

when analyzing target miRNAs with high sequence similarities.

Chapter 4 and 5 illustrate an electrochemical bioassay for miRNAs. Before

demonstrating the assay, Chapter 4 first discusses a novel and environmentally

friendly reaction – the synthesis of polyaniline (PANI) via DNAzyme-catalyzed

polymerization of aniline, which was firstly thoroughly investigated and then

applied to the assay for signal output. The electrochemical impedance

spectroscopy (EIS)-based bioassay described in Chapter 5 utilized charge

neutral peptide nucleic acid (PNA) as capture probes (CPs) to hybridize with

target miRNA, which in turn guided the deposition of insulating PANI on the

surface of a substrate electrode. Consequently, an electrochemical impedimetric

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signal was produced. The signal amplification and sensitivity improvement

were conveniently achieved by the catalytic reaction and excellent mismatch

discrimination capability was observed due to the excellent hybridization

selectivity of PNA CPs.

Chapter 6 gives a summary of the above-mentioned projects with some

suggestions of future research directions in this field.

IX

List of Tables

Table 1-1. Classification of optical detection and electrochemical detection

methods for nucleic acids based on the final signal output.

Table 2-1. Synthetic oligonucleotides (5'→3') used in the bioassay.

Table 2-2. Comparison of several literature methods for SNP detection.

Table 3-1. Sequences of synthetic oligonucleotides used in this bioassay.

Table 3-2. Composition of all buffer solutions used in this bioassay.

Table 3-3. Comparison with representative fluorescent detection methods for

miRNA.

Table 3-4. Analysis of let-7a in total RNA samples extracted from cultured cells.

Table 5-1. MicroRNA sequences of let-7 family.

Table 5-2. Comparison with other electrochemical detection methods for

miRNA

X

List of Figures

Figure 1-1. Scheme illustration of the process of the production of mature

miRNAs.

Figure 1-2. MicroRNAs function as biological regulators through their

incorporation into the RISC: (a) degrade the perfectly matched mRNAs and (b)

inhibit the translation of imperfectly matched mRNA.

Figure 1-3. Unique reverse transcription strategies for miRNA to generate

cDNA.

Figure 2-1. Schematic of (a) the working principle the ferrofluid-based

homogeneous assay for SNP (b) sequences of the FNPs and target DNA.

Figure 2-2. TEM image of the highly water soluble MNP-PAA prepared. The

magnetic nanoparticles were synthesized with a particle diameter of 6–8 nm.

Figure 2-3. TGA curve of the highly water soluble MNP-PAA prepared.

Figure 2-4. FT-IR spectrum of the highly water soluble MNP-PAA prepared.

Insert shows ferrofluidic behavior of MNP-PAA.

Figure 2-5. UV-Vis spectra of TMB solution treated by 10 nM MNPs after

hybridizing with (1) control, (2) 2.0 and (3) 6.0 nM SNP target. Inset:

Photographs of the FNP solution in the without/with applying a permanent

magnet.

Figure 2-6. Buffer optimization: effect of monovalent cation (Na+)

concentration ranging from 50 mM to 2.0 M on the stability and hybridization

efficiency of the FNPs. Curve marked with 2 is the sample and curve marked

with 1 is the control.

Figure 2-7. Buffer optimization: effect of pH ranging from 5.5 to 9.5 on the

stability and hybridization efficiency of the FNPs. Curve marked with 2 is the

sample and curve marked with 1 is the control.

Figure 2-8. Calibration curves () for the response of unbound probe-catalyzed

colorimetric TMB oxidation in the presence of target KRAS MT-C after LCR

(15-thermal cycles). Femtomolar samples are marked with (). Error bars stand

for the standard deviations in the measurements.

Figure 2-9. UV-Vis absorbance spectra of the unbound probe-catalyzed

colorimetric TMB oxidation when 0, 3, 8 16, 25 and 35 pM SNP target present

in the assay.

XI

Figure 2-10. SNP discrimination of the assay with large excess mismatch

sequences. Comparing to the control set (MT-to-WT = 1:0), no significant

change in signal was observed even when the WT concentration was 1000 times

higher. Similarly, SNP detection was carried out with KRAS mutation-to-wild

type target (MT-to-WT) ratios up to 1:1000. 15.6 pM MT-C target was used.

Figure 3-1. TEM images of the streptavidin-coated magnetic beads (Dynabeads

M-280) with diameters of 2.8 µm.

Figure 3-2. Schematic of the proposed DSN-based fluorescent bioassay for

miRNA.

Figure 3-3. The fluorescence intensities of the remained BF oligonucleotides

solution before and after linking BF oligonucleotides to the MBs.

Figure 3-4. Optimization of the experimental conditions. 2.0 nM target miRNA

let-7a, 100 nM probes, and 0.4 U DSN.

Figure 3-5. The dependence of the fluorescence signal on the DSN incubation

time. 2.0 nM target miRNA let-7a, 100 nM probes, 40 ºС, 0.4 U DSN.

Figure 3-6. (a) The calibration curve of let-7a (100 nM probes, 0.4 U DSN and

a period of 2 hrs incubation at 40 ºС) and (b) fluorescence spectra of varied

concentrations of let-7a.

Figure 3-7. Responses of the let-7a assay to 2.0 nM of let-7a, let-7f, and let-7d.

Experimental conditions are as for Figure 3.

Figure 4-1. (A) Photos of the reaction mixtures (50 mM aniline/PAA + 50 mM

hydrogen peroxide + 2.0 μM the DNAzyme) at different pH values and (B)

Different structures of PANI: (a) Leucoemeraldine, (b) Emeraldine base (EB),

(c) Emeraldine salt (ES), (d) Pernigraniline, (e) Ortho- and para-substituted

carbon-carbon, carbon-nitrogen bond structure. (C) DNAzyme-catalyzed

polymerization of aniline in the presence of hydrogen peroxide.

Figure 4-2. UV-Vis spectra of the polymerization of aniline oxidized by

hydrogen peroxide in the presence of the DNAzyme. The working solution

contained 50 mM aniline/PAA (1:1), 2.0 μM the DNAzyme and 50 mM

hydrogen peroxide in pH 3.0 0.10 M PB buffer. From bottom to top: 0, 15, 30,

45, 60, 75, 90, 120, 150, 180, 210, 240, 270, 300, 330 and 360 s after starting

the polymerization.

Figure 4-3. (A) pH optimization of aniline polymerization. Working solution:

50 mM aniline/PAA, with () and without () 2.0 μM the DNAzyme, and 50

mM hydrogen peroxide in phosphate buffer with different pH values ranging

from 2.0 to 5.0. UV-Vis measurements were carried out 6 min later after starting

XII

the polymerization. (B) The effect of pH on the extinction coefficient of the

synthesized PANI.

Figure 4-4. Effect of the amount of hydrogen peroxide on the reaction rate of

aniline polymerization. Working solution: 50 mM aniline/PAA, 2.0 μM

DNAzyme and different concentrations of hydrogen peroxide in the phosphate

buffer (pH 3.0).

Figure 4-5. Double-reciprocal plots of the catalytic activity of 2.0 μM

DNAzyme at a fixed concentration of one substrate and varying concentration

of the second substrate.

Figure 4-6. FT-IR spectrum of the proton doped half-oxidized PANI

(emeraldine salt).

Figure 4-7. A cyclic voltammogram of PANI on GCE in 2.0 M HCl aqueous

solution from 0 to 0.8 V at a scan rate of 50 mV/s.

Figure 4-8. Morphological characterization (SEM image) of the synthesized

PANI.

Figure 5-1. EIS spectra of (1) the control and the miRNA hybridized biosensor

before PANI deposition. 100 fM let-7b and 60 min hybridization at 50 ºС. Inset:

Scanning tunneling microscopic image of the gold bead electrode.

Figure 5-2. Schematic of the working principle of the label-free miRNA

biosensor. (A) miRNA hybridization, (B) hybridized miRNA-guided PANI

deposition, and (C) EIS measurement.

Figure 5-3. (A) EIS spectra and (B) the corresponding voltammograms at

100 mV/s of a let-7b biosensor to (1) 100 fM let-7b and (2) 100 fM control RNA.

60 min hybridization at 50 °C and 30 min incubation in pH 3.0 of 0.10 M

potassium phosphate containing 10 μM DNAzyme, 20 mM aniline, and

200 mM hydrogen peroxide. Insets: (top) Bode plot of the biosensor and

(bottom) Randle equivalent circuit used to fit the EIS data: Rs – solution

resistance, Cdl – double layer capacitance, Rct – charge-transfer resistance, W

– Warburg impedance element.

Figure 5-4. Dependence of Rct on (a) pH, (b) aniline, (c) DNAzyme, and (d)

hydrogen peroxide; after 60 min hybridization of 250 fM let-7b at 50 °C. Data

points represented six replicates.

Figure 5-5. Dependence of Rct of (1) 10 fM, (20) 250 fM, and (3) 5.0 pM let-

7b after 60 min hybridization at 50 °C on the incubation time in pH 3.0 of

0.10 M potassium phosphate containing 10 μM DNAzyme, 20 mM aniline, and

200 mM hydrogen peroxide. Data points represented six replicates.

XIII

Figure 5-6. Calibration curves for let-7b. 60 min hybridization at 50 °C and 30

min incubation in pH 3.0 of 0.10 M potassium phosphate containing 10 μM

DNAzyme, 20 mM aniline, and 200 mM hydrogen peroxide . Data points

represented six replicates.

Figure 5-7. EIS responses (Rct) of a let-7b biosensor to 100 fM of let-7b, let-

7c, let-7a, let-7e, and pre-let-7b, respectively. 60 min hybridization at 50 °C and

30 min incubation in pH 3.0 of 0.10 M potassium phosphate containing 10 μM

DNAzyme, 20 mM aniline, and 200 mM hydrogen peroxide. Data points

represented six replicates.

XIV

List of Abbreviations

AgNP Silver nanoparticle

AuNP

CL

Gold nanoparticle

Chemiluminescence/Chemiluminescent

cNCB Chameleon NanoCluster Beacon

CPs Capture probes

dsDNA Double-stranded DNA

DEG Diethylene glycol

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DSN Duplex-specific nuclease

EDC 1-Ethyl-3-3-dimethylaminopropyl carbodiimide

hydrochloride

EDTA Ethylenediaminetetraacetic acid

EIS Electrochemical impedance spectroscopy

FRET Fluorescence resonance energy transfer

FNP Ferrofluidic nanoparticle probe

FT-IR Fourier transform infrared

GCE Glassy carbon electrode

GMNP

GO

Gold-coated magnetic nanoparticle

Graphene oxide

GPC Gel permeation chromatograph

HCR Hybridization chain reaction

HRP Horseradish peroxidase

LCR Ligase chain reaction

LDR Ligase detection reaction

LNA Locked nucleic acid

Let-7 Lethal-7

XV

LED Light emitting diode

LOD Limit of detection

MB Magnetic bead

MNP Magnetic nanoparticle

miRNA MicroRNA

mRNA Messenger RNA

MT Mutant target

NHS N-hydroxysuccinimide

NGS Next-generation sequencing

OD Optical density

PAA Polyacrylic acid

PANI Polyaniline

PB Phosphate buffer

PBS

PCB

Phosphate buffered saline

Printed circuit board

PCR Polymerase chain reaction

PNA

QDs

Peptide nucleic acid

Quantum dots

qPCR Quantitative polymerase chain reaction

qRT-PCR Quantitative reverse transcription polymerase chain reaction

RCA

Rct

Rolling circle amplification

Charge-transfer resistance

RISC RNA-induced silencing complexes

RNA Ribonucleic acid

SBE Single-base extension

SCE Saturated calomel electrode

SDR Strand-displacement reaction

XVI

SEM Scanning electron microscopy

SMRT Single molecule real-time

SNP Single-nucleotide polymorphism

SOLiD Supported oligonucleotide ligation and detection

TEM Transmission electron microscope

TMB 3,3’,5,5’-Tetramethylbenzidine

TGA Thermogravimetric analysis

Tris Tris(hydroxymethyl) aminomethane

UP Ultrapure

UV-Vis Ultraviolet-visible

WT Wild-type

1

Chapter 1. Introduction

It has been found that nucleic acids, including deoxyribonucleic acid (DNA)

and ribonucleic acid (RNA), provide genomic templates1 to determine the

amino acid sequences along the polypeptide chain of protein,2 and thus play a

critical role in all activities of living beings. For example, nucleic acids encode

genetic instruction and regulate the cell growth, cell development, cell

proliferation and differentiation, cell apoptosis, and neuronal patterning etc.3

Recent studies have revealed that numerous diseases,4, 5 including various types

of cancer like breast cancer6 and lung cancer,7, 8 and metabolic syndromes like

cardiovascular disease9 and diabetes,10 originate from the abnormalities of

nucleic acids. These facts intrigued scientists to find methods to qualitatively

and quantitatively detect nucleic acids, especially the disease-related ones.

1.1 Disease-related nucleic acids

1.1.1 Single-nucleotide polymorphisms in DNA

Single-nucleotide polymorphisms (SNPs) belong to DNA sequence variations11

and are defined as single base-pair positions where variable alleles exist.12 They

are referring to single base transversion (switch among purines and pyrimidines)

as well as base transition (switch between purines or pyrimidines only), but

normally do not include single base insertion or deletion. Because DNA are

made of four kinds of nucleobases i.e. A, T, C and G, SNPs theoretically could

be up to tetra-allele polymorphisms; while in fact, tri- or tetra-allele

polymorphisms almost do not exist in human DNA. This is the reason that SNPs

2

are regarded as one type of bi-allele polymorphisms and can be simply noted as

+/− during a certain SNPs genotyping and quantification.

During the 1990s, as the most abundant DNA variations,13 SNPs attracted wide

attention along with the progress of Human Genome Project. In the past two

decades, intensive research efforts have been devoted to the study of SNPs.

Computational analysis has predicted that there are over 15 million SNPs in the

human genome occurring on an average of one SNP in every 200 nucleotides.14

SNPs are believed to be closely associated with various medical conditions and

genetic diseases although the majority of them have little impact on human

health.5, 15, 16 Information gathered to date suggests that SNPs may serve as a

new generation of biomarkers for cancer diagnosis and prognosis,5, 15, 16

presenting clinical scientists with new perspectives in diagnostics and a

promising therapeutic frontier.

Because of their prominent relevance to human health, the identification of

clinically relevant SNPs has become a major initiative in molecular biology

research. The establishment of the relationship between typical SNPs with

diseases has prompted research on SNP genotyping and quantification

techniques. Since SNPs are considered as the next generation of biomarkers,

there is growing demand for researchers to develop simple and robust

techniques that allow rapid, sensitive and selective genotyping, and

quantification of SNPs.

3

1.1.2 Abnormal expression of miRNA

MicroRNAs (miRNAs) are a class of single-stranded, highly conserved,17 non-

coding, regulatory RNAs derived from an approximately 70-nucleotide long

hairpin stem-loop precursor known as pre-miRNAs.18 After the pre-miRNAs

are cleaved by the ribonuclease Dicer, mature miRNAs with lengths of 22

nucleotides are formed.19, 20 The whole process that how mature miRNAs

formed can be seen in Figure 1-1.21

Figure 1-1. Schematic of the process of the production of mature miRNAs.

(Reprinted from reference 21, with permission from Elsevier)

4

MiRNAs exist in a wide range of living creatures including animals, plants and

even viruses. The first miRNA, lin-4, was discovered in C. elegans by Victor

and colleagues in 1993;22 while it has not been regarded as a distinct class of

biological regulator until the early 2000s when miRNAs were found playing a

vital regulatory role in gene expression20 and cell differentiation through their

incorporation into the RNA-induced silencing complexes (RISC), which either

inhibit the translation of imperfectly matched messenger RNAs (mRNAs) to

protein or degrade the perfectly matched mRNAs as illustrated in Figure 1-2.20,

21

Figure 1-2. MicroRNAs function as biological regulators through their

incorporation into the RISC: (a) degrade the perfectly matched mRNAs and (b)

inhibit the translation of imperfectly matched mRNA. (Reprinted from

reference 21, with permission from Elsevier)

Along with the continuing identification of many more miRNAs, it is believed

that over 50% of all protein encoding genes in humans are regulated by

miRNAs.23, 24 Increasing evidence has shown that, in addition to their regulatory

roles in gene expression, miRNAs are linked to various physiological and

pathological processes, and human malignancies in particular25, 26 For example,

it was observed that in the serum of ovarian cancer patient, miRNA-21, -29a, -

92, -93, and -126 are obviously over-expressed compared with a healthy

5

person.27 In the plasma of gastric cancer patients, the expression level of let-7a

is lower whereas the miRNA-17-5p, -21, -106a and -106b are significantly

higher than the a normal person.28 Lu et al. observed that a down-regulation of

miRNA expression is an emerging feature in cancer and specific dysregulation

of certain miRNAs is seen in specific cancer types.29 What is more, miRNA

expression profiles can be used to classify poorly differentiated tumor

successfully while the mRNA profile counterparts are highly inaccurate under

the same conditions.29 It was also believed that miRNAs are directly associated

with medical conditions of patients including their individual responses to

diseases, drugs, and other environmental factors. Therefore, studies of miRNAs

expression levels have great potential in therapeutics, drug discovery, and

molecular diagnostics.29, 30 The function of miRNA in the regulation and the

corresponding mechanism are summarized and discussed in detail in two

recently published review articles.31, 32

Provided that a correlation between the miRNA expression level and the state

of an illness can be established, the expression levels of miRNAs can be used

to indicate if a person is not in a good health condition and advance treatments

can be applied before disease deterioration. Thus, similar to the SNPs in DNA,

miRNAs can also be considered as the biomarkers for early disease diagnosis,

prognosis and in-time therapy. Because of their close association with human

health, abnormal levels of miRNA expression must be reliably identified and

quantified as the first step toward improving human health.

6

As aforementioned, diagnosis and prognosis of disease-related nucleic acids at

early stage before symptoms appear will enable in time prevention and

treatment to minimize the impairment may be induced by the diseases and could

help to reduce the mortality rate of patients. This is the ultimate goal that

researchers are pursuing and during the past few decades, it continuously guides

researchers to investigate and develop effective methods to realize the early

diagnosis and prognosis of disease-related nucleic acids.

As we know, the simplest method to detect the presence or the amount of nucleic

acids is to measure the ultraviolet-visible (UV-Vis) absorbance at a wavelength

of 260 nm, which is usually noted as optical density at 260 nm (OD260).

However, the abnormalities of nucleic acids, including single-nucleotide

mutation (which results in SNPs) within a specific type of nucleic acids as well

as the abnormal expression level of a kind of regulating nucleic acids (such as

miRNAs) with specific sequence, are usually in rather low concentrations. The

basic UV-Vis method is far from sensitively detecting nucleic acids in low

concentration even with quality control OD260/280 ≥ 1.8 and OD260/320 ≥ 1.8

considering effect of pH and ionic strength33 as well as the interference from

numerous substances that also absorb UV-Vis light around 260 nm. More

importantly, it is incapable of detecting nucleic acids with a particular sequence,

not to mention the ability to selectively genotype SNPs or discriminate a certain

miRNA from its similar family members. Therefore, nucleic acid bioassays with

adequate sensitivity and sequence selectivity/specificity are urgently needed.

More sensitive and selective bioassays/techniques with superior sequence

7

discrimination ability need to be developed for the detection of disease-related

nucleic acids.

1.2 General principle of nucleic acid detection

During the past few decades, numerous bioassays for the detection of nucleic

acids have already been developed. Generally, the assay procedure consists of

two main steps. First, under proper conditions of salt concentration and

temperature, the nucleic acids of interest (i.e. target) hybridize to a recognition

bioreceptor/probe (usually an oligonucleotide with sequence fully

complementary to the target) through Watson-Crick base pairing and

subsequently result in a signal. Second, this signal will then be amplified and

transduced to another measurable signal that can be collected by a readout

device in a customer-desired way, sometimes in real time.13 As for quantitative

detection, the final output signal is proportional to the amount of the original

target nucleic acids present in the sample solution, and thus the concentration

of target can be quantified.

1.3 Detection techniques of disease-related nucleic acids

According to the detection techniques used for the final output signal

measurement, widely used nucleic acids detection methods can be classified to

optical detection, electrochemical detection, gravimetric detection,

piezoelectric detection, thermometric detection, etc.34, 35 Currently, the

detection methods for nucleic acids are mainly optical detection and

electrochemical detection. Their more detailed classification 21, 34 based on the

final signal output can be seen in Table 1-1.

8

Table 1-1. Classification of optical detection and electrochemical detection

methods for nucleic acids based on the final signal output.

Optical detection

Colorimetric detection36

Fluorescent detection 37

Surface Plasmon Resonance (SPR) detection38

Surface Enhanced Raman Scattering (SERS)

detection39

Chemiluminescent (CL) detection40

Electrochemical detection

Impedimetric detection41

Voltammetric detection42

Amperometric detection43

Potentiometric detection44

Herein, the discussion in the following sections will focus on colorimetric

detection, fluorescent detection, and impedimetric detection which are closely

relevant to the projects in this thesis. The advantages and limitations of these

detection methods will be discussed accordingly in the following sections. And

possible measures to establish novel, highly sensitive and selective bioassays

for nucleic acids will be proposed.

1.3.1 Colorimetric detection

Colorimetric detection is one of the two most prevailing optical detection

methods for nucleic acids. It is a method based on Beer-Lambert law which

establishes a direct linear correlation between the absorbance and the

concentration of a substance within a certain range.45 The final signal can be

distinguished by naked eye or more commonly by a UV-Vis spectrometer.

9

At the beginning of 1980s, most of the colorimetric detections relied on biotin-

labeled oligonucleotides as detection probes and gel electrophoresis as the final

signal output device.46 It was Murasugi47 who firstly detected target DNA with

the biotin-labeled oligonucleotides. Later, this method was followed and further

developed by Riley and his colleagues.48, 49 However, gel electrophoresis is

labor-intensive and time-consuming that are not welcomed by researchers.

What is more, detection of nucleic acids at this stage cannot unambiguously

determinate DNAs with different sequences.

The colorimetric detection has not been developed at a large scale until 1990s,

when Travascio50, 51 revealed the intrinsic catalytic activity of DNA and

designed a DNAzyme (PS2.M), which can catalyze a series of oxidation

reactions and produce UV-Vis spectrometer-detectable signals. The DNAzyme

was then used for developing colorimetric bioassays for DNA.52, 53 The final

signal readout platform, i.e. UV-Vis spectrometer, became much simpler and

faster, and sequence distinguishability was also achieved with an intelligent

design of DNAzyme at that time.

The advantages that colorimetric bioassays exhibited are simple mechanism to

be understood, inexpensive instrument, easy operation, stable signal, high

repeatability, fast detection, and a wide range of applications.34 However, the

sensitivity of direct colorimetric bioassays is relatively low and the limit of

detection (LOD) is not low enough. Advanced materials and/or pre-signal

amplification need to be coupled into this type of bioassays to improve their

sensitivity and decrease the LOD. In 1996, Mirkin and co-workers54 judiciously

10

designed the first colorimetric DNA detection method coupling with gold

nanoparticles (AuNPs). This milestone work enabled the AuNPs to become the

most popularly used nanomaterials in colorimetric bioassays to improve the

sensitivity and decrease the LOD.55 Nonetheless, gold is an expensive material

that makes the bioassays unfavorable to some extent. Consequently, cheaper

nanomaterials have been explored to replace the AuNPs while still retaining

their attractive features.

1.3.2 Fluorescent detection

Fluorescent detection is the other one of the two most prevailing optical

detection methods for nucleic acids. It is based on the fact that electrons in a

fluorophore can absorb photons and be excited from the ground electronic state

(lower energy level) to excited states (higher energy levels), and when the

electrons relax to the ground state, light is emitted and it can be collected by a

spectrofluorometer.

During early years, fluorescent dyes were always used as probes for the

fluorescent detection of nucleic acids.56 However, the use of large dose of dyes

to stain the target nucleic acids is quite toxic and environmentally unfriendly.

Besides, the dyes are not sequence selective and thus cannot be used for the

detection of nucleic acids with certain sequences. Molecular beacons that have

nucleic acid sequence discrimination ability designed by Tyagi and Kramer in

1996 brought great progress for the fluorescent detection of nucleic acids.57

After that, a new generation of fluorescent bioassays was established and

fluorescent detection of nucleic acids became much more sensitive and

11

sequence distinguishable.58, 59 Soon, various designs of molecular beacon-based

probes were created and utilized in fluorescent bioassays to decrease the LOD

as well as to enhance the detection sensitivity and specificity of nucleic acids.60

In recent years, fluorescent detection was coupled with polymerase chain

reaction (PCR) and it is now the gold standard for nucleic acids detection due

to its high sensitivity and selectivity as well as ultralow LOD.61

However, fluorescent bioassays have their own limitations like photo bleaching,

the need of additional fluorescent dyes which would expose high toxicity to live

samples, complex operation and expensive equipment.62, 63 The gold standard,

i.e. PCR-based fluorescent detection of nucleic acids, needs quite complicated

pre- and post-PCR treatment procedures, expensive equipment, and is labor-

intensive. Thus, cost-control and procedure-simplification are essential

regarding this type of detection.

1.3.3 Impedimetric detection

Impedimetric detection is one of the most important electrochemical detection

methods. The earliest electrochemical nucleic acids detection strategy should

be traced back to 1960s when Palecek64 found that nucleic acids can be analyzed

based on the redox reaction of nucleic acids at a mercury electrode. Later,

electrochemical methods for the detection of nucleic acids received substantial

attention and numerous electrochemical bioassays have been developed during

the past few decades.65

12

Impedimetric detection of nucleic acids involves electrochemical impedance

spectroscopy (EIS) for the signal collection. It measures the charge-transfer

resistance (Rct) of an electrochemical system and the data are fitted to an

electrical circuit – usually Randle equivalent circuit – and produce an EIS

spectrum.66, 67 The semicircle in the EIS spectrum is closely associated with the

charge transfer of the probe and its diameter indicated the Rct. The value of Rct

can thus be conveniently obtained from the EIS spectrum. Large Rct indicated

that a large amount of charge-transfer impeding material is present at the

electrode surface. Base on this mechanism, Peng et al. established an

impedimetric detection method for disease-related nucleic acids by the

deposition of an insulating polymer film on the surface of the electrode.41 The

excellent sensitivity, ultralow LOD, inherent miniaturization, and good

compatibility with standard microfabrication and semiconductor technologies

make this method quite appealing for the detection of disease-related nucleic

acids.

Homogenous bioassays such as some colorimetric bioassays and fluorescent

bioassays can always achieve good reproducibility; while in contrast, as a kind

of typical heterogeneous bioassays, electrochemical bioassays are inferior when

referring to this aspect. Nevertheless, the main advantage of electrochemical

bioassays is their good sensitivity and low LOD compared to other bioassays.

Furthermore, low power requirement, less cost, portable system, and good

compatibility also endow electrochemical bioassays a significant position in the

area of nucleic acids bioassays.

13

According to the various requirements of the detection techniques, numerous

bioassays have already been established for the detection of nucleic acids.

Nevertheless, each of them has its own disadvantages that limit its wide-spread

applications. None of the current bioassays can cover all aspects including high

sensitivity, low LOD, high selectivity, low cost, concise procedure, rapid

detection, good repeatability, etc. One or more aspects would be sacrificed when

a particular detection needs to meet a specific criterion. Further development

for each type of bioassays is necessary to address its own weaknesses and make

full use of its advantages and benefits. The gaps and aims of this thesis are

summarized as the following:

Colorimetric bioassays have relatively inferior sensitivity and LOD.

Thus advanced technology and effective signal amplification strategies have to

be introduced to improve its sensitivity and decrease the LOD.

Fluorescent bioassays are more expensive and involve more

complicated procedures. In this thesis, judicious design will be made to simplify

the assay process and decrease the cost of the assay.

Impedimetric bioassays have relatively inferior repeatability but have

good sensitivity and low LOD. Generally the worse the repeatability will be

when more steps are involved in a bioassay. Project in this thesis will develop

an assay with simplified procedure, so that better repeatability can be obtained

and meanwhile the good sensitivity as well as low LOD can be remained.

14

1.4 Detection of SNPs in DNA

1.4.1 Difficulties in the detection of SNPs in DNA

As the first step toward improving human health, SNPs must be reliably

identified and quantified. However, the intrinsically subtle difference between

wild-type and mutant genes – a single-base variation – makes it a challenging

task specifically to detect low-abundance SNPs out of a large amount of a wile-

type gene. Ideally, SNP genotyping and quantification techniques should be

rapid, robust, fully automatic, highly accurate, and affordable.68

1.4.2 Developed SNP genotyping and quantification techniques

To date, three groups of SNP genotyping and quantification techniques have

been developed. They are allele-specific hybridization-based techniques, allele-

specific enzymatic techniques, and sequencing techniques.

1.4.2.1 Allele-specific hybridization-based techniques

Allele-specific hybridization-based techniques leverage on the subtle difference

in affinity for the formation of SNP target-capture probe duplexes. In other

words, it relies on the thermal stability arising from the single-nucleotide

variation between a wild-type and a mutated gene.69 In most cases, the

hybridization of allele-specific hybridization bioassays is firstly performed on

solid substrates, which will benefit the separation of interfering species, and

enable construction of high throughput multiplexed SNP genotyping and

quantification platforms.

15

For example, Lambert and his colleagues used a commercial SPR sensor and

modified DNA probes on it for rapid detection of a 21-base long DNA target.

One-base mismatch can be distinguished (without the co-existing interfering

mismatches) and the detection limit was 100 pM.70 Since this type of techniques

does not require the involvement of complex enzymatic reactions, nor the use

of detection probes, it is straightforward, label-free, fast, cost-effective,

convenient, and easy to be conducted. Nevertheless, simply relying on the

detection of subtle difference in stability caused by a single base variation

suffers from the problems of low selectivity and inferior sensitivity on the other

side.71 LOD at picomolar level is not good enough and the selectivity when

interfering mismatches exist is far from satisfactory for SNP genotyping and

quantification.

In order to achieve an acceptable selectivity and better sensitivity as well as

lower LOD, great care must be taken in the optimization of the hybridization

conditions such as salt concentration and incubation temperature. Besides,

secondary structure in the DNA probes, including molecular beacons, Y-shape

junction, toeholds, and G-quadruplexes were designed and employed to achieve

this goal.

Molecular beacons are stem-loop structured oligonucleotides that contain both

fluorescent reporters and quenchers. The fluorescence of the reporters is

quenched when the oligonucleotides fold into stem-loops by fluorescence

resonance energy transfer (FRET) between the reporters and quenchers. The

fluorescence of the reporters is restored when complementary target strands

16

hybridize and unfold the stem-loops.57 The first application of molecular

beacons in SNP genotyping was reported by Kramer and co-workers.72 Two

molecular beacons respectively labelled with fluorescein and tetramethyl

rhodamine were employed to detect wild-type and mutant genes selectively.

With the continuing progress of research, many derivatives of molecular

beacons have been developed, such as super-quencher molecular beacons,73

quencher-free molecular beacons,74 and stemless molecular beacons.75 All of

the derivatization strategies are used to increase the sensitivity and specificity

of SNP genotyping.

Y-shaped junction probes are specifically-designed DNA strands that form Y-

shaped junctions upon folding. Based on a template-enhanced hybridization

process, Y-shaped junction probes are utilized for SNP genotyping

isothermally. For example, Zhang et al. reported an electrochemical SNP

genotyping strategy for oral cancer employing the Y-shaped junction probes and

deoxyinosine nucleoside substitution on SNP sites. An LOD at 130 fM was

obtained by linear sweep voltammetry.76 Yeh et al. also developed an assay for

the detection of SNP based on the termed Chameleon NanoCluster beacon

(cNCB) probes. When a target DNA was exposed to the two segments of the

cNCB probes, the Y-shaped junction DNA structure was generated; while one-

base mismatched strand presented, a frame shift in one of the arms of the Y-

shape junction caused a color change of silver nanocluster, which can be

detected by fluorometry or by the naked eye.77

17

A toehold is a short, single-stranded, overhanging region in double-stranded

DNA (dsDNA).71 A fast strand-displacement reaction (SDR) occurs when a

totally-complementary oligonucleotide binds to the toehold with replacement of

the original short double strand by the oligonucleotide.78 Application of toehold

in SNP detection was first proposed by Zhang and his colleagues in 2010 using

a DNA-origami platform. The DNA origami was constructed in a way that it

contained a line of protruding linear capture probes, capable of forming toeholds

with biotinylated, partially complementary reporting probes. Streptavidin was

used as a contrast label to reveal the position of the reporting probes. When

target DNA was employed, SDR occurred and reflected by the disappearance

of the streptavidin feature on the DNA-origami platform.79 Later, based on

locked nucleic acid (LNA) and the toehold structure, Gao et al. developed and

ON-OFF switching SNP assay.80 The immobilized probes first hybridized with

methylene blue-tagged capture probes and the signal was ON. After introducing

the target DNA, the toehold on the capture probes initiate the SDR and switch

OFF the signal; while when the one-base mismatch presented, SDR was

forbidden and the signal is still ON. By replacing the normal nucleotide on

capture probes with LNA at the SNP site, the ability to discriminate SNP mutant

sequence significantly improved. The discrimination factor of the assay was as

high as 6000 and the LOD was found to be 58 pM.

A G-quadruplex is a four-stranded structure assembled from a guanine-rich

oligonucleotide.81 It is likely to coordinate with metal ions and can be bound by

hemin to form a peroxidase-like complex – DNAzyme.82 Kolpashchikov et al.

reported an SNP assay based on DNAzyme, which was separated into two

18

segments with four triple-guanine sequences split in a 2 + 2 pattern. In the

presence of a target DNA, the binding arms formed a DNAzyme with hemin for

the consequent DNAzyme-catalyzed colorimetric signal output.83 The

mismatch cannot induce the formation of DNAzyme and thus no signal was

produced. This concept of G-quadruplex was further developed by Wang and

his co-workers.84, 85 The performance of this type of SNP genotyping assay was

significantly improved by incorporating more complex procedures.

Even so, it remains a formidable task to detect SNPs selectively in the presence

of a large excess of the wild-type gene, which poses a considerable threat to the

target. After all, hybridization is thermodynamics driven. Much effort, like

powerful target amplification and/or signal amplification strategies fully

compatible with the allele-specific hybridization, has therefore been devoted to

overcome these problems. For example, Gao et al. introduced mismatch-

eliminating nucleases and used an electrochemical platform for signal

transduction as well as signal amplification. It was demonstrated that nucleases

are capable of eliminating all imperfectly-hybridized interfering DNA strands

as well as unhybridized probes. The hybridized SNP target-capture probe

duplexes can serve as the templates to guide the deposition of polyaniline

(PANI); and the deposited PANI then served as a signal generator in the

construction of an electrochemical biosensor for highly sensitive and selective

SNP genotyping. What needs attention here is that the nucleases are only used

to clean up, not for signal transduction and amplification. This should not be

confused with the allele-specific enzymatic techniques in the next section.

19

1.4.2.2 Allele-specific enzymatic techniques

Allele-specific enzymatic techniques rely on the discrimination and

amplification power of the utilized enzymes to achieve better selectivity and

sensitivity as well as lower LOD. Similar to the allele-specific hybridization-

based techniques, most of the allele-specific enzymatic techniques are also

performed on solid supports in order to separate enzymatic reaction products

conveniently from the reaction mixture and for the easy configuration of

multiplexed assays. It is also desirable that the enzymatic reactions be carried

out in a homogeneous or nearly homogeneous manner to have a good control

over the whole assay process and obtain a better repeatability.

Generally, this type of techniques is much better than that of their allele-specific

hybridization-based counterparts with respect to selectivity, which primarily

originates from the high selectivity of the enzymatic reactions and two

recognition events, i.e. the hybridization of the allele-specific probes and

common probes to adjacent positions with SNP targets. The major downsides

of the allele-specific enzymatic reaction-based SNP genotyping and

quantification techniques are their high cost and sometimes time-consuming

multistep protocols. This is the price needed to pay when superior sensitivity

and selectivity as well as lower LOD are pursued. Widely employed allele-

specific enzymatic techniques are enzymatic ligation, primer extension, and

enzymatic cleavage which are discussed in the following sections.

The principle of the allele-specific enzymatic ligation technique lies in the

covalent ligation of two adjacent probes that are connected at or near an SNP

20

site by a ligase. The ligase was first introduced to SNP genotyping by Landegren

et al. in 1988 and one-base mismatch was successfully detected by

fluorometry.86 This technique provided a novel and rapid way to discriminate

SNP in genomic DNA. In order to significantly enhance the sensitivity and

decrease the LOD, it was further developed through coupling to amplification

techniques like ligase chain reaction (LCR), and advanced materials like silver

nanoparticles (AgNPs) and AuNPs. For example, He et al. showed that

picomoles to nanomoles of DNA with single-base mismatches can be visualized

with a lateral flow strip biosensor by using AuNPs and hairpin oligonucleotides

with double-target DNA binding sequences87 or hairpin-functionalized

AuNPs.88 Since non-functionalized AuNPs used for the colorimetric detection

of DNA sequences was first reported by Li et al.,89 AuNPs were continuously

investigated and coupled to allele-specific enzymatic ligations to improve the

performance of SNP genotyping. Liu and colleagues designed an SNP chip

utilizing the optical properties of AuNPs, which were further promoted by silver

enhancement and a ligation reaction.90 When a target DNA was introduced,

capture probes on the chip and the probes on the AuNPs were ligated.

Discrimination by the naked eye or flatbed scanner was executed after the silver

enhancement and non-stringency wash. More recently, Gao et al. achieved real-

time PCR-like sensitivity and LOD in the detection of SNP by incorporating

AuNPs in LCR.91, 92 A nicked duplex was first formed when the two types of

AuNP-tagged capture probes were brought together by the target during

hybridization at 45 °C. The two capture probes that were fully complementary

to the target (template) can be covalently connected by ligation at the mutation

site, whereas the other capture probes remained unaffected because of the

21

mismatch at the mutation site. The subsequent denature process separate the

SNP target and the ligated strands, both of which served as templates for the

next round LCR. Exponential amplification of the ligation product can be

achieved and irreversible change of the color of the reaction mixture from wine

red to grey can be detect to confirm the existence of the target DNA down to 20

aM. Aside from AuNPs, AgNPs were also coupled to ligation-based technique

for the SNP genotyping and quantification. Erickson and his co-workers utilized

AgNPs as signal enhancers of SERS and by engaging ligase detection reaction

(LDR), SNPs in human KRAS oncogene were successfully identified.93, 94

Ordinary PCR utilizes primer pairs for extension and amplification of target

genes.95 However, to detect the existence of SNPs, primers with single-base

variations at the 3’ ends, deliberately designed at SNP sites, are engaged in an

allele-specific PCR so that the extension reaction of PCR occurs with only

perfectly matched sequences. The first successful application of SNP

genotyping by the allele-specific PCR was realized by Gibbs et al.,96 in which

a third primer (common primer) was introduced to identify the products with

different “allele-specific primers”. A high discrimination factor of 100 proved

the robustness and reliability of this technique. Another technique termed

single-base extension (SBE) was firstly proposed by Anderson et al.97 Primers

with their 3’ ends immediately adjacent to query sites of mutations were

designed to hybridize to target DNA. An SBE reaction occurred with the

introduction of four dideoxynucleotides tagged with distinctive reporting

molecules,98 which can be detected by various techniques like fluorometry99 and

matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

22

(MALDI-TOF MS).100 Afterwards, Steemers et al. proposed a whole-genome

genotyping technique using a two-color SBE reaction to score the SNPs on a

BeadArray platform.101 Target DNA was hybridized with primers pre-

immobilized on the beads and SBE reaction processed subsequently. Based on

the same concept, instead of polymeric beads, gold-coated magnetic NPs

functionalized with streptavidin (SA-GMNPs) were used for the immobilization

of the primers.102 After the SBE reaction, the GMNPs were loaded with

fluorophore-tagged SBE products and the SNP genotyping can be accomplished

by comparing the output color. The use of the GMNPs offers a very convenient

means for separating the SBE products from the reaction mixture, thus

alleviating the requirement for PCR-product purification and producing an

ultralow background.

Restriction endonucleases are a class of enzymes that specifically recognize and

cleave certain DNA sequences.103 In contrast, they are unable to cleave their

targets if their recognition regions contain SNPs or other types of mutations.104

Leveraging on this property, SNPs within the recognition regions of the

restriction endonucleases can be detected. Invader assay is a SNP genotyping

technique using a restriction endonuclease towards the DNA triplex among a

target DNA strand, an allele specific probe and an invader probe.105 If the allele

specific probe perfectly matches the target DNA, the restriction endonuclease

cleaves off the invader probe from the DNA triplex; while the triplex containing

an imperfectly matched DNA strand, the invader probe in the triplex cannot be

cleaved off. SNP genotyping is realized through the fluorescent tag on the

released invader probe. Further to this concept, Hall and colleagues developed

23

an advanced version of the invader assay in which two invasive reactions were

used for the amplification of the target DNA.106 Consequently, the

amplification power of the invader assay was enhanced by three orders of

magnitude, i.e. from 104 to 107.

In order to achieve the desired sensitivity and LOD, the allele-specific

enzymatic reaction-based techniques, for example LCR and invader assays, rely

on enzymatic amplifications of the targets. Since these assays normally engage

several enzymes (such as ligase and restriction nucleases) and signal generating

tags (such as AuNPs and fluorophores) incorporated in the amplification

products, it remains to be seen whether they are able to outperform PCR-based

techniques in terms of speed, cost, LOD, sensitivity and specificity.

1.4.2.3 Sequencing techniques

The above mentioned two techniques are based on known SNPs (sequences) in

order to specifically design the SNP probes with certain sequences to achieve

the allele-specific discrimination, thus implying that these SNP genotyping and

quantification techniques are not applicable for genotyping and quantification

of new or unknown SNPs. In contrast, sequencing techniques are a group of

powerful tools in the discovery of new SNPs.

The gold standard of DNA sequencing is the Sanger sequencing technique,

which is a chain-termination-based method. It was widely and almost

exclusively adopted during the early stage of genomic research. The most

successful application of Sanger sequencing technique is the Human Genome

24

Project, during which the first collection of SNPs was actually accomplished by

Sanger sequencing. Unfortunately, the main drawbacks of this technique are its

low throughput and formidable high cost, which hinder its further applications

in diagnosis and prognosis in our everyday life.

In order to overcome the drawbacks of the traditional Sanger sequencing

technique, various next-generation sequencing (NGS) techniques sprang up

around the world with the advent of microfabrication techniques.

Representative NGS techniques that have been developed include

pyrosequencing which is the first commercialized NGS technique,107 single

molecule real-time (SMRT) sequencing,108 Illumina,109 supported

oligonucleotide ligation and detection (SOLiD),110 and Ion Torrent which is the

latest entry of the NGS family.111 These NGS techniques dramatically increased

the throughput and offer the possibility to genotype unknown SNPs at large

scales.112, 113 Further detailed discussion on this type of techniques can be found

involved in a published review article114 and will not be given here because

sequencing techniques for genotyping unknown SNPs is beyond the scope of

this thesis. Nonetheless, this does not deny the significance of the NGS

techniques. On the contrary, NGS techniques will provide the basis for

developing SNP bioassays since it makes the unknown SNPs in DNA to be

known and then let the other SNP genotyping and quantification techniques

follow up. Thus, NGS techniques surely hold their irreplaceable positions in

genomic research despite some of them are still expensive and time-consuming

to some extent. The other two categories of techniques can be chosen once the

25

SNPs are identified by sequencing. In this way, it will be much more economic

both in time and financial resources for SNP genotyping and quantification.

1.4.3 Detection techniques developed in this thesis

In Chapter 2 of the thesis, an allele-specific enzymatic technique for SNP

genotyping and quantification is demonstrated. By employing nucleases for

LCR amplification, the bioassay exhibited excellent sensitivity and selectivity

for genotyping and quantification of the disease-related SNPs.

1.5 Detection of abnormal expression levels of miRNAs

1.5.1 Difficulties in the detection of miRNAs

Precise discrimination and quantification of miRNA with a specific sequence

that has high clinical relevance to disease is essential to human health. On one

hand, it is necessary to investigate the regulatory functions of miRNAs and

further the drug discovery. On the other hand, it is critical for early diagnosis,

prognosis, and in-time therapy. Nonetheless, unlike DNA and message RNA

(mRNA), the unique attributes of miRNAs, such as their short lengths, low

abundance, and high sequence similarities among members of a miRNA family,

make miRNA detection with high sensitivity and specificity technically

challenging and demanding.115

The short lengths of target miRNAs bring difficulty to the hybridization-based

detection since the melting temperature of the duplex formed between a probe

and a target miRNA is low, which reduces the stringency of hybridization and

is more prone to induce imperfect match and subsequently a false response.

26

Moreover, the short lengths of miRNAs will also obstruct the using of general

PCR-based assays since the primers in PCR have similar sizes with miRNAs.

In order to carry out the PCR-based assays, much shorter primers are desired.

However, shorter primers have quite low melting temperatures and low

specificity, which will severely affect the efficiency and specificity of PCR as

well as the assay.116

The concern of low abundance of miRNAs demands that the detection

techniques of miRNAs should be able to obtain low background signals (high

S/N ratios), high sensitivity and low LOD; otherwise a minute amount of target

miRNA cannot be assuredly identified and quantified.

As for the issue of high sequence similarities among miRNA family members,

it needs to be addressed by designing and fabricating assays with high

selectivity so that different miRNAs with down to one-base difference can be

undoubtedly distinguished.

1.5.2 Developed techniques for miRNA detection

Up to the present moment, various classical techniques for miRNA detection

including Northern Blotting and quantitative reverse transcription PCR (qRT-

PCR), as well as recently emerged techniques including PCR-free molecular

biology-based techniques and nanotechnology-based techniques have been

developed for the detection of disease-related miRNAs.117

27

1.5.2.1 Classical techniques for miRNA detection

Northern blotting was developed by Alwine and colleagues in 1977 at Stanford

University.118 It employs electrophoresis to separate RNA by size, and then

transfer the separated RNA to a membrane (the actual northern blotting step),

followed by the hybridization with detection probes and final detection.

Northern blotting was the primary choice for the investigation of miRNAs

during the early days.119, 120 It was widely adopted because it is suitable to detect

miRNAs with different sizes121, 122 and has provided significant values in studies

that how miRNAs play crucial roles in the gene regulation. However, the

laborious procedure, time-consuming approach, large sample-amount demand,

toxic chemical involvement, low throughput, and inferior sensitivity of

Northern blotting motivated scientists to explore better alternatives.123-125

Figure 1-3. Unique reverse transcription strategies for miRNA to generate

cDNA. (Reprinted with permission from reference 123. Copyright 2013

American Chemical Society)

As the gold standard for profiling gene expression, qRT-PCR is commonly used

for the detection of abnormal expressed disease-related miRNAs. The reverse

transcription refers to transcribing RNA to its complementary DNA (cDNA),

which then undergoes the general PCR procedure. As mentioned above,

miRNAs are so short that it is difficult to find proper primers and then go

28

through the reverse transcription. Thus, as can be seen from Figure 1-3, unique

reverse transcription methodologies,115, 123, 126 including the widely used

poly(A) polymerase extension method, and stem-loop primer/liner specific

primer extension method are used to successfully generate a cDNA with proper

length for the subsequent PCR. The qRT-PCR techniques involving TaqMan

probes or SYBR green as signal generators have achieved excellent

performance, and corresponding assays have already been commercialized.

Nonetheless, qRT-PCR is even more complicated than the ordinary PCR. It is

more expensive, time-consuming and labor-intensive, and technically

demanding since specialists with professional knowledge and skills are

necessary. Besides, the whole process needs to be operated cautiously

considering that any minor contamination may lead to large variations after the

PCR amplification.

1.5.2.2 Recently emerged techniques for miRNA detection

Due to the limitations of the existing classical techniques mentioned above, and

meanwhile the urgent demand of establishing high-performance bioassays to

profile disease-related miRNA expression, several novel techniques such as

PCR-free molecular biology methodology and advanced nanotechnology have

been introduced to miRNA bioassays.

In recent years, PCR-free molecular biology methodologies like LCR, 127, 128

rolling circle amplification (RCA), 129, 130 hybridization chain reaction (HCR),

131, 132 and duplex-specific nuclease (DSN)-cleavage reaction133, 134 have not

been successively developed. The common property among these

29

methodologies is the signal amplification ability which is realized by target

reusing and recycling, so that good sensitivity and low LOD can be achieved.

For example, Zhang et al. described a sensitive and specific method to quantify

multiple miRNAs simultaneously by using ligation and LCR techniques

together. The ligation of DNA probes were first completed in the presence of

target miRNAs (templates), and subsequently the ligated DNA sequences

instead of target miRNAs underwent LCR for the signal amplification.

Electrophoresis was used for the final detection and the LOD was pushed down

to 0.2 fM.128 The thermal technique LCR can achieve outstanding sensitivity

and selectivity as well as ultralow LOD and it has already gotten one step closer

to point-of-care since it is much simplified compared with PCR. Nevertheless,

the main drawback of LCR is the need of a thermal cycler which is not preferred

at point-of-care.

HCR is an enzyme-free and isothermal technique that can circumvent the

thermal cycler. Yang et al. developed a graphene oxide (GO)-based fluorescent

bioassay for sensitive detection of miRNA coupled with HCR.131 In the

presence of a target miRNA, HCR was initiated and two species of fluorescent

hairpin probes were triggered to form DNA duplex products. All the original

probes and final products were then anchored on GO and only the fluorescence

of the upright stood DNA duplexes were quenched by GO and thus their

fluorescence was detected. The HCR-based miRNA bioassays are cost-effective

and can achieve a good sensitivity as well as low LOD, but the selectivity is not

ideal considering that it is thermodynamically driven.

30

Recently, Yin et al. engaged a DSN cleavage-based technique coupled with

Taqman probes to sensitively and selectively detect miRNAs.133 The Taqman

probes were cleaved by DSN when strands of a target miRNA perfectly

hybridized to the probes, and the previously quenched fluorophores were then

released and emitted a detectable fluorescent signal. Compared with LCR, DSN

cleavage reaction is more attractive since it is isothermal and completely

removes the requirement of a thermal cycler, but still can realize the target

recycling and signal amplification. And compared with HCR, it employs the

enzyme DSN that possesses excellent sequence-distinguish ability and thus can

acquire superior selectivity. Therefore, DSN cleavage-based techniques has a

good prospect regarding disease-related miRNA detection.

During the past two decades, nanotechnology has gained significant progress

and numerous nanomaterials are prepared for scientific research and

applications. Nanomaterials, especially the zero dimensional nanoparticles and

one dimensional nanowires, exhibited attractive properties for uses in miRNA

bioassays. Firstly, due to their nanosize comparable to nucleic acids,

nanomaterials can be engaged to miRNA detection in multiple ways, for

example acting as the probe carrier, tag, catalyst, signal generator, or quencher;

and ensure an even interaction with the miRNA. Secondly, the small dimension

also minimizes the whole size of the bioassay, which is beneficial to the

development of miRNA bioassays for uses at point-of care. Thirdly, different

nanomaterials have their own specific properties that can be utilized and thus

31

widen the scope for developing various nanotechnology-based miRNA

detection techniques.

For example, AuNPs have many attractive properties such as good water

dispensability, biocompatibility, catalytic ability, and an ultrahigh extinction

coefficient (108–109 M-1cm-1). A real-time colorimetric bioassay for the

detection of miRNA was established by making use the ultrahigh extinction

coefficient of the AuNPs.135 AuNP networks were first fabricated and the

presence of a target miRNA released the AuNPs and generated a colorimetric

signal. Ruthenium oxide nanoparticles (RuO2 NPs) were found to have good

peroxidase-like activity and thus can be used as an enzyme mimic for signal

generation and amplification in the detection of miRNA. Peng et al. used RuO2

NPs to tag miRNA as well as the enzymatic property to catalyze the

polymerization of 3,3’-dimethoxybenzidine in order to achieve the amplified

electrochemical detection of disease-related miRNAs.41 Other nanoparticles

like quantum dots (QDs)136 and carbon nanoparticles137 were also utilized to

fabricate bioassays for miRNA detection.

Proton doped PANI has a metal-like electronic conductivity and was thus

involved in electrochemical bioassays for miRNA. For instance, Fan et al.

demonstrated a nanogap-based technique for miRNA detection by employing

electron-conductive PANI nanowires. After hybridization to capture probes, the

target miRNA guided the deposition of PANI nanowires and thus signal

amplification as well as the signal output were achieved. The conductivity of

32

the deposited PANI was then used to indicate the amount of target miRNA in a

sample solution.138

1.5.3 Detection techniques developed in this thesis

In Chapter 3, one of the PCR-free molecular biology methodologies – DSN

cleavage-based technique – is presented for the detection of target miRNAs.

The engagement of DSN successfully realized target recycling and thus signal

amplification, and meanwhile achieved the discrimination of target miRNA

with specific sequence.

In Chapter 4 and 5, a nanotechnology-based technique is demonstrated for the

sensitive detection of miRNA. In Chapter 4, polymer nanowires were first

synthesized in aqueous solution through a novel, simple and green method. This

new synthetic method was then employed in Chapter 5 as the signal generating

process and signal amplifier of the bioassay for disease-related miRNA.

1.6 Purpose, significance and scope of this thesis

The purpose of the studies reported in this thesis is to overcome the

disadvantages of current bioassays and to develop novel, simple, cost-effective,

highly sensitive, and selective bioassays for the detection of disease-related

nucleic acids.

Considering the relatively inferior sensitivity and LOD of colorimetric

bioassays, a novel colorimetric bioassay for the detection of DNA with high

sensitivity and selectivity is established by coupling with advanced

nanomaterials. In Chapter 2, a very cost-effective nanomaterial − ferrofluidic

33

nanoparticles which are made of iron oxide − will be introduced to bioassays

for the genotyping and quantification of disease-related SNPs in DNA.

Regarding the complicated procedures and high cost of fluorescent

bioassays, a simple fluorescent bioassay evading PCR amplification procedure

for the detection of miRNA without losing good sensitivity is designed. In

Chapter 3, an enzyme-based isothermal signal amplification method could be

exploited instead of the tedious PCR and a much simplified fluorescent bioassay

for the detection of disease-related miRNA is explored.

Bearing in mind that the impedimetric bioassays always can achieve

excellent sensitivity and low LOD but are somewhat poor in repeatability, in

Chapter 4 and 5, a highly sensitive and reproducible impedimetric miRNA

bioassay on the basis of target-guided deposition of an insulating polymer is

developed for the disease-related miRNA profiling.

By overcoming the weaknesses and making full use of the strength of the

existing bioassays, newly developed bioassays extend the application range of

each method. They also open new perspectives in developing nucleic acid

detection tools for diagnosis and prognosis of genetic diseases. Once the

genotyping and profiling platform for disease-related nucleic acids is applied to

clinical practice, timely prevention and early treatment can thus be carried out

and the mortality rate of patients will be reduced.

The studies in this thesis focus on establishing two new optical bioassays and

one electrochemical bioassay since these two types of bioassays are among the

most promising bioassays for the detection of disease-related nucleic acids. The

34

mechanisms within the established bioassays are also explored and discussed in

each chapter.

35

Chapter 2. A Ferrofluid-Based Homogeneous Bioassay for Highly Sensitive

and Selective Detection of Single-Nucleotide Polymorphisms

36

2.1 Introduction

Nucleic acids provide the genomic templates1 that encode for proteins essential

for all cellular functions. Ample evidence has revealed that diseases like cancer

are closely associated with mutations in gene sequences and single-nucleotide

polymorphisms (SNPs) in particular.4 The importance of SNPs has urged

researchers to develop SNP detection methods for their sensitive and accurate

identification in the presence of excessive wild-type (WT) genes. By knowing

these SNPs, population screening and disease diagnosis can be designed and

performed accordingly. Therefore, SNPs have become an important class of

biomarkers in diagnosis after the completion of the Human Genome Project.

Unlike other DNA assays, the intrinsically subtle difference between WT and

mutant genes – a single base variation – makes it a challenging task to

specifically detect low abundant SNPs out of large amounts of co-existing WT

genes. Conventional techniques for SNP detections rely mostly on polymerase

chain reaction (PCR) and fluorescence detection. Unfortunately, this technique

is highly time-consuming and cost intensive as the fluorescence readout requires

expensive instrumentation and sophisticated algorithms.62 Owing to the desire

for SNP detection devices with high specificity, high sensitivity, low LOD and

low-cost operation, enormous efforts have been invested in research to improve

analytical performance of SNP assays.

With the advancement in nanotechnology over the past decade, materials with

dimensions in scale of 1 to 100 nm are playing a significant role in the

development of SNP assays.139 With unique size-dependent physical and

chemical properties such as high surface-to-volume, high surface energy,

37

surface plasmon resonance, and quantum effect, nanoparticles allow stable

immobilization of bioreceptors and being coupled to many new signal

transduction and amplification schemes.140, 141 To date, many heterogeneous

SNP screening assays using nanoparticles in conjunction with colorimetry,

fluorometry or electrochemical methods have been proposed.142 Heterogeneous

detection, however, has low hybridization efficiency due to steric hindrance and

limited number of probes coated on the solid support further restricts the

performance of the assays.142 To realize SNP assays that offer the simplicity of

the heterogeneous approach along with high hybridization efficiency and

reproducibility of homogeneous assay, magnetic nanoparticles (MNPs) were

used to facilitate homogeneous hybridization and provide simple and quick

separation of the hybridized products.143 With appropriate surface coating,

MNPs can be solubilized in water and provide functional moieties for

bioreceptors attachment to design the capture and/or detection probes. Spatial

hybridization between probes and target can also be achieved with the reaction

being performed in a homogeneous manner. The ease of magnetic separation is

frequently used to overcome the shortcoming of homogeneous ligation by

removing reacted probes from unreacted ones which would otherwise pose as a

threat to the detection.143

In view of the urgent need for simple and inexpensive SNP assays, this report

presented a novel SNP assay that for the first time exploited the intrinsic

properties of ferrofluid (colloidal MNPs). Efficient homogeneous hybridization

was carried out by oligonucleotide-coated ferrofluidic nanoparticle probes

(FNPs). The amplification power of the assay was demonstrated by the

38

incorporation of a ligase chain reaction (LCR) in which a minute amount of

target crosslink millions the FNPs together forming an aggregate of the FNPs.

Separation of the reacted (aggregate) from the unreacted (free) FNPs was

achieved simply based on their drastic difference in magnetic field strength

under an external magnetic field. Finally, SNP is conveniently genotyped and

quantified by colorimetry.

2.2 Materials and methods

2.2.1 Reagents and apparatus

Unless otherwise stated, reagents were obtained from Sigma-Aldrich (St Louis,

MO, USA) and used without further purification. 1-Ethyl-3-3-

dimethylaminopropyl carbodiimide hydrochloride (EDC) and N-

hydroxysuccinimide (NHS) were acquired from Pierce (Rockford, IL, USA).

Diethylene glycol (DEG) and tris (hydroxymethyl) aminomethane (Tris) were

from Alfa Aesar (Heysham, UK). Ampligase® thermal stable ligase kit was

brought from Epicenter Biotechnologies (Madison, WI, USA). Hydrogen

peroxide (QRec, 30–32%, AR) was obtained from ERC Sdn Bhd (Malaysia).

Dimethyl sulfoxide (DMSO) was gotten from MP Biomedicals LLC (Solon,

OH, USA). H3PO4 (85%) was provided by Fisher Scientific PTE LTD

(Singapore). HCl (37%) was bought from Schedelco Pte Ltd (Singapore).

NaOH (Chemi-con, 99–100%) was from Dickson Instrument & Reagent Store

(Singapore). Unmodified oligonucleotides were custom made by 1st Base

(Singapore) and the modified oligonucleotides were custom made by Integrated

DNA Technologies Incorporated (Coralville, IA, USA). All the reagents were

without further treatment unless specified. All aqueous solutions were freshly

39

prepared with nuclease-free ultrapure (UP) water with an electrical resistance

of 18.3 MΩ·cm. The pH of the Phosphate buffered saline (PBS) was adjusted

by adding an appropriate amount of NaOH solution or HCl solution.

UV-Vis absorption measurements were carried out using an Agilent Cary 60

UV-Vis spectrophotometer which was from Agilent Technologies Singapore

PRE LTD (Singapore). Fourier transforms infrared (FT-IR) spectroscopic

experiments were performed with a Bio-Rad Excalibur Series FTS 3000

spectrometer (Hercules, CA, USA) using KBr pallets and the spectra were

collected after 20 scans between 4000‒400 cm-1 at a resolution of 4 cm-1 for

characterization of poly acrylic acid (PAA) coated MNPs. Transmission

electron microscope (TEM) was conducted on FEI Tecnai F20 which is

provided by FEI Company (Hillsboro, OR, USA) using a 200kV acceleration.

Thermogravimetric analysis (TGA) was performed on SDT-2690 Simultaneous

DTA-TGA which is provided by TA Instruments (New Castle, DE, USA) from

room temperature to 800 ºС with a heating rate of 10 ºС /min under air

atmosphere.

2.2.2 Preparation of ferrofluidic MNP-PAA

The magnetic nanoparticles coated with poly acrylic acid (MNP-PAA) were

synthesized following the protocol by Ge et al.144 with slight modifications to

control the particle size. The NaOH/DEG stock solution was prepared by

dissolving 50 mmol NaOH in 10 mL DEG at 120 °C for 1 hr under argon before

cooling down and then kept at 70 °C. In the synthesis, 2 mmol FeCl3 was first

dissolved in 8 mL DEG before adding 4 mmol PAA. The mixture was heated to

40

220 °C under argon and vigorous stirred for 30 min until a transparent light-

yellow solution was obtained. A 1 mL of NaOH/DEG stock solution was then

injected rapidly into the above hot mixture and heated for another 10 min to

induce the formation of black solution containing MNP-PAA with desired size.

The final product was washed with repeating precipitation with ethanol and re-

dispersion in UP water for three times, and finally dispersed in 1 mL UP water

for further use.

2.2.3 Prepare ferrofluidic nanoparticle probes

The FNPs were prepared by functionalizing colloidal MNP-PAA with the

amine-terminated oligonucleotide 1 and 2 (in Table 2-1), respectively.

Carboxyl-terminated MNP-PAA were pretreated in 10 mL of 10 mM PBS (0.5

M NaCl, pH 5.5) containing fresh-prepared 3 mM EDC and 5 mM NHS for 1

hr at room temperature. The suspension was then washed three times with PBS

buffer to remove the excess reagents before addition of the oligonucleotides.

After gently shook at room temperature for 3 hrs, the oligonucleotide-attached

MNPs were obtained by centrifugation and the probe content was analyzed.

Generally, there were 20 oligonucleotides strands on the surface of each FNP.

The unreacted carboxyl groups on the MNP were blocked by incubating with 1

M glycine for 2 hrs. After thorough washing with PBS buffer, the FNPs were

kept at 4 °C and ready for use.

41

Table 2-1. Synthetic oligonucleotides (5'→3') used in the bioassay

Name Sequence

Oligonucleotide 1 NH2-TTT TTT TTT TT GCC TAC GCC AC-OH

Oligonucleotide 2 PO4-G AGC TCC AAC TAC CA TTT TTT

TTT-NH2

MT-C Half Target A TG GTA GTT GGA GCT C

MT-C Half Target B PO4-GT GGC GTA GGC AA

KRAS Wild-Type Target

(KRAS WT)

AAA CTT GTG GTA GTT GGA GCT G|GT

GGC GTA GGC AAG AGT GCC TTG

KRAS Mutant Target-C

(KRAS MT-C)

AAA CTT GTG GTA GTT GGA GCT C|GT

GGC GTA GGC AAG AGT GCC TTG

2.2.4 Buffer optimization for hybridization

Presence of cations and pH in the buffer would affect the stability of FNPs and

the hybridization efficiency with target DNA. To determine the stability of the

FNPs, the 90 nM FNP-1 and 90 nM FNP-2 and 15 nM KRAS MT-C target were

dispersed into a series of 1.6 mL 50 mM tris-HCl buffers varying the sodium

(Na+) salt concentrations (50 mM, 500 mM, 1000 mM, 1500 mM and 2000 mM)

and pH values (5.5, 6.5, 7.5, 8.5 and 9.5). The UV-Vis absorbance of the

unbound FNPs was observed at 260 nm before and after magnetic separation.

To determine the hybridization efficiency, the FNPs were incubated with the

totally complementary template, KRAS MT-C target, in the same series of

buffer at room temperature for 1.5 hrs. Similarly, the UV-Vis absorbance of

unbound FNPs was observed at 260 nm before and after magnetic separation.

2.2.5 TMB-based signal amplification

After separating out the hybridized FNPs by permanent magnet, a colorimetric

detection was performed subsequently on the unbound FNPs following the

protocol by Gao et al.145 The assay was carried out at room temperature in 1 mL

of reaction buffer (0.2 M HAc/NaAc, pH 3.5) containing 530 mM hydrogen

42

peroxide, 81.6 μM TMB and 40 μL of the remaining unbound FNPs. The

reactions were allowed to incubate at room temperature for 1 hr before

recording the UV-Vis absorbance of product at 652 nm.

2.2.6 Ligation chain reaction

To further improve the sensitivity and push down the LOD of assay, ligation

chain reaction (LCR) was employed to amplify the signal as previous report.143

Ampligase® thermal stable ligase was used to facilitate the exponential

amplification in the LCR. In a typical reaction, a 100 μl of reaction solution

containing 1 × Ampligase reaction buffer, 50 U Ampligase, 1.5 μM FNP-1, 1.5

μM FNP-2, 1.5 μM MT-C target half A, 1.5 μM MT-C target half B, and KRAS

MT-C target was subjected to 15 thermal cycles of LCR. Each thermal cycle

composed a 2-min ligation at 45 °C and a 1-min denaturation at 90°C. After the

thermal cycling, the ligated FNPs was dispersed in 900 μL of the optimized

hybridization buffer and separated by the magnet. Subsequently, TMB-based

signal amplification was performed on the unbound FNPs to determine the

original target. The calibration curve was depicted accordingly.

2.3 Results and discussion

2.3.1 Principle of the assay

As illustrated in Figure 2-1, the SNP assay comprises of three steps:

(i) capturing of target SNP strands via hybridization and LCR with the

FNPs to form a FNP aggregate

(ii) magnetic separation of the FNP aggregate from unreacted FNPs

43

(iii) amplified colorimetric detection of the unreacted FNPs through the

catalytic oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by the

FNPs.145

Figure 2-1. Schematic of (a) the working principle the ferrofluid-based

homogeneous assay for SNP (b) sequences of the FNPs and target DNA.

Highly water-soluble FNPs were prepared and discretely functionalized with

the two types of amine-terminated oligonucleotides (FNP-1 and FNP-2) via

amide coupling reaction with the carboxylic groups of PAA that encapsulated

the FNPs. The FNP-1 will align with FNP-2 when both of them are annealed

onto a target SNP template i.e. KRAS, a mutation (MT-C) as depicted in Figure

2-1. Amplification is attained by the LCR in which multiple hybridizations

44

occur among the FNPs with minute amount of the target to form the FNP

aggregate. Under an external magnetic field, each FNP will act as single

magnetic domain and when numerous FNPs aggregated during hybridization,

the large FNP aggregate behaves much more strongly towards the magnetic

field. Separation of the FNP aggregate from the unreacted FNPs is achieved by

using a permanent magnet to pull and pack the FNP aggregate to the bottom of

the reaction vessel.

To ensure the success of assay, it is crucial that there a substantial difference

between individual FNPs and the FNP aggregate so that the unreacted FNPs

stay in the reaction mixture when a magnetic field is applied through a

permanent magnet. In other words, the FNP aggregate should set out easily in a

magnetic field whereas the free FNPs remain in solution. It is well-known that

when MNPs are sufficiently small ( 10 nm) and each MNP is thoroughly

covered with an anti-clumping coating, the magnetic attraction of MNPs is weak

enough that the Van der Waals force of the anti-clumping coating is sufficient

to prevent magnetic clumping or agglomeration – forming a ferrofluid.146 It was

reported that poly acrylic acid (PAA) coated MNPs (6–8 nm) show ferrofluidic

behavior when the colloidal solution of the PAA coated MNPs is subjected to

an external magnetic field, due to their small size and superparamagnetic

properties.144 Therefore, the PAA coated MNPs were employed in this work.

Two types of the FNPs (FNP-1 and FNP-2) were prepared by functionalizing

the PAA coated MNPs with two types of oligonucleotide probes, respectively.

45

Figure 2-2. TEM image of the highly water soluble MNP-PAA prepared. The

magnetic nanoparticles were synthesized with a particle diameter around 6–8

nm.

As shown in Figure 2-2, TEM image showed that the diameter of MNP-PAA is

around 6–8 nm. To verify the MNP-PAA prepared, other characterizations

including TGA and FT-IR were also carried out. TGA curve in Figure 2-3

indicated that the main weight loss is around 300 ºС and 320 ºС, which should

be due to the dehydration and decarboxylation of PAA. The weight ratio of the

MNPs is about 58% and PAA is about 38%. The FT-IR spectrum in Figure 2-4

confirmed the existence of PAA on MNPs. Strong band at 588 cm-1 belongs to

the Fe-O vibration of the MNPs. Three peaks located at 1404, 1455 and 1526

cm-1 were assigned to symmetric C-O stretching of -COO- groups, asymmetric

C-O stretching of -COO- groups and CH2 bending mode of coated PAA.

46

Figure 2-3. TGA curve of the highly water soluble MNP-PAA prepared.

Figure 2-4. FT-IR spectrum of the highly water soluble MNP-PAA prepared.

Moreover, functionalization of the MNPs with anionic oligonucleotides further

stabilized the MNPs, forming a very stable ferrofluid (Figure 2-5 inset). Apart

from the formation of ferrofluid, another critical factor is the functionality of

47

the FNPs. The FNPs must be freely accessible to both the SNP target and ligase,

and particularly to the ligase as it is much bulkier than the SNP target. As can

be seen in Figure 2-1(b), the two amine-terminated capture probes were

therefore designed with a (T)9 spacer to eliminate any possible steric effect.

Furthermore, surface densities of the probes were kept low by controlling the

concentrations of the oligonucleotide probes. It was estimated that on average

20 oligonucleotide probes were attached onto each FNP.

Figure 2-5. UV-Vis spectra of TMB solution treated by 10 nM MNPs after

hybridizing with (1) control, (2) 2.0 and (3) 6.0 nM SNP target. Inset:

Photographs of the FNP in the solution without/with applying a permanent

magnet.

To demonstrate the accessibility of the immobilized probes on the FNPs, a

complementary KRAS MT-C target and a control were introduced to the FNPs,

respectively. As revealed in Figure 2-5, the control showed practically identical

UV-Vis responses before and after hybridization and magnetic separation, thus

suggesting that there is no FNP aggregate formation and the free FNPs remain

in solution even subjected to the permanent magnet. This is evidently due to

48

their ultra-small size and high dispensability after functionalization.144 On the

other hand, after hybridization with the KRAS MT-C target, a significant

change in absorbance was observed after magnetic separation, indicating that a

large amount of FNPs was removed by the magnet. The results suggest that the

functionalized FNPs are capable of capturing the SNP target and hybridizing

into a FNP aggregate that can be separated out by the permanent magnet.

2.3.2 Optimization of the conditions

To achieve highest possible analytical signal, there should be efficient

hybridization of FNPs with the SNP target to form the FNP aggregate that will

be first set out by the permanent magnet and leaving the unreacted FNPs in the

solution. One key factor that greatly influences the hybridization efficiency is

the presence of cations. In general, cations are added into the hybridization

buffer to compensate for the negatively-charged phosphate groups on the

oligonucleotide backbone and stabilize the hybridized duplexes.147 However, an

increase in ionic strength of the buffer often leads to colloid coagulation (self-

aggregation) as the double layer thickness decreases. Self-aggregation of the

FNPs would pose a threat in distinguishing the unreacted (free) and hybridized

(aggregate) FNPs. Furthermore, pH-induced conformational changes in the

oligonucleotide probes may hinder the accessibility and binding affinity of

target to the immobilized probes.148 Optimization of the hybridization buffer is,

therefore, a critical step to establish good stability of the FNPs and at the same

time promote hybridization with the SNP target.

49

Figure 2-6. Buffer optimization: effect of monovalent cation (Na+)

concentration ranging from 50 mM to 2.0 M on the stability and hybridization

efficiency of the FNPs. Curve marked with 2 is the sample and curve marked

with 1 is the control.

In this work, tris-based buffers were used to ensure that the hybridization

process will be solely influenced by the free cations coming from the added salts.

Monovalent salts were found to be much more efficient in duplex stabilization

in solution than divalent salts.147 Thus, the cationic shielding effect influencing

the stability and hybridization efficiency of the FNPs was studied with

concentration of the monovalent sodium in range of 50 mM to 2 M (Figure 2-

6). The negligible change in absorbance for FNPs in the absence of the SNP

target (trace 1) showed relatively good stability of the FNPs in the buffer. A

slight increase in A observed when sodium concentration increased above 1

M suggest that coagulation of the FNPs occurs due to the decrease in

electrostatic repulsion between FNPs as ionic strength of buffer was further

increased. Fortunately, such effect did not obstruct the hybridization process.

The change in absorbance for the FNPs after reacting with the SNP target was

50

significantly larger than that of the control (trace 2). This indicates that the FNPs

have hybridized with the SNP target to form the FNP aggregate which is then

separated out quickly from the solution by the magnet. Thus, the amount of free

FNPs in solution decreases substantially and leads to the significant change in

absorbance. It was also observed that the hybridization efficiency increases with

increasing concentration of sodium, a trend found in several other DNA sensing

systems.149, 150 On the other hand, a reverse effect started to show when the salt

concentration was increased beyond 1 M. Again, this could be due to the

decrease in electrostatic repulsion, causing the FNPs to aggregate and hinder

the hybridization process. Nonetheless, the hybridization was shown to be most

efficient at 1 M of sodium concentration.

The accessibility and binding affinity of target to the FNPs were studied in 1 M

of sodium buffers varying in pH range from 5.5 to 9.5 (Figure 2-7). The FNPs

remained considerably stable in the pH range studied (trace 1) while the

hybridization process was significantly affected by the acidity of the

hybridization buffer (trace 2). The largest response was observed at pH 7.5, thus

implying that there is minimal steric hindrance between neighboring FNPs and

the hybridization process was most efficient. At lower pH, protonation of the

base groups on phosphate backbone decreases electrostatic repulsion between

the FNPs and hindered the hybridization processes. Similarly, low hybridization

efficiency observed at higher pH could be due to extensive deprotonation of the

base such that the probes bend away from each other. Therefore, the optimal

conditions that allow efficient hybridization and differentiation of the

51

hybridized FNP aggregate from the unreacted FNPs were 1 M of sodium at pH

7.5.

Figure 2-7. Buffer optimization: effect of pH ranging from 5.5 to 9.5 on the

stability and hybridization efficiency of the FNPs. Curve marked with 2 is the

sample and curve marked with 1 is the control.

2.3.3 LOD of the assay

With this distinct change associated with the SNP target and the catalytic

properties of the FNPs toward the oxidation of TMB,145 additional experiments

were conducted to establish the relationship between the colorimetric signal and

the target concentration. It was found that the FNPs can be directly utilized for

the detection of SNPs in the range of 1.0 to 90 nM. However, such an LOD is

far below the requirement for SNP detection and an in situ amplification

mechanism is therefore inevitable to make this assay more appealing.

To improve the assay sensitivity and obtain a lower LOD, the minute amount

of target captured by the FNPs has to be amplified and this could be fulfilled by

performing PCR before hybridization. However, the cost and time consumed

52

would gradually increase for such multiple-steps assay despite the exceptional

amplifying power of PCR. An alternative way is to employ LCR. This allele-

specific enzymatic ligation provides similar amplification power as PCR and is

able to amplify the target and ligate the MNP probes simultaneously under

specific thermal cycling conditions.93, 143 More importantly, through judicious

design of the two probes with SNPs located at the ligation sites, LCR is much

more specific than PCR for SNPs genotyping.91 To significantly improve the

sensitivity and decrease the LOD of the assay, LCR was therefore utilized to

amplify minute amounts of the target and ligate the FNPs to form the FNP

aggregate.

Figure 2-8. Calibration curves () for the response of unbound probe-catalyzed

colorimetric TMB oxidation in the presence of target KRAS MT-C after LCR

(15-thermal cycles). Femtomolar samples are marked with (). Error bars stand

for the standard deviations in the measurements.

53

Figure 2-9. UV-Vis absorbance spectra of the unbound probe-catalyzed

colorimetric TMB oxidation when 0, 3, 8 16, 25 and 35 pM SNP target present

in the assay.

As can be seen in Figure 2-8, a calibration curves for the response of unbound

probe-catalyzed colorimetric TMB oxidation in the presence of target KRAS

MT-C after 15-thermal cycles LCR was depicted with a dynamic range from 0

to 35 pM. A linear coefficient 0.99 validated that this assay is reliable for SNP

discrimination. The corresponding UV-Vis absorbance spectra of each point on

the calibration curve were shown in Figure 2-9, and the assay was shown to be

able to distinguish the SNP target at concentration as low as 3 pM after only 15

thermal cycles. This LOD was three orders of magnitude lower than that without

target amplification and comparable with the best colorimetric SNP assays.

Moreover, femtomolars of SNP targets were detected when extensive thermal

cycling (up to 20 thermal cycles) was performed. As indicated in Figure 2-8,

two red points in the calibration curve referred to that the concentrations of

targets was 300 fM and 600 fM before LCR, and after 5 thermal cycles of LCR,

54

the concentrations of the targets were theoretically increased to 9.6 pM and 19.2

pM respectively. Thereafter, they were subjected to the same experimental

procedure as the samples for calibration curve above-mentioned. Since it was

found that the final signals detected can be fit to the calibration curve, the

femtomolar LOD was regarded as achieved.

2.3.4 SNP discrimination of the assay

Figure 2-10. SNP discrimination of the assay with large excess mismatch

sequences. Comparing to the control set (MT-to-WT = 1:0), no significant

change in signal was observed even when the WT concentration was 1000 times

higher. Similarly, SNP detection was carried out with KRAS mutation-to-wild

type target (MT-to-WT) ratios up to 1:1000. 15.6 pM MT-C target was used.

The capability of the FNPs to discriminate mutants is particularly important in

SNP genotyping. To determine the ability of selectively detecting the SNP

target in sample matrix containing large excess mismatch sequences, SNP

detection was carried out with KRAS mutation-to-wild type target (MT-to-WT)

ratios ranging from 1:0 to 1:1000. Comparing to the control set (MT-to-WT =

55

1:0), no significant change in signal was observed even when the WT

concentration was 1000 times higher (Figure 2-10). This implies that the

nonspecific binding of mismatch sequence to the FNPs is negligible. The

excellence analytical performance of this assay strongly supports that the

specific-designed FNPs are highly sensitive and selective in the screening for

SNP target even in complex matrixes.

Table 2-2. Comparison of several literature methods for SNP detection

Method LOD SNP

Discrimination

Homo/

Heterogeneous Detection Ref.

LCR+Magnetic

separation+Catalytic

amplification

300

fM 1000:1 Homogeneous UV-Vis

This

work

Target-recycled

ligation+MNP

capture+Enzymatic

amplification

600

fM 1000:7.5 Homogeneous Fluorescence 141

LCR+Magnetic beads

separation+Enzymatic

amplification

30

pM 1000:1 Homogeneous UV-Vis 143

LCR+Gold nanoparticle 20

aM 1500:1 Homogeneous UV-Vis 92

Ligation mediated strand

displacement+DNAzyme

amplification

0.1

fM 1:1a Homogeneous Fluorescence 85

Graphene Oxide absorption

capacity+Single base

extension

3 nM 10:1 Homogeneous Fluorescence 99

Circular strand-displacement

amplification+Magnetic

beads separation

0.13

fM 400:1 Heterogeneous CL 151

Ligase+Molecular beacon 1 pM 10:1 Heterogeneous Cyclic

Voltammetry 152

a Only single base mismatch target as control was provided.

56

The proposed bioassay here for SNP genotyping and quantification was also

compared with its counterparts reported previously. As summarized in Table 2-

2, compared to the previously published SNP discrimination method, the

present work exhibited its advantageous LOD as well as SNP discrimination

ability.

2.4 Conclusion

In conclusion, the proposed assay revealed the versatility and power of FNPs as

the target capturer and signal generator for SNP genotyping and quantification.

The assay was able to discriminate one copy of the SNP target in 1000 copies

of WT gene. The demonstrated attractive characteristics of simple, rapid and

low cost colorimetric SNP genotyping could therefore open a new perspective

in the development of SNP genotyping tools for uses at point-of-care. Ongoing

efforts may apply this assay to screen for SNPs in real samples and expand its

capability to the detection of other nucleic acids such as methylated DNAs and

microRNAs.

57

Chapter 3. A Simple and Highly Sensitive Fluorescence Assay for

MicroRNAs

58

3.1 Introduction

MicroRNAs are highly conserved17 small RNAs that regulate the expression of

genes by binding to the 3’-untranslated regions of messenger RNAs (mRNAs)

through a dual-mechanism: translational repression and target degradation.3, 153

A representative family of miRNAs is the lethal-7 (let-7), the first known human

miRNA family that has ten mature sequences in humans, i.e. let-7a, 7b, 7c, 7d,

7e, 7f, 7g, 7i, mir98 and mir202.154 They were discovered for the first time in

the nematode as crucial developmental regulators.155 It has been observed that

numerous diseases such as various types of cancer like lung cancer and breast

cancer, metabolic syndromes like diabetes,10 are closely associated with

abnormal expression levels of miRNAs. Increasing evidence has suggested that

miRNAs are potentially diagnostic as well as prognostic biomarkers. For

example, several studies have indicated that the expression levels of let-7

miRNAs are frequently lower in tumor tissues than those in normal tissues.156

Removal of let-7 binding site on mRNA causes over expression of protein and

produce tumor157 since let-7 miRNAs acting as tumor suppressors can no longer

function. As for the metabolic syndromes, an essential role of let-7 family plays

is that it regulates glucose metabolism in multiple organs. Over expression of

let-7 in pancreas will lead to impaired glucose tolerance and reduced pancreatic

insulin secretion, which may result in diabetes and ectopic fat deposition in the

liver. Studies have indicated that let-7 inhibition not only prevents from diabetes

and fatty liver but also provides a new therapy to reverse and cure them.158

Considering that the alteration of miRNA expression level is closely related to

the diseases as well as the survival of patients,159 the techniques that are capable

59

of profiling the expressions of miRNAs are important and urgently demanded

as early diagnosis could help to reduce the mortality rate of patients. Fortunately,

coupled with their regulatory roles, miRNAs are an extremely stable form of

nucleic acids which are immune to endogenous RNase degradation.160 Besides,

they can be easily obtained through body fluids such as blood and the levels of

miRNAs in blood are stable, reproducible and consistent among individuals of

the same species,161 hence making miRNAs ideal biomarkers for diagnosis and

prognosis. However, miRNAs are generally in short length, low cellular

abundance and there is an extremely high level of homology of down to one

base difference within a miRNA family. These characteristics of miRNAs make

their detection challenging as the detection method has to be both highly

sensitive and selective. Currently, there are already several techniques for

miRNA detection such as conventional Northern blotting,119, 162 microarray,163

(RCA),164 and quantitative polymerase chain reaction (qPCR).165 All these

techniques have made great contributions to miRNA research in the early days

and some are still popular tools in miRNA research. Nonetheless, each of them

has its own limitations like tedious procedure, high cost, time consuming,

inferior selectivity, or poor sensitivity and LOD. For example, Northern blotting,

whereby the detection of isolated RNAs allows the study of the level of gene

expression in cells and also size determination, is the most established nucleic

acid assay and still considered as the “gold standard” of miRNA expression

analysis. However, its poor sensitivity and LOD in conjunction with its

inherently tedious procedure and long assay time makes Northern blotting non-

ideal for routine miRNA expression profiling.

60

More recently, several homogeneous amplification strategies for miRNAs that

mainly rely on target recycling amplification to improve the sensitivities and

obtain lower LODs of miRNA assays have been developed.133, 135, 166-169 Unlike

heterogeneous assays which intrinsically suffer from poor reproducibility, the

homogeneous miRNA assays are expected to greatly improve the

reproducibility and simplify the assay procedure. Among them, the use of a

duplex-specific nuclease (DSN) to amplify miRNA hybridization events

appeared very attractive since the amplification process was carried out

isothermally. As compared to other commonly used enzymatic amplification

methods such as qPCR and ligase chain reaction (LCR)170 utilizing polymerase

and ligase respectively, the isothermal miRNA amplification realized by DSN

completely removes the requirement of a thermal cycler.171 This greatly

simplifies point-of-care diagnostics. It also implies that the assays are easier to

use, cost effective and more tolerant to inhibitory components from crude

samples. Ye’s group133 firstly utilized the DSN signal amplification strategy

coupled with the Taqman probes to sensitively detect miRNAs. Soon after, this

DSN amplification strategy has been further developed by other researchers.

For instance, Lin et al.172 employed backbone-modified molecular beacons for

miRNA detection with reasonably good sensitivity and satisfactory selectivity.

Despite achieving an LOD of 0.5 pM within 40 min and the advantages with

the use of molecular beacons, it also has its limitations such as possible false

positive signals due to the co-existing of the unhybridized molecular beacons.

Moreover, the molecular beacons needed to be modified on their backbones,

making the preparing steps more complex and costly. In another report, Deng

and co-workers173 proposed a dual-amplification strategy by making use of

61

DNAzyme and achieved excellent sensitivity, LOD and selectivity.

Unfortunately, dual amplification also means one more catalytic step before

final detection, making it somehow cumbersome. Degliangeli and his

colleagues134 recently published a miRNA quantification method by using

DNA-gold nanoparticle conjugates as detection probes. Nevertheless, the LOD

of the assay – 5.0 pM after 5 hrs incubation – is not entirely satisfactory as

compared to other miRNA assays. Meanwhile, a more sensitive miRNA assay

was reported by Xi et al.174 Through exploiting the differential affinity of WS2

nanosheets toward short and long oligonucleotides and their efficient

fluorescence-quenching ability, a miRNA assay was developed. The assay was

highly sensitive and selective with an LOD of 300 fM. Nonetheless, simple,

rapid, low cost and ease-to-use miRNA assays with high sensitivity and

selectivity as well as low LOD suitable for uses at point-of-care are still in great

demand. To significantly enhance the sensitivity, decrease the LOD and

simplify the assay procedure, in this report, we proposed a simple, rapid, highly

sensitive and selective homogeneous miRNA assay that couples magnetic beads

(MBs) with the DSN for background reduction and signal amplification. By

conveniently and completely removing unreacted detection probes, minute

changes in fluorescence intensity of the analyzed solution could be

unmistakably detected, thereby producing an LOD of 60 fM and a very broad

linear dynamic range from 100 fM to 2 nM.

62

3.2 Materials and methods

3.2.1 Reagents and apparatus

The DNA detection probes, synthetic miRNAs, and all other oligonucleotides

were custom-made by Integrated DNA Technologies and their respective

sequences are listed in Table 3-1.

Table 3-1. Sequences of synthetic oligonucleotides used in this bioassay

Name Sequence (5’- 3’)

Probe 5’-/5Biosg/T9 AAC TAT ACA ACC TAC TAC CTC

AT9/36-FAM/-3’

Let-7a (miRNA) 5’-rUrGrA rGrGrU rArGrU rArGrG rUrUrG rUrArU

rArGrU rU-3’

Let-7f (miRNA) 5’-rUrGrA rGrGrU rArGrU rArGrA rUrUrG rUrArU

rArGrU rU-3’

Let-7d (miRNA) 5’-rArGrA rGrGrU rArGrU rArGrG rUrUrG rCrArU

rArGrU rU-3’

dT21 5’-TTT TTT TTT TTT TTT TTT TTT-3’

The DSN was purchased from Evrogen through Genomax (Singapore).

Dynabeads (M-280 streptavidin-coated), monodispersed with < 5% batch-to-

batch variations, were bought from Life Technologies (Singapore), and their

TEM images were shown in Figure 3-1.

Ethylenediaminetetraacetic acid (EDTA), disodium salt was obtained from 1st

Base. The composition of buffer solutions used in this work, including 1 ×, 2 ×

binding and washing (B&W) buffer, and hybridization buffer, was listed in

Table 3-2.

63

Figure 3-1. TEM images of the streptavidin-coated magnetic beads (Dynabeads

M-280) with diameters of 2.8 µm.

Table 3-2. Composition of all buffers used in this bioassay

Buffer Composition

1× Binding and Washing Buffer

(1× B&W)

5.0 mM Tris-HCl, 0.5 mM EDTA, 1.0 M NaCl,

pH 7.5

2× Binding and Washing Buffer

(2× B&W)

10 mM Tris-HCl, 1.0 mM EDTA, 2.0 M NaCl, pH

7.5

Hybridization Buffer

(for Mg2+ Optimization)

50 mM Tris-HCl, pH 7.54:

Vary concentration of MgCl2 in buffer from 5.0 to

40 mM

Hybridization Buffer

(for pH Optimization)

50 mM Tris-HCl, 25 mM MgCl2:

Vary pH of buffer from 6.5 to 9.0

Optimized Hybridization

Buffer 50 mM Tris-HCl, 25 mM MgCl2, pH 8.0

All solutions were prepared using nuclease-free UP water. The pH values of all

buffer solutions were adjusted with appropriate amounts of HCl or NaOH.

Nuclease-free centrifuge tubes (0.65 mL and 2.0 mL) were purchased from

Scientific Specialties Incorporated (Lodi, CA, USA). Microfuge tubes (0.2 mL)

were from Thermo Fisher Scientific Incorporated (Waltham, MA, USA) and

were certified free of DNase, RNase, human and genomic DNA contaminations.

64

Fluorescence measurements were carried out on a Nanodrop 3300 from Thermo

Fisher Scientific Incorporated. All fluorescence data were collected at 519 nm

as the fluorescein-capped DNA detection probes have the maximum emission

wavelength of 519 nm. The excitation wavelength for fluorescein was 495 nm.

3.2.2 Preparation and quantification of DNA detection probe-MB

conjugates

The conjugation of the fluorescein-capped DNA detection probes to the

streptavidin-coated MBs was performed according to the recommended

procedure. The feeding ratio between the MBs and fluorescein-capped DNA

detection probes was estimated based on the information provided in the user

manual of Dynabeads M-280 streptavidin MBs. First, a desired volume of M-

280 streptavidin MBs was placed into a centrifuge tube and the solvent was

discarded with the help of a permanent magnet. After three careful washings

with the 1 × B&W buffer, the MBs were dispersed to the 2 × B&W buffer and

a required amount of dually tagged (biotin at 5’ end and fluorescein at 3’ end)

DNA detection probes was added. The mixture was gently vortexed for 15 min

at room temperature to ensure the completion of the interaction between biotin

and streptavidin. The supernatant was then separated out; its fluorescent

intensity was determined and was used to estimate the coupling efficiency of

the DNA detection probes to the MBs. Finally, the DNA detection probe-MB

conjugates were washed three times with the hybridization buffer, dispersed in

the hybridization buffer, and stored at 4 ºС for later use. It was estimated that

4.1 × 105 DNA detection probes were coupled onto each MB, which occupied

19% of the total capacity of the MBs, leaving enough space for target miRNA

65

hybridization and DSN cleavage. All the fluorescein-related steps were

conducted in aluminum foil-wrapped centrifuge tubes to prevent from the

exposure of the content to illumination which may adversely affect the

fluorescence properties of the fluorescein tags.175

3.2.3 Optimization of experimental conditions and miRNA detection

All experimental conditions, including the dosage of the DSN, concentration of

Mg2+, pH of buffer solution, incubation temperature, and incubation time were

optimized before the detection of the target miRNAs. The concentration of the

DNA detection probes used in optimization experiments was 100 nM and the

concentration of the target miRNA was 2.0 nM. For the target miRNA detection,

30 µL of 100 nM probes were used in the assay with varying concentration of

the target miRNA. An appropriate amount of the DSN was added to each

microfuge tube followed by vortexing to thoroughly mix the reaction mixture.

After incubation, the DSN cleavage process was stopped when the MBs

together with unreacted DNA detection probes were separated out by the

permanent magnet. The supernatant then underwent fluorescence measurement.

The selectivity of the assay was studied by comparing the responses of the target

miRNA (let-7a) with both one and two-base mismatched miRNA in the let-7

family, namely let-7f and let-7d, respectively. The concentrations of let-7f, and

let-7d were all kept at 2.0 nM and the comparison was conducted under

optimized conditions.

66

3.2.4 Analysis of miRNA in total RNA extracted from cultured cells

The celles were cultured under the following culture condition: 5% CO2/95%

air, 37 ºС. The growth medium for each cell line is as follows.

HeLa: ATCC-formulated Eagle's Minimum Essential Medium (Catalog No. 30-

2003) + 1% (vol/vol) penicillin-streptomycin + 10% (vol/vol) fetal bovine

serum (FBS). H1299 (Lung cancer cell): ATCC-formulated RPMI-1640

Medium (Catalog No. 30-2001) + 1% (vol/vol) penicillin-streptomycin + 10%

(vol/vol) FBS. MRC-5 (Normal cell): ATCC-formulated Eagle's Minimum

Essential Medium (Catalog No. 30-2003) + 1% (vol/vol) penicillin-

streptomycin + 10% (vol/vol) FBS.

First, remove and discard the culture medium. Second, rinse the cell with 0.25%

(w/v) Typsin-0.53mM EDTA solution. Add 3 mL of Trypsin-EDTA solution to

75 mL flask and observe cells under an inverted microscope until cells are

dispersed (~ 10 min). Then, add 7 mL of growth medium and aspirate cells by

gently pipetting. Lastly, transfer aliquots of the cell suspension to new flask and

incubate at 37 ºС.176-178

Total RNA of cultured cells was extracted using a miRNeasy RNA extraction

kit (Qiagen V.V., Hilden, Germany) according to the recommended procedure

and miRNAs in the total RNA were enriched by using a clean-up kit. The yield,

quality, and concentration of total RNA were routinely examined by UV-Vis

spectrophotometry. As a model miRNA, let-7a detection in the extracted total

RNA was performed. Reference values of let-7a were obtained by qPCR.

67

3.3 Results and discussion

3.3.1 Assay principle

DSN is an endonuclease isolated from the hepatiopancreas of Paralithodes

camtschaticus (the red king crab) Among single-stranded DNAs, DNA

homoduplexes, single-stranded RNAs, RNA homoduplexes, DNA-RNA

heteroduplexes, the DSN displays a strong preference for cleaving DNA

homoduplexes and DNA in DNA-RNA heteroduplexes, i.e. DSN is only active

towards DNA strands in duplexes particularly. Moreover, the DSN is able to

discriminate imperfectly matched duplexes from perfectly matched ones.179

These unique attributes offer new possibilities for the development of

ultrasensitive nucleic acid assays and are the basis of the proposed miRNA

assay. As illustrated in Figure 3-2, when the target miRNA strands and the DNA

detection probes form heteroduplexes, the DSN selectively cleaves the DNA

detection probes while retains the target miRNA strands intact and release them

back to solution for next cycle. The cleaved DNA detection probes together with

their fluorescence tags are dispersed to the solution. A cumulative signal can,

therefore, be obtained after numerous rounds of the hybridization and DSN

cleavage cycles after a sufficiently long period of incubation. After the

incubation, the unreacted DNA detection probes on the MBs are completely

removed from the reaction mixture by using a permanent magnet. All the

reaction mixture containing the cleaved DNA detection probes is pipetted out

while holding the MBs at the bottom of the microfuge tube by the permanent

magnet, thereby leaving only the cleaved DNA detection probes together with

their fluorescein tags in the solution for detection. Hence, a negligible

68

background is obtained and the fluorescence intensity of the solution is directly

associated with the concentration of the target miRNA.

Figure 3-2. Schematic of the proposed DSN-based fluorescent bioassay for

miRNA.

3.3.2 Optimization of conditions

Contrary to the chemical coupling which may suffer from relatively large batch-

to-batch variations in coupling efficiency, the dually tagged DNA detection

probes offered a simple and highly efficient route to conjugate them onto the

MBs through strong interaction between the biotin moieties on the DNA

detection probes and the streptavidin moieties on the surface of the MBs. The

dissociation constant of biotin-streptavidin conjugates Kd (4 × 10-14 M) is

regarded as small enough to warrant robust attachment to the MBs and high

stability of the attached DNA detection probes on the MB surface.180 Figure 3-

3 indicated that during the first 15 min of incubation, the MBs were already

completely coated with the DNA detection probes. Before conjugating BF

oligonucleotides to the MBs, the original BF oligonucleotides solution has quite

69

a high fluorescent intensity as the blue curve indicated; while after conjugation,

the MBs with the conjugated BF oligonucleotides on their surface were

separated out by a permanent magnet, and the orange curve indicated the

fluorescent intensity of the solution containing the excess (free) BF

oligonucleotides.

Figure 3-3. The fluorescence intensities of the remained BF oligonucleotides

solution before and after linking BF oligonucleotides to the MBs.

In addition, the utilization of streptavidin molecules as anchoring sites ensures

appropriate spacing of the DNA detection probes for easy access of the DSN. It

was estimated that the distance between adjacent DNA probes on MBs is about

6 nm while the DSN has a size around 4.7 nm calculated from its molecular

mass of 40 kD.181 In principle, the DSN should be able to freely access to the

DNA probes, taking into consideration the flexibility of the immobilized probes.

70

To exploit the DSN cleavage process as a powerful amplification strategy, the

cleavage of the DNA detection probes from the MBs should be exclusively

associated with the concentration of the target miRNA or the fluorescence

intensity must be solely controlled by the hybridization efficiency of the target

miRNA. Therefore, to have the highest possible sensitivity and lowest LOD as

well as a straightforward correlation (linear dependence) between the analytical

signal and the target miRNA concentration, the concentrations of the DSN

should be kept sufficient in order to capitalize on its amplification power. The

high concentrations of the DSN imply that the DSN cleavage proceeds much

more rapidly than the miRNA hybridization process and the DNA detection

probes are cleaved instantly as soon as they are hybridized with the target

miRNA strands, thus ensuring that the analytical signal is directly associated

with the target miRNA concentration and its hybridization time. Insufficient

amounts of the DSN will likely result in a non-linear behavior between the

fluorescence intensity and the target miRNA concentration, whereas a high

amount of the DSN will not only increase the detection cost, but also increase

the possibility of false positive detection. To maximize the sensitivity and

minimize the LOD of the assay while keeping its cost low, our attention had to

be focused on the detection of traces of miRNAs where reasonable amounts of

the DSN should be sufficient. Indeed, using let-7a as a model target miRNA,

our experiments showed that the fluorescence intensity increases rapidly with

the increase in the dosage of the DSN from 0.01 U to 0.30 U and approaches a

steady-state in the presence of > 0.40 U DSN. It was found that 0.40 U DSN is

sufficient for the detection of miRNAs down to femtomolar levels. Likewise,

the concentration of the DNA detection probes should also be kept sufficient to

71

make certain that the cleavage of the DNA detection probes from the MBs is

solely controlled by the target miRNA hybridization process. Consequently, a

considerably long period of hybridization, which favors the detection of

miRNAs at ultralow levels, can be employed. Again, any notable depletion of

the DNA detection probes will probably result in a non-linear dependence of

the fluorescence intensity on the target miRNA concentration. Our experiments

showed that 100 nM of the DNA detection probes is sufficient for the detection

of target miRNA from femtomolar to nanomolar concentration.

To take full advantage of the DSN cleavage process as a powerful amplification

strategy for maximizing the assay sensitivity and minimize the LOD, besides

the dosage of the DSN, all other experimental variables must be optimized. The

concentration of Mg2+ is crucial to this assay because on one side, a certain

amount of Mg2+ is necessary to sustain the activity of the DSN as well as the

efficiency of the miRNA hybridization; while on the other side, the DSN is

sensitive to the presence of high concentrations of salts. A 10-fold decrease of

the DSN activity was reported in the presence of 0.20 M NaCl.182 Therefore, an

appropriate amount of Mg2+ should be present in the solution. From the

experimental results shown in Figure 3-4, 25 mM of Mg2+ was found to be

optimal for the assay to maintain efficient miRNA hybridization and maximal

activity of the DSN.

72

Figure 3-4. Optimization of the experimental conditions. 2.0 nM target miRNA

let-7a, 100 nM probes, and 0.4 U DSN.

The pH of the reaction medium is another important variable for the success of

the assay. It has been observed that the activity of the DSN strongly depends on

solution pH even though it has a relatively broad pH working range from 3.5 to

8.5 with the pH optimum at 6.6. Extreme pH values below 3.0 or above 9.0

will completely inactive the DSN.183 More importantly, the pH value of the

buffer solution strongly affects the fluorescence of the fluorescein tags.

Fluorescein is deprotonated in alkaline medium and protonated in acidic

solution. In either case the conformation of fluorescein molecule is altered and

subsequently the fluorescent efficiency (quantum yield) of fluorescein is

significantly diminished. Taking these two aspects into consideration, it was

73

found that pH 8.0 was optimal for the proposed assay, as reflected by the green

curve in Figure 3-4.

In addition, the incubation temperature is expected to strongly affect both the

hybridization efficiency and the DSN activity. Generally, the hybridization

temperature should be 10 to 15 ºС lower than the melting temperature to have

high hybridization efficiency while maintaining good selectivity. The estimated

melting temperature of let-7a was 55 ºС under the present experimental

conditions. In principle, the assay should be performed at temperatures 45 ºС.

However, the DSN prefers a higher temperature than 45 ºС to attain a better

activity. The optimal temperature for the DSN to reach its highest activity is 60

ºС.183 Temperature higher than 60 ºС adversely affects both the activity of the

DSN and the hybridization efficiency. The blue curve in Figure 3-4 displayed

the effect of the incubation temperature on the sensitivity and LOD of the assay.

As seen in Figure 3-4, the lowest LOD was achieved at 40 ºС.

We also investigated the dependence of the fluorescence intensity of the assay

on the DSN incubation time. As represented in Figure 3-5, the fluorescence

intensity increased rapidly with the DSN incubation time in the first 60 min and

then slowly leveled off with prolonged incubation. Our experiments indicated

that a period of 2 hrs incubation is sufficient although longer incubation time,

for example 240 min, could further decrease the LOD of assay by 20%.

Longer incubation time can be employed when even lower detection limit is

required.

74

Figure 3-5. The dependence of the fluorescence signal on the DSN incubation

time. 2.0 nM target miRNA let-7a, 100 nM probes, 40 ºС, 0.4 U DSN.

3.3.3 Analytical performance

Utilizing the optimized experimental conditions, the correlation between the

fluorescence intensity and the target miRNA concentration was investigated. As

demonstrated in Figure 3-6, the calibration curve showed a linear dynamic range

from 100 fM to 2.0 nM with a coefficient of 0.9885. The relative standard

deviation was less than 10% throughout the linear range from 100 fM to 2.0 nM.

Figure 3-6b displayed the fluorescence spectra acquired when varied

concentrations of the target miRNA let-7a used in the study. As can been seen

from Figure 3-6b, the background signal of the assay, corresponding 0.0 nM of

the target miRNA, was negligibly small, barely above zero. Obviously, the

simple magnetic separation of the unreacted DNA detection probes from the

reaction mixture was very efficient. The engagement of the MBs as the DNA

detection probe carriers and the two-step procedure – amplification and

magnetic separation – in this assay had a much lower non-target miRNA-related

75

fluorescent responses simply due to the fact that practically all unreacted

fluorescence tags are removed before fluorescence measurements, thus

producing an extremely low background and providing substantial

improvement in signal/noise ratio. The LOD, estimated as three times of the

standard deviation of the background, was 60 fM or 1.8 amol. In previous

reports where the unreacted detection probes co-existed with cleaved ones,

noticeable fluorescence responses were observed, evidently owing to the

incomplete quenching the fluorescence tag.133, 134, 174 Therefore, the separation

of the unreacted DNA detection probes is of paramount importance in

generating a sensitive fluorescence signal and in significantly reducing

background fluorescence. The sensitivity and LOD obtained in this assay was

much better than those of similar assays and comparable to that of qPCR

miRNA assay. More importantly, the much reduced background fluorescence,

only 1/10 – 1/100 of those observed in other fluorescence miRNA assays,

produced an extremely wide linear calibration curve, extending from 100 fM to

2.0 nM. As comparing to qPCR miRNA assays, through the DSN-mediated

target cycling, the amplification is accomplished isothermally at 40 ºС,

suggesting that the proposed assay is easier to implement, more cost effective,

and more tolerant of inhibitory components from crude samples than qPCR.

76

Figure 3-6. (a) The calibration curve of let-7a (100 nM probes, 0.4 U DSN and

a period of 2 hrs incubation at 40 ºС) and (b) fluorescence spectra of varied

concentrations of let-7a.

The selectivity of this assay was investigated through comparing the responses

of the target miRNA let-7a to the one-base mismatched miRNA let-7f and two-

77

base mismatched miRNA let-7d. As shown in Figure 3-7, both one-base

mismatched let-7f and two-base mismatched let-7d were clearly discriminated

from let-7a. The signal of the perfectly matched let-7a was 14 times higher than

that of the one-base mismatched let-7f and 24 times higher than that of the two-

base mismatched let-7d. The excellent selectivity is probably due partly to the

mismatch discriminability of the DSN and partly to the fact that the DSN

requires at least 15 perfectly paired bases in the DNA-RNA heteroduplex for

cleavage. In our assay, the one-base mismatch of let-7f is in the center of the

total 22 bases. Thus, only 10 or 11 perfectly matched bases are there in the

DNA-RNA duplex, which effectively decreases the efficiency of the DSN

cleavage.

Figure 3-7. Responses of the let-7a assay to 2.0 nM of let-7a, let-7f, and let-7d.

Experimental conditions are as for Figure 3-6.

The proposed bioassay for miRNA here was also compared with its counterparts

currently exist. As summarized in Table 3-3, compared with the previously

78

published miRNA detection methods, the present work exhibited its superior

selectivity toward one-base mismatched miRNA family member. Some of the

works have achieved ultralow LOD down to several attomolar for miRNA

detection, however, they can not distinguish a specific miRNA among its family

members with high sequence similarity, which is a significant issue regarding

to miRNA detection. Meanwhile, the present work is advantageous at the

simplicity, low cost and repeatability, which are quite attractive for further real

sample applications.

Table 3-3. Comparison with representative fluorescent detection methods for

miRNA

Method LOD

Selectivity for

one-base

mismatch

Detection

time Simplicity Low cost Repeatability Ref.

DSN+MB 60

fM 14:1 2 hrs + + + + + + + + + + + + + +

This

work

DSN+Perylene 5 pM 4:1 3.5 hrs + + + + + + + + + 184

DSN+Taqman probe 100

fM Not realized 30 min + + + + + + + + + + + + + + 133

DSN+AuNP 5 pM Not realized 5 hrs + + + + + + + + + 134

Phage+AuNP+

magnetic microparticle 3 aM Not realized > 5 hrs + + + + + + 185

Toehold-mediated

strand displacement

reaction+DNA

tetrahedron

5 fM 11:1 4.5 hrs + + + + + + + + 186

Silver nanocluster+

DNA scafford

100

pM Not realized 1 hr + + + + + + + + 187

Graphene quantum

dots+pyrene-

functionalized

molecular beacons

100

pM Not realized 2 hrs + + + + + + + + + 188

79

3.3.4 Sample analysis

To evaluate its practicability, the proposed assay was applied to analyze let-7a

in three total RNA samples extracted from cultured cells. The results were

summarized in Table 3-4. As seen in Table 3-4, the expression levels of let-7a

obtained by the proposed assay agreed well with those obtained by qPCR. The

relative errors associated with the detections were found to be less than 20%.

As compared to PCR-based assays, the isothermal amplification via the DSN

facilitated target cycling together with its excellent selectivity is very valuable

since the freedom from thermal cycling would greatly improve the adaptability

of the assay for direct miRNA expression profiling with minimal requirement

of sample preparation.

Table 3-4. Analysis of let-7a in total RNA samples extracted from cultured cells

Lung cancer cells

(106 copy μg-1)

HeLa cells

(106 copy μg-1)

Normal cells

(106 copy μg-1)

This method 1.60.30 3.80.80 5.30.92

qPCR 1.80.32 3.30.68 5.60.80

3.4 Conclusion

In conclusion, we have developed a simple and highly sensitive assay for

miRNAs by using MB-carried DNA detection probes coupled with DSN-

facilitated isothermal target cycling amplification. The accumulative nature of

the target cycling amplification offered a powerful and yet flexible means to

significantly improve the sensitivity and decrease the LOD of the assay. The

isothermal amplification strategy greatly improved the suitability in direct

profiling miRNA expressions with minimal or no sample pretreatments. The

MBs ensured a complete removal of possible interference from unreacted DNA

80

detection probes before fluorescence measurements, thus producing an ultralow

fluorescence background and an extended dynamic range. The simplicity, high

sensitivity, low LOD and the freedom from thermal amplification and miRNA

tagging are some of the interesting features for the development of a simple,

robust, low cost, and highly sensitive miRNA expression profiling tool uses at

point-of-care.

81

Chapter 4. Application of Catalytic Nucleic Acids in Bioassay for

MicroRNA Part I: Synthesis of Polyaniline via DNAzyme-Catalyzed

Polymerization of Aniline

82

4.1 Introduction

As one of the important classes of conducting polymers, polyaniline (PANI) has

been extensively studied due to its high conductivity in its doped (oxidized)

state189 and its intriguing electrochemical, electronic, optical, and electro-

optical properties.190 The conjugation mechanism of PANI is unique among the

conducting polymers owing to a combination of benzenoid and quinoid rings

leading to three different oxidation states, namely leucoemeraldine, emeraldine,

and pernigraniline.191 It has been found that PANI exhibits insulator-to-

conductor transitions and multiple color changes depending on its oxidation

states and solution pH. PANI can be doped either by protonation with a protonic

acid or by charge-transfer with an oxidation agent and its electronic and optical

properties may be controlled reversibly by varying the doping level. Technical

applications of PANI include biosensors,192-194 corrosion protection,195 energy-

storage devices,196 and antistatic coating.197 PANI has also been commonly

utilized to fabricate nanoelectronics including printed circuit boards (PCB),198

organic light emitting diodes (LEDs)199 and organic lightweight batteries.200

PANI is commonly synthesized by oxidative polymerization of aniline in

strongly acidic media either chemically201-205 or electrochemically.206-208

Chemical polymerization of aniline is performed using strong oxidants such as

ferric salts,202 bichromate,203 permanganate,204 or peroxydisulfate.205 These

oxidants are able to oxidize aniline under highly acidic conditions, thus leading

to the formation of chemically active cation radicals of aniline. The cation

radicals subsequently react with aniline molecules, yielding oligomers and

finally PANI. The reaction is far from being environmentally friendly because

83

of high acid and oxidant concentrations, and the use of these oxidants also

generates significant amounts of by-products which require further steps of final

product purification. Moreover, the reaction of chemical polymerization of

aniline is not kinetically controllable. On the other hand, electrochemical

polymerization of aniline and its derivatives is typically carried out in highly

acidic aqueous solutions by the application of a constant voltage/current or by

potential ramping.207 In comparison, electrochemical polymerization of aniline

has several advantages over chemical polymerization since the process is clean

and no toxic oxidants involved, and the synthesized PANI possesses flexible

electrical properties.208 Electrochemical polymerization can also be used to coat

electrodes because electrochemical polymerization/deposition enables coating

of PANI on rather complex geometries. However, the electrochemical methods

have some limitations. For instance, it is difficult to prepare a large amount of

PANI in a short period of time because the polymerization is carried out only

on the surface of the electrode. In addition, PANI synthesized by both chemical

and electrochemical approaches is often confronted with over-oxidation state

that may result in the degradation of PANI.209

In recent years, PANI synthesis via biocatalytic approaches using enzymes has

become more and more attractive as an alternative synthetic route.210 Unlike the

chemical approaches where a large amount of toxic materials are released as by-

products, biocatalytic polymerization of aniline using enzymes is advantageous

since it is a very simple one-step process carried under mild conditions211 and

does not require strong acidic media and highly toxic oxidizing reagents or

additional purification steps. It is therefore an environmentally friendly

84

approach with high potential for industrial scale production in high yield

without contaminant by-products of oxidant degradation due to the efficiency

of the biocatalyst.212, 213 Moreover, the structure and solubility of PANI can be

greatly improved by optimizing the reaction conditions, which is beneficial for

further processing. Horseradish peroxidase (HRP) was first used for the

synthesis of PANI.210, 214 HRP is an oxidoreductase which catalyzes the radical

polymerization of aniline in the presence of a mild oxidizing agent such as

hydrogen peroxide. In the presence of hydrogen peroxide, HRP catalyzes the

oxidation of aromatic amines and phenols to generate free radicals. In the case

of aniline, the aniline free radicals coupling to produce dimers and further

oxidation and coupling reactions result in the formation of PANI. Other

enzymes such as laccase,215 palm tree peroxidase,216 bilirubin oxidase,217

soybean peroxidase,218 and glucose oxidase219 were also used as catalysts for

aniline polymerization under mild conditions in aqueous media. It should be

noted that enzymes are very sensitive to hydrogen peroxide concentration which

usually requires stepwise addition of diluted hydrogen peroxide to the reaction

medium. Also, enzymes can be easily denatured by environmental changes

because a minor alteration of their native protein conformation could result in

complete loss of their catalytic activity.

More recently, it has been observed that single-stranded nucleic acids with

specific sequences coupled with cofactors are catalytic through forming higher

order structures – the so called G-quadruplexes ‒ DNAzymes. DNAzymes are

DNA molecules that have the ability to perform chemical reactions, such as

catalytic action. One interesting example of DNAzymes that reveals peroxidase-

85

like activity is a supramolecular complex between hemin and a single-stranded

guanine-rich nucleic acid.51 It was suggested that the intercalation of hemin into

the guanine quadruplex docked layers of the nucleic acid leads to the

biocatalytic functionality.50 It has also been observed that this G-quadruplex

DNAzyme can catalyze the polymerization of several aromatic compounds and

produce according polymers.220, 221 In the present work, a novel approach for

the synthesis of PANI based on the G-quadruplex DNAzyme was developed.

The proposed approach addresses the limitations of both enzymatic and

chemical polymerization of aniline. Comparing to natural peroxidases, the

DNAzyme is much more stable and less expensive to produce by DNA

synthesis. More importantly, unlike enzymes, the DNAzyme remains active in

a wide range of hydrogen peroxide concentration and can be reversibly

denatured and renatured many times without losing its activity. Therefore, the

use of the DNAzyme as catalyst for aniline polymerization represents a viable

alternative route in comparison with peroxidase-based synthesis. It is

environmentally friendly and does not require a stepwise addition of diluted

hydrogen peroxide to a reaction medium in contrast to the peroxidase-based

approaches. The application of this synthesis in bioassay will be demonstrated

in Chapter 5.

4.2 Experimental section

4.2.1 Reagents and apparatus

Aniline (> 99.5%), Polyacrylic acid (2100 sodium salt) (PAA), and hemin (>

98%, HPLC) were purchased from Sigma-Aldrich. Oligonucleotide (5’-GGT

GGT GGT GGT TGT GGT GGT GGT GG-3’) for construction of DNAzyme

86

was custom made by Integrated DNA Technologies Incorporated. All the

reagents were from Sigma-Aldrich and used without further purification unless

specified. A 0.10 M phosphate buffer (PB) containing 0.10 M potassium

chloride (KCl) was used in the aniline polymerization study and its pH was

adjusted by adding an appropriate amount of sodium hydroxide (NaOH) or

phosphoric acid (H3PO4) solution. All aqueous solutions were freshly prepared

with UP water.

FT-IR spectroscopic experiments were performed with a Bio-Rad Excalibur

Series FTS 3000 spectrometer and the spectra were collected after 20 scans

between 4000‒400 cm-1 at a resolution of 4 cm-1. Cyclic voltammetric tests were

conducted with a CHI 660D electrochemical analyzer (CH Instrument, Austin,

TX, USA). A three-electrode system was used with a glassy carbon electrode

(GCE) as the working electrode, saturated calomel electrode (SCE) as the

reference electrode, and platinum wire as the auxiliary electrode, respectively.

All potentials reported in this work were referred to the SCE. The standard four-

probe technique using an Agilent 4157 parameter analyzer from Agilent

Technologies (Santa Clara, CA, USA) in conjunction with a probe station was

employed to measure the electrical conductivity of compressed pellets of

polyaniline samples. A gel permeation chromatograph (GPC) comprising

Waters Model 515 HPLC pumps and a Waters Model 2414 refractive index

detector (Milford, MA, USA) was used to determine the molecular weight of

the PANI. Scanning electron microscopic (SEM) image was collected on a

JEOL JSM-6701F Field Emission Scanning Electron Microscope (FESEM)

provided by JEOL USA Incorporated (Peabody, MA, USA).

87

4.2.2 Preparation of the G-quadruplex DNAzyme

5.0 mM stock solution of hemin was prepared by dissolving 50 µmol of hemin

in 10 mL DMSO and stored at -20 ºС in dark. The working solution of hemin

(100 μM) was prepared by diluting the stock solution with UP water. 100 µM

the oligonucleotide solution was prepared by dissolving an appropriate amount

of the oligonucleotide in 0.10 M KCl and then heated to 88 ºС for 10 min; after

which the solution was gradually cooled down to room temperature in 20 min.222

The DNAzyme solution (50 µM) was prepared by adding dropwise 1.0 mL of

the 100 μM hemin solution to 1.0 mL of the oligonucleotide solution. The

mixture was incubated for 40 min at room temperature to fold the G-quadruplex

and form the DNAzyme (G-quadruplex hemin complex).

4.2.3 DNAzyme-catalyzed polymerization of aniline

50 mM aniline/PAA (1:1) solutions with different pH values ranging from 2.0

to 5.0 were prepared in 0.10 M PB buffer. 2.0 μM the DNAzyme and 50 mM

hydrogen peroxide (final concentration) were then added to the aniline/PAA

solutions to initiate aniline polymerization. PAA in the solutions was used as a

polyelectrolyte to procure a more ordered para-linked PANI with fewer

branches and increase the solubility of the synthesized PANI in aqueous

solution.223 UV-Vis spectra of the reaction mixture at 750 nm were recorded

with the proceeding of the polymerization. The same mixture without the

DNAzyme was used as control and its UV-Vis spectra were also recorded. In

the mechanistic study of the polymerization reaction, the concentration of one

substrate was fixed and that of the second substrate was varied. UV-Vis spectra

were collected immediately with a 15-s interval. The reciprocal of the initial

88

reaction rate versus the reciprocal of substrate concentration was plotted. Each

point was repeated for three times.

4.3 Results and discussion

4.3.1 DNAzyme-catalyzed polymerization of aniline

It was reported that certain DNAzymes, such as the G-quadruplex-hemin

DNAzyme, exhibit peroxidase-like activities in the presence of peroxidase

substrates and hydrogen peroxide, thus offering new possibilities to engage

DNAzymes in biocatalytic applications.51, 52, 224 Compared to the bulky and

fragile peroxidases, their small size and high chemical stability make them

attractive candidates to be used as efficient catalysts in organic synthesis and

polymer synthesis. In addition, it is critical that the polymerization of aniline

should be carried out in an acidic medium with a pH value below the pKa value

of aniline (pKa 4.63), to effectively utilize the polyanionic template by

forming electrostatic adducts along PAA between cationic aniline molecules

and anionic acrylic acid moieties and to minimize the parasitic branching of

PANI.225 Unfortunately, most natural enzymes are active and stable only around

neutral pH values. In contrast, the chemical nature of the DNAzyme may be

stable and remain active in a much wider pH range.

89

Figure 4-1. (A) Photographs of the reaction mixtures (50 mM aniline/PAA +

50 mM hydrogen peroxide + 2.0 μM the DNAzyme) at different pH values and

(B) Different structures of PANI: (a) Leucoemeraldine, (b) Emeraldine base

(EB), (c) Emeraldine salt (ES), (d) Pernigraniline, (e) Ortho- and para-

substituted carbon-carbon, carbon-nitrogen bond structure. (C) DNAzyme-

catalyzed polymerization of aniline in the presence of hydrogen peroxide.

To demonstrate this concept, we first evaluated the catalytic activity of the

DNAzyme in the oxidation and polymerization of aniline. It was observed that

in accordance with the HRP-catalyzed polymerization of aniline, the reaction

between aniline and hydrogen peroxide in the presence of the DNAzyme

90

produced exactly the same colored products (Figure 4-1). As seen in Figure 4-

1A, similar to the HRP-catalyzed oxidation and polymerization of aniline,226 a

blue-green color developed immediately after spiking a tiny amount of the

DNAzyme ( 2.0 M) into a mixture of 50 mM aniline/PAA and 50 mM

hydrogen peroxide at pH < 4.0; while the color of the reaction mixture at pH >

4.0 was yellow. The blue-green product is believed to be emeraldine base, which

is half-oxidized state of PANI (Figure 4-1B (b)). This emeraldine base can be

easily doped by proton and consequently transformed to the emeraldine salt

(Figure 4-1B (c)), which is the most popular state of PANI since it has an

excellent electrical conductivity. The emeraldine base can also be further

oxidized to the fully-oxidized pernigraniline (Figure 4-1B (d)) after a prolonged

period of polymerization. In addition, before forming emeraldine base, there is

a state of PANI that cannot be recognized by naked eye ‒ leucoemeraldine

(Figure 4-1B (a)). It is the fully-reduced state of PANI with white or clear in

color. As to the yellow product, it is likely the mixture of ortho- and para-

substituted carbon-carbon, carbon-nitrogen bond structure23 and aniline

oligomers227 (Figure 4-1B (e)).

As shown in Figure 4-2, the catalytic polymerization of aniline in the presence

of the DNAzyme has two UV-Vis absorption peaks, one sharp peak at 410

nm and another broad peak at 750 nm. As the polymerization proceeded, the

absorbances for both peaks intensified. The UV-Vis spectra are in good

agreement with those found in literature.226 Unfortunately, the DNAzyme used

in this polymerization system has a sharp UV-Vis adsorption peak at 410 nm

due to the chromogenic hemin, which may interfere with the analysis of the

91

polymerization process. This interference was circumvented by using the

absorbance data at 750 nm. Nonetheless, all the data induced from the

absorbance at 750 nm were generally consistent with those obtained at 410 nm.

The absorbance at 750 nm was therefore utilized to investigate the kinetics of

the polymerization of aniline in the following sections.

Figure 4-2. UV-Vis spectra of the polymerization of aniline oxidized by

hydrogen peroxide in the presence of the DNAzyme. The working solution

contained 50 mM aniline/PAA (1:1), 2.0 μM the DNAzyme and 50 mM

hydrogen peroxide in pH 3.0 0.10 M PB buffer. From bottom to top: 0, 15, 30,

45, 60, 75, 90, 120, 150, 180, 210, 240, 270, 300, 330 and 360 s after starting

the polymerization.

92

4.3.2 Optimization

Figure 4-3. (A) pH optimization of aniline polymerization. Working solution:

50 mM aniline/PAA, with () and without () 2.0 μM the DNAzyme, and 50

mM hydrogen peroxide in phosphate buffer with different pH values ranging

from 2.0 to 5.0. UV-Vis measurements were carried out 6 min later after starting

the polymerization. (B) The effect of pH on the extinction coefficient of the

synthesized PANI.

From the pH optimization experiments depicted in Figure 4-3A, pH 3.0 was

found to be optimal for the DNAzyme-catalyzed polymerization of aniline.

When the solution pH was higher than 4.0, the polymerization rate was

93

substantially suppressed, judging by the much slower change in the absorbance

at 750 nm. Meanwhile, to rule out the possibility of the effect of pH on the

extinction coefficient of PANI, the optical properties of the synthesized PANI

was thoroughly investigated. PANI synthesized at pH 3.5 was separated by

centrifugation at 15000 rpm. Then it was dispersed to 0.10 M PB buffers with

different pH values. As can be seen in Figure 4-3B, the influence of pH on the

extinction coefficient of PANI was negligible comparing to the drastic changes

in the polymerization of aniline under different pH values. The average

molecular weight of the synthesized PANI under the optimal conditions was

found to be 51300.

As shown in Figure 4-4, the reaction rate increased with the increase of the

concentration of the oxidizing agent hydrogen peroxide. It was shown that the

reaction rate approached to its maximum in the presence of 50 mM of

hydrogen peroxide. Also, it was shown that the reaction rate can be conveniently

controlled at desired levels just by simply adjusting the concentration of

hydrogen peroxide. However, further increasing in hydrogen peroxide

concentration was little beneficial to the polymerization of aniline. On the

contrary, high concentrations of hydrogen peroxide may be detrimental since a

considerable amount of the PANI may be over-oxidized or undergo the

undesired ortho-branching which will have a negative impact on the

conductivity of the aniline. On the other hand, in the commonly used HRP-

catalyzed polymerization of PANI, the concentration of hydrogen peroxide can

never reach such a high level as HRP will be quickly deactivated in the presence

of 10 mM hydrogen peroxide.

94

Figure 4-4. Effect of the amount of hydrogen peroxide on the reaction rate of

aniline polymerization. Working solution: 50 mM aniline/PAA, 2.0 μM

DNAzyme and different concentrations of hydrogen peroxide in the phosphate

buffer (pH 3.0).

4.3.3 Kinetics of the DNAzyme-catalyzed polymerization of aniline

It was found that the initial rate of polymerization varied linearly with the

concentration of the DNAzyme, suggesting that the polymerization of aniline is

first-order with respect to the DNAzyme. This is a good indication that the

catalytic process may obey the Michaelis-Menten kinetics.228 And more

importantly, the linear dependence on the DNAzyme concentration strongly

suggests that the DNAzyme is stable in the reaction medium. To gain insight in

the mechanism of the catalytic polymerization of aniline in the presence of the

DNAzyme, further investigations were conducted over wide ranges of aniline

and hydrogen peroxide concentrations. Varying the concentrations of aniline

and hydrogen peroxide, a series of experiments were carried out to explore the

kinetics of the DNAzyme-catalyzed polymerization of aniline.

95

Figure 4-5. Double-reciprocal plots of the catalytic activity of 2.0 μM

DNAzyme at a fixed concentration of one substrate and varying concentration

of the second substrate.

Figure 4-5 shows double reciprocal plots of initial polymerization rate versus

one substrate concentration, obtained at predetermined concentrations of the

second substrate. Good linearity (R2 > 0.99) was obtained throughout the

experiments. These linear plots strongly suggest that the polymerization process

96

obeys the Michaelis-Menten kinetic mechanism as it accords with the classical

Michaelis-Menten equation (Eq. 1):

1

v=

Km

vmax[S]+

1

vmax (Eq. 1)

The linear correlation between the initial polymerization rate and the

concentration of substrate will bring great benefit for its future applications in

chemical sensors and biosensors. That is because such a feature will simplify

the relationship between the analytical signal and the analyte concentration,

making the data processing concise and easily understood.

In addition, when the concentration of one substrate was preset and the

concentration of the other substrate was varied, it was found that these linear

double-reciprocal plots are parallel to each other, i.e. the slopes of these plots

are the same. This is the typical characteristic of the ping-pong mechanism,229

demonstrating that the DNAzyme will bind and react with the first substrate and

release the first product before binding and reacting with the second substrate.

4.3.4 Characterization of the polymerization product

FT-IR spectroscopy was utilized to characterize the synthesized PANI. Figure

4-6 depicts the FT-IR spectrum of the proton doped half-oxidized PANI, which

was green in color. Proton doping was accomplished by treating the as-

synthesized PANI in 2.0 M HCl. As seen in the spectrum, the adsorption peaks

at 3450 and 3235 cm-1 can be assigned to the asymmetric and anti-symmetric –

NH+– stretching of PANI.230 The absorption band at 2920 cm-1 belongs to C–H

vibration.231 The two very sharp bands at 1572 and 1500 cm-1 are due to quinone

and benzene ring deformation.232 The C–N stretching of the secondary aromatic

97

amine is located at 1300 cm-1. The band at 1133 cm-1 is originated from

Q=NH+–B or B–NH+–B after doping (Q = quinoid unit, B = benzoid unit) and

the band at 820 cm-1 is assigned to C–H out of plane bending of para-

substitution pattern, indicating a head-to-tail coupling of aniline.233 The FT-IR

spectrum confirms that the reaction product between aniline and hydrogen

peroxide catalyzed by the DNAzyme is PANI in its emeraldine salt form.

Figure 4-6. FT-IR spectrum of the proton doped half-oxidized PANI

(emeraldine salt).

Supportive evidence of the formation of PANI can be found in cyclic

voltammogram of the reaction product coated electrode. As mentioned early,

PANI exists in three different oxidation states which can be conveniently

interconverted by electrochemical means and cyclic voltammetry in

particular.234 Moreover, an understanding of inter-conversion of various

oxidation states of PANI produced can also be conveniently studied by cyclic

98

voltammetry. A cyclic voltammogram of the synthesized PANI is shown in

Figure 4-7. Similar to PANI prepared by conventional methods,235, 236 two pairs

of current peaks centered at 0.20 and 0.49 V were observed. These peaks can be

assigned to the oxidation of PANI from leucoemeraldine to emeraldine and to

further oxidation from emeraldine to pernigraniline.234-236 At potentials more

cathodic that the first oxidation peak of 0.26 V, PANI is in its non-conducting

leucoemeraldine state. As the potential is swept more anodically, the current

rises sharply and peaks at 0.26 V due to the partial oxidation of PANI to yield

the emeraldine salt form. The complete oxidation peak of the polymer is then

obtained at 0.51 V at which point the emeraldine salt is converted to the non-

conducting pernigraniline salt. Upon potential reversal, two reduction peaks

appeared at 0.47 and 0.15 V, corresponding to the reduction of pernigraniline to

emeraldine and emeraldine to leucoemeraldine, respectively. The inter-

conversion of different structural forms could be done repetitively without any

shifts in peaks or changes in the repetitive cyclic voltammetric scans. It is the

partially oxidized, protonated form of PANI, known as the emeraldine salt that

is of particularly interest as it is responsible for PANI’s electron conductivity.

Furthermore, SEM images of the PANI were collected in order to characterize

the morphology of the synthesized PANI. As seen in Figure 4-8, the

morphological characteristic of the synthesized PANI is quite similar to those

obtained in a in previous study.237 The linear structure with an average diameter

at around 90 nm can be clearly identified, thus validating that linear PANI

molecules were well formed along the PAA template.

99

Figure 4-7. A cyclic voltammogram of PANI on GCE in 2.0 M HCl aqueous

solution from 0 to 0.8 V at a scan rate of 50 mV/s.

Figure 4-8. Morphological characterization (SEM image) of the synthesized

PANI.

100

4.3.5 Proton doping

According to previous studies, the PANI synthesized at pH 3.0 was estimated

to have an electrical conductivity around 10-6 S/cm,238 which was in the

semiconductor regime; while after doping, the electrical conductivity can be

increased to as high as 10‒103 S/cm,237 closing to that of conducting materials.

Indeed, the conductivity of the as-synthesized PANI in the pH 3.0 PB buffer

was found to be 3.8 × 10-6 S/cm, whereas the conductivity of the PANI after

doping in 2.0 M HCl was significantly enhanced to 180 S/cm approaching that

of metal conductors. Such a characteristic of the adjustable electrical

conductivity offers ample opportunities to construct electrical/electrochemical

bioassays considering that materials with designated electrical conductivities

can now be fabricated instead of finding various materials to fit in different

devices. Furthermore, since H+ doping is reversible,239 PANI may also act as an

“on-off” switch, i.e. when PANI is doped, it is conductive and thus turns on the

switch that allows electric current to flow through; while after dedoping, it is

insulating and turns off the switch that would block the current at that point.194

Based on such a simple mechanism, interesting technical applications such as

environmental monitoring or biosensing are expected.

4.4 Conclusion

In summary, it was demonstrated for the first time that the G-quadruplex-hemin

DNAzyme effectively catalyzes the polymerization of aniline under acidic

conditions. The DNAzyme in this study consists of single-stranded DNA and

hemin mimicking that of peroxidase. Such a peroxidase-mimicking catalyst is

more tolerant to the environmental conditions than its natural peroxidase

101

counterparts. For example, HRP will lose its activity once the concentration of

hydrogen peroxide in working solution is ≥ 10 mM or the pH is ≤ 4.5. In contrast,

the DNAzyme can still maintain a good activity under acidic conditions (e.g.

pH 2.5) or a much higher hydrogen peroxide concentration up to several

hundred millimolars. Due to its robustness, the DNAzyme can be employed as

an attractive alternative to peroxidases under extreme conditions.

Predominantly para-coupled PANI with minimal branching can be conveniently

synthesized in pH 2.5‒3.0 phosphate buffer in the presence of a stoichiometric

amount of hydrogen peroxide. Since the reaction conditions are mild and no

toxic by-products are released during the oxidative polymerization of aniline,

the proposed DNAzyme-catalyzed approach is environmentally friendlier than

the conventional chemical routes. The much higher concentration of hydrogen

peroxide allowed in the system implies a simpler and more efficient PANI

synthesis route. In principle, this approach may be extended to the synthesis of

other conducting polymers such as polypyrrole, polythiophene, and poly(3,4-

ethylenedioxythiophene).

102

Chapter 5. Application of Catalytic Nucleic Acids in Bioassay for

MicroRNA Part II: A Highly Sensitive MicroRNA Biosensor Based on

Hybridized MicroRNA-guided Deposition of Polyaniline

103

5.1 Introduction

Because of the emerging diagnostic and prognostic values of miRNAs,

intensive research efforts have been devoted to the development of miRNA

assays for uses at point-of-care. It is of paramount importance that the method

for miRNA expression profiling should be highly sensitive, selective, and only

analyze mature miRNAs as they are the active form of miRNAs. Toward this

goal, label-free miRNA assays represent a group of attractive candidates for

miRNAs expression analysis, enabling multiplexed miRNA expression

profiling with much simplified protocols and with minimal sample preparation.

So far, several label-free approaches, such as Taqman probe-based

fluorometry,133 silver nanorod-based SERS,240 nanomechanic atomic force

microscopy,241 dynamic piezoelectric cantilever,242 stacking-hybridized

universal tag,243, 244 single-stranded DNA binding protein-assisted capillary-

electrophoresis168, 245, nanoparticle-based colorimetry,135, 246 and

electrochemical biosensors220, 247 have been reported. Among them, the

development of label-free electrochemical biosensors appears especially

appealing, particularly electrochemical impedance spectroscopy (EIS)-based

label-free biosensors. The inherent miniaturization of electrochemical

biosensors and their compatibility with standard microfabrication and

semiconductor technologies, coupled with high sensitivity and low LOD

assured by chemical/electrochemical amplification strategies, provide

tremendous opportunities for the electrochemical biosensors to play a

significant role in future miRNA expression analysis. Their high portability and

affordability could allow miRNAs expression to be profiled in a decentralized

setting such as at point-of-care.

104

To date, several EIS approaches have been attempted in the development of

miRNA biosensors. For example, based on ruthenium oxide (RuO2)

nanoparticle-initiated deposition of an insulating film, EIS detection of miRNAs

can be conveniently performed after the miRNAs are tagged with

RuO2 nanoparticles.41 However, RuO2-tagging procedure is a major drawback.

More recently, on the basis of catalyzed deposition of poly(3,3-

dimethoxybenzidine) by horse reddish peroxidase,247 label-free EIS biosensors

for miRNAs were reported. Thereafter, LODs down to 2.0 fM were observed.

The multistep procedure in the preparation of biosensors or in the detection of

miRNAs may hinder its further development.

To further enhance the sensitivity, decrease the LOD and simplify the detection

protocol, a simple and yet highly sensitive biosensor for label-free detection of

miRNAs was described in this report. Through incorporating a cumulative

signal amplification mechanism – the hybridized target miRNA-guided

deposition of polyaniline (PANI), label-free detection of miRNAs was realized

by measuring the charge transfer resistance (Rct) in EIS. As compared to

previous reports, the procedure was simplified and the much higher efficiency

in the polymerization of aniline and the subsequent deposition of PANI resulted

in a sub-femtomolar LOD.

5.2 Experimental section

5.2.1 Materials and reagents

105

Cysteine-terminated PNA CPs were custom-made by Biosynthesis Incorporated.

(Lewisville, TX, USA) and used as received for the construction of the

respective miRNA biosensors. Synthetic let-7 miRNAs (see in Table 5-1) and

oligonucleotides for construction of the DNAzyme were from Integrated DNA

Technologies. Freshly-distilled aniline was used in the deposition of PANI. All

other chemicals of certified analytical grade were obtained from Sigma-Aldrich

and used without further purification. All aqueous solutions were freshly

prepared with UP water. A pH 8.0 of 0.10 M phosphate buffer was used as the

hybridization and washing buffer. A pH 3.0 of 0.10 M potassium phosphate

buffer containing 50 mM aniline, 400 mM hydrogen peroxide, and 10 µM

DNAzyme was used as the PANI deposition cocktail. The PNA CP solution

used for the preparation of the biosensor was typically 2.0 μM.248

Table 5-1. MicroRNA sequences of let-7 family

Name Sequence (5’- 3’)

Let-7a (miRNA) 5’-rUrGrA rGrGrU rArGrU rArGrG rUrUrG

rUrArU rArGrU rU-3’

Let-7b (miRNA) 5’-rUrGrA rGrGrU rArGrU rArGrG rUrUrG

rUrGrU rGrGrU rU-3’

Let-7c (miRNA) 5’-rUrGrA rGrGrU rArGrU rArGrG rUrUrG

rUrArU rGrGrU rU-3’

UV−Vis experiments for the quantification of RNA samples were conducted on

a NanoDrop 1000 spectrophotometer provided by Thermo Scientific

Incorporated. All electrochemical experiments were performed with a Zennium

electrochemical workstation supplied by Zahner Elektrik (Kronach, Bavaria,

Germany) using a conventional three-electrode system consisting of the

106

biosensor, a nonleak Ag/AgCl (3.0 M NaCl) reference electrode from Cypress

Systems (Lawrence, KS, USA), and a coiled platinum wire counter electrode.

All electrochemical experiments were carried out at room temperature. All

potentials reported in this work were referred to the Ag/AgCl reference

electrode.

EIS experiments were carried out at conducted at E0’ of Ru(NH3)63+/2+ (-0.11 V)

over a frequency range from 10 mHz to 1.0 MHz with a 5-mV sinusoidal voltage

perturbation. The EIS testing solution was made of pH 12.0 0.20 M phosphate

buffer containing 2.5 mM of Ru(NH3)6Cl3/2.5 mM Ru(NH3)6Cl2. Ru(NH3)6Cl3/

Ru(NH3)6Cl2 in the phosphate buffer served as redox probes so as to perform

EIS.

5.2.2 Biosensor fabrication

A cleaned gold bead electrode with a predominant (111) faceting of the surface

(Figure 5-1 insert) was incubated in 100 µL of 2.0 μM PNA solution in a sealed

centrifuge tube for 4 hrs at room temperature. After the incubation, the electrode

was washed with 1:1 (v/v) mixture of water and acetonitrile, water, respectively,

and dried in a stream of Ar. The PNA CP coated electrode was further incubated

in 1.0 mg/mL cysteine for 2 hrs during which PNA molecules realign their

molecular axes with the normal surface. The surface coverage of the PNA CP

monolayer, estimated electrochemically using a published procedure,249 was

found to be in the range of 11.8–13.5 pmol/cm2. The biosensor was stable for at

least two months when stored in a clean environment.

107

Figure 5-1. EIS spectra of (1) the control and (2) the miRNA hybridized

biosensor before PANI deposition. 100 fM let-7b and 60 min hybridization at

50 ºС. Inset: Scanning tunneling microscopic image of the gold bead electrode.

5.2.3 MicroRNA hybridization and EIS detection

MicroRNA hybridization, DNAzyme-catalyzed polymerization of aniline, and

the hybridized miRNA-guided deposition of PANI were performed as follows:

The PNA CP coated gold bead electrode (biosensor) was immersed in 100 μL

of the hybridization buffer containing a target miRNA and incubated in a

controlled environment at 50 ºС for 60 min. After a stringent wash at 60 ºС with

the hybridization buffer, the hybridized biosensor was then incubated in 100 μL

of the PANI deposition cocktail for 30 min at room temperature. The biosensor

was thoroughly rinsed with the pH 3.0 0.10 M potassium phosphate buffer and

water to remove any residual DNAzyme and aniline monomer. Finally, EIS

measurements were performed in the pH 12 0.20 M phosphate buffer containing

2.5 mM Ru(NH3)6Cl3/2.5 mM Ru(NH3)6Cl2. Data were reported as an average

of six replicates.

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5.3 Results and discussion

5.3.1 Detection scheme

The principal steps in the operation of the biosensor are schematically shown

in Figure 5-2. A monolayer of charge neutral PNA CPs, acting as a bioaffinitive

interface, is self-assembled on the surface of the gold bead electrode. To

improve the quality and stability of the PNA CP monolayer and to minimize

non-specific deposition of PANI, cysteine is used to fill in the defects of the

PNA monolayer, forming a mixed monolayer. Incubation of the biosensor in

the hybridization buffer containing the target miRNA brings the target miRNA

strands together with a high density of negative charges onto the biosensor (step

A). A subsequent incubation of the hybridized biosensor in a cocktail consisting

of the DNAzyme, aniline, and hydrogen peroxide results in the oxidative

polymerization of aniline catalyzed by the DNAzyme and the deposition of

PANI guided by the anionic target miRNA strands hybridized to the PNA CPs.

Strong electrostatic attraction between anionic miRNA strands and protonated

aniline molecules produces a high concentration of protonated aniline and

creates a high concentration of protons (acidity) at the biosensor surface. The

high concentration of aniline and high acidity greatly facilitate the

polymerization aniline and the deposition of PANI in the presence of a catalyst

(the DNAzyme) and an oxidant (hydrogen peroxide). Oxidative polymerization

of aniline is catalytically initiated by the DNAzyme and the deposition of PANI

is guided by the hybridized target miRNA strands at the biosensor surface (step

B). The excellent electron-transfer impeding power (insulating power) of the

deposited PANI in alkaline medium allows EIS detection of the target miRNA

(step C). High sensitivity and low LOD can be conveniently realized after a

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sufficiently long period of incubation in the PANI deposition cocktail. The

utilization of neutral PNA instead of anionic DNA in the biosensor effectively

alleviates the undesired (unrelated to miRNA hybridization) adsorption of

aniline molecules and their subsequent polymerization and deposition, thus

producing a minimal background and facilitating the detection of miRNA at

ultralow levels.

Figure 5-2. Schematic of the working principle of the label-free miRNA

biosensor. (A) miRNA hybridization, (B) hybridized miRNA-guided PANI

deposition, and (C) EIS measurement.

5.3.2 Feasibility study

It was reported that in the presence of polyanionic templates such as nucleic

acid molecules, the formation of PANI occurs exclusively along the polyanionic

molecules.250 It was also reported that many G-rich DNA sequences are able to

form G-quadruplexes and display peroxidase-like activities after complexing

with hemin, known as DNAzyme.50 Similar to HRP, DNAzyme is capable of

initiating the polymerization and subsequent deposition of PANI guided by

prefabricated DNA nanostructures.251 The time-dependent study of the

deposition of PANI indicated that the growth of PANI occurs exclusively along

the DNA templates.251 On the other hand, it sounds a bit peculiar that PANI is

utilized to create an electron-impeding layer on the biosensor surface since

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being a well-studied conducting polymer PANI is best known for its electron-

conducting properties. However, as we know, PANI can exist in insulating,

semiconducting, and conducting state depending on its doping state (usually

H+ doping in a strong acid). It was reported that the conductivity of PANI drops

precipitately with increasing pH and it is converted to an excellent insulator in

alkaline medium.252 Instead of further exploiting the most popular conducting

and electroactive properties of PANI for the development of chemical and

biosensing devices as it has been demonstrated in numerous reports,253 this work

for the first time exploited the possibility of leveraging on the excellent

insulating power of PANI to develop an EIS biosensor for miRNA expression

profile analysis.

To test the feasibility of utilizing the electron insulating property of PANI as a

sensitive signal generator for the EIS biosensor, a synthetic miRNA let-7b was

examined at a biosensor coated with PNA CPs fully complementary to let-7b.

For comparison, a totally non-complementary synthetic RNA of the same length

as let-7b (control) was also tested. As seen in Figure 5-3A, after a period of

60 min hybridization and subsequent 30 min incubation in the PANI deposition

cocktail, a distinct difference between let-7b and the control was obtained. The

EIS spectrum of the let-7b biosensor was made of a semicircle at high

frequencies and a straight line with a phase angle of 45° at low frequencies

(Figure 5-3A trace 1). The straight line with 45° phase angle is less important

because it is related to the solution diffusion process of the redox probes, known

as Warburg impedance.67 In the case of EIS-based biosensors, characteristics of

the diffusion process of the redox probes are largely independent of the

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biosensing processes. The semicircle in the EIS spectrum, on the other hand, is

closely associated with the charge-transfer of the redox probes and its diameter

is Rct – a direct measure of the charge-transfer process.67 The large semicircle

observed at the let-7b biosensor indicated that a layer of charge-transfer

impeding material exists at the biosensor surface. In contrast, a straight line with

a phase angle of 45° was obtained in the whole frequency range at the control

(Figure 5-3 A trace 2),67 implying that Rct in this case is largely insignificant.

In addition, a much less significant difference was observed between the control

and the biosensor after hybridization and before PANI deposition (Figure 5-1).

Because the control is completely non-complementary to the PNA CPs, none of

the miRNA strands is expected to be brought to the biosensor. On one hand, in

the presence of deprotonated cysteine on the biosensor surface at pH 8.0

(hybridization buffer), nonspecific uptake of the control miRNA is effectively

minimized. On the other hand, instead of a layer of anionic target miRNA

strands, the protonated cysteine in the pH 3.0 PANI deposition cocktail largely

retards the deposition of PANI. Therefore, the control biosensor should remain

as original – the same as the blank biosensor. In addition, before the deposition

of PANI, when the EIS spectrum is collected in the pH 12.0 phosphate buffer

cationic Ru(NH3)63+/2+ alleviate electrostatic repulsion between now anionic

miRNA and the redox probes Ru(NH3)63+/2+; and Ru(NH3)6

3+/2+ probes are able

to approach the electrode surface closely. Therefore, there are no noticeable

changes in the reversibility or Rct of Ru(NH3)63+/2+ between the control and the

hybridized biosensor before PANI deposition. Apparently, the

large Rct observed with the let-7b miRNA is closely associated with the target

hybridization and PANI deposition processes – a key parameter (analytical

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signal) in EIS biosensors. Values of Rct can be conveniently extracted from the

EIS spectrum by either direct analysis of the spectrum or by fitting the spectrum

to a Randle equivalent circuit (Figure 5-3 A inset). The large Rct indicated that

a large amount of charge-transfer impeding material is present at the biosensor

surface as a direct consequence of let-7b hybridization and it is possible to

realize sensitive miRNA detection via the DNAzyme-catalyzed polymerization

of aniline and hybridized miRNA strand-guided deposition of PANI. Upon

hybridization, the target let-7b strands are brought to the biosensor surface by

their complementary CPs. A subsequent incubation in the PANI deposition

cocktail results in the formation of a thin PANI film alongside the hybridized

miRNA strands, akin to that of HRP-catalyzed polymerization of aniline and

DNA-guided deposition of PANI.250, 251 Supportive evidence of the formation

of the charge-transfer impeding PANI film on the biosensor surface was found

in voltammetric test of the biosensor (Figure 5-3 B). Unlike the control which

displayed the usual reversible voltammogram of Ru(NH3)63+/2+ with a peak-to-

peak potential separation (ΔEp) of 59 mV (Figure 5-3 B trace 2), a quasi-

reversible voltammogram with smaller peak currents and a larger ΔEp of 95 mV

was obtained with let-7b (Figure 5-3 B trace 1), again suggesting the presence

of a charge-transfer impeding layer on the biosensor surface. The smaller peak

currents and the larger ΔEp are direct consequence of the resistance to charge

transfer between the substrate gold electrode and Ru(NH3)63+/2+ redox probes.249

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Figure 5-3. (A) EIS spectra and (B) the corresponding voltammograms at

100 mV/s of a let-7b biosensor to (1) 100 fM let-7b and (2) 100 fM control RNA.

60 min hybridization at 50 °C and 30 min incubation in pH 3.0 of 0.10 M

potassium phosphate containing 10 μM DNAzyme, 20 mM aniline, and

200 mM hydrogen peroxide. Insets: Randle equivalent circuit used to fit the EIS

data: Rs – solution resistance, Cdl – double layer capacitance, Rct – charge-

transfer resistance, W – Warburg impedance element.

5.3.3 Optimization

Owing to unique attributes of miRNAs, there is little room to fine-tune the

miRNA hybridization conditions. Our efforts were therefore focused on the

optimization of the signal amplification (PANI deposition) process. In the case

of PANI deposition, the deposition rate must be determined by one or a

combination of the following: (i) mass-transport processes of aniline and

hydrogen peroxide in solution, (ii) DNAzyme-catalyzed polymerization of

aniline, which is dependent on the concentrations of DNAzyme, aniline, and

hydrogen peroxide, and (iii) miRNA-guided deposition of PANI. Under

selected experimental conditions, when both the mass-transport and aniline

polymerization are much faster than the PANI deposition process, the PANI

deposition rate is then solely controlled by the number of the hybridized miRNA

strands on the biosensor surface, and thus, by the concentration of the target

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miRNA. This sole miRNA-controlled process can be achieved by “speeding up”

polymerization and mass transport. High mass-transport rates are obtainable

when working with high concentrations of the reactants. It is critical that the

polymerization of aniline should be carried out in an acidic medium with a pH

value far below then pKa of aniline (pKa = ~ 4.6),254 so as to create a favorable

environment for the polymerization of aniline (fast polymerization rate), to

effectively utilize the hybridized miRNA strands as templates to guide the

deposition of PANI, and to minimize indiscriminative deposition of PANI by

forming electrostatic adducts along miRNA strands between cationic aniline

molecules and anionic phosphate moieties. Unfortunately, most natural

enzymes are active and stable only around neutral pH values. In contrast, the

chemical nature of the DNAzyme may be stable and remain active in a much

wider pH range, thus well-suited for this purpose. To verify this hypothesis, we

first evaluated the catalytic activity of the DNAzyme in the polymerization of

aniline and the deposition of PANI. It was found that the catalytic activity

toward the polymerization of aniline and deposition of PANI is strongly pH

dependent, attaining its maximal activity in the pH range of 2.5–3.0 (Figure 5-

4 A), reflected by the largest Rct observed. The amount of PANI found at the

biosensor surface dropped sharply to a negligible level once the solution of pH

was higher than 5.0, evidently due to a much lower polymerization rate and

weaker interaction between aniline and hybridized miRNA strands. No PANI

deposition was detected when the pH of the PANI deposition cocktail was > 6.0.

Figure 5-4 B shows the effect of the DNAzyme on Rct of the biosensor. It was

noted that as the DNAzyme concentration was increased, Rct increased rapidly

and almost linearly at first, but then slowly became practically independent of

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DNAzyme concentration at ≥ 10 µM, implying that the deposition of PANI is

now solely controlled by the amount of hybridized miRNA strands on the

biosensor surface. Further increase in DNAzyme resulted in excessive

polymerization of aniline and an appreciable increase in Rct of the control,

probably due to nonspecific deposition of PANI in the presence of large

excesses of PANI in the cocktail. As seen in Figure 5-4 B, Rct of the control

increased significantly from ~ 5 to 25 Ω cm2 in the PANI deposition cocktail

containing 20 µM DNAzyme, 20 mM aniline, and 200 mM hydrogen peroxide,

thus deteriorating the LOD from femtomolar to picomolar levels. Therefore, 5–

10 µM DNAzyme was chosen to ensure sufficiently high sensitivity and good

signal-to-noise ratios. Unlike HRP which can be easily deactivated by pH and

high concentrations of hydrogen peroxide, as a small HRP-mimic, the

DNAzyme is expected to be very robust. Indeed, the DNAzyme was found to

be much more tolerant than HPR in terms of reaction conditions. For example,

it was observed that the DNAzyme remains active in a much wider range of

hydrogen peroxide concentration of 10 μM to 400 mM, where enzyme like HRP

is quickly deactivated at hydrogen peroxide concentrations > 10 mM.

In addition, it was observed that a moderate amount of aniline (20 mM)

produces the best result Figure 5-4 C. Too low an aniline concentration resulted

in a slow polymerization and possibly a quick depletion of aniline. Too much

aniline in the PANI deposition cocktail may lead to the formation of highly

soluble oligomers and low molecular weight of PANI, thus reduced the PANI

deposition rate and adversely affect the sensitivity and LOD of the biosensor.

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Figure 5-4. Dependence of Rct on (A) pH, (B) DNAzyme, (C) aniline, and (D) hydrogen peroxide; after 60 min hybridization of 250 fM let-7b at 50 °C. Data points represented six replicates.

As for hydrogen peroxide, a high enough concentration of hydrogen peroxide is

the prerequisite for maximizing the amplification power of the DNAzyme. The

high concentration of hydrogen peroxide ensures that there is no substantial

depletion of hydrogen peroxide and the PANI deposition is totally independent

of hydrogen peroxide, which in turn infers that the amount of PANI deposited

on the biosensor surface would scale up with the amount of the number of

hybridized miRNA strands and the incubation in the PANI deposition cocktail.

As shown in Figure 5-4 D, Rct of the biosensor was maximized in the presence

of ≥ 100 mM hydrogen peroxide.

Since the hybridized target miRNA-guided deposition of PANI is

cumulative, Rct is also closely associated with the incubation time in the PANI

deposition cocktail. High sensitivity and low LOD are expected if the deposition

of PANI is allowed to proceed for a sufficiently long period of time. To leverage

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on the PANI deposition for sensitivity improvement, a series of EIS tests were

carried out with different incubation time in the PANI deposition cocktail.

Figure 5-5. Dependence of Rct of (1) 10 fM, (20) 250 fM, and (3) 5.0 pM let-

7b after 60 min hybridization at 50 °C on the incubation time in pH 3.0 of

0.10 M potassium phosphate containing 10 μM DNAzyme, 20 mM aniline, and

200 mM hydrogen peroxide. Data points represented six replicates.

A plot of Rct at different incubation time is shown in Figure 5-5. In all cases,

an immediate increase in Rct was observed due to the formation of the charge-

transfer impeding PANI films on the biosensor. At 10 and 250 fM let-7b, Rct of

the biosensor was found to be proportional to the incubation time throughout

(Figure 5-5 traces 1 and 2), suggesting that there is very little activity loss of the

DNAzyme during the incubation; whereas at a much higher concentration of

5.0 pM let-7b, Rct of the biosensor gradually leveled off after an initial linear

increasing phase (Figure 5-5 trace 3). The deviations from linearity after a

prolonged period of incubation is probably due to the fact that Rct of the

biosensor has reached such a high value that further deposition of PANI has

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significantly less impact on it. Eventually, Rct of the biosensor may reach a

steady state when the target concentration is sufficiently high and the incubation

time is sufficiently long. It was found that a period of 30 min incubation is

sufficient for the detection of femtomolars of miRNAs.

5.3.4 Analytical performance of the biosensor

Figure 5-6. Calibration curves for let-7b. 60 min hybridization at 50 °C and 30

min incubation in pH 3.0 of 0.10 M potassium phosphate containing 10 μM

DNAzyme, 20 mM aniline, and 200 mM hydrogen peroxide. Data points

represented six replicates.

Under optimized experimental conditions, standard solutions of synthetic

miRNA let-7b were used to evaluate the performance of the biosensor. As

shown in Figure 5-6, a linear relationship between Rct and let-7b concentration

was observed from 1.0 fM to 5.0 pM with a regression coefficient of 0.97 and a

relative standard deviation of ~ 14% at 100 fM. The LOD of the biosensor was

estimated to be 0.50 fM at a signal/noise ratio of 3.0 (the standard deviation

in Rct of six independent control biosensors was taken as the noise). As

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compared to previously reported EIS miRNA biosensors, the improved

sensitivity and decreased LOD are likely originated from the highly efficient

polymerization and deposition of PANI. Lower LOD was attainable with a

longer hybridization and PANI deposition time.

The capability of the biosensor in selectively detecting specific miRNAs in a

miRNA family was evaluated by analyzing three representative members of let-

7 family, namely fully complementary (let-7b), one-base-mismatched (let-7c),

and two-base-mismatched (let-7a). As seen in Figure 5-7, a 95% and 99% drop

in Rct were observed when let-7c and let-7a were tested on the let-7b biosensor,

respectively. In other words, selectivity factors of 20 and 100 were observed

with one-base mismatched and two-base mismatched miRNAs, respectively.

Negligible responses, indistinguishable from that of the control, were observed

when miRNAs with three or more bases are mismatched. This level of mismatch

discrimination ability between complementary and mismatched sequences is

better than those of many hybridization-based biosensors,35 most probably due

to the much higher sequence selectivity of PNA.255 Unlike thermal

amplifications employed in ligase chain reactions and qPCR, without a

denaturing step the proposed biosensor is less prone to interferences from pre-

miRNAs since their highly stable hairpin structures remain intact under the

hybridization conditions, making miRNA regions inaccessible for hybridization

to the PNA CPs. A selectivity factor of ~ 40 was obtained when equal molars

of let-7b and pre-let-7b were analyzed at the let-7b biosensor.

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Figure 5-7. EIS responses (Rct) of a let-7b biosensor to 100 fM of let-7b, let-7c, let-7a, let-7e, and pre-let-7b, respectively. 60 min hybridization at 50 °C and 30 min incubation in pH 3.0 of 0.10 M potassium phosphate containing 10 μM DNAzyme, 20 mM aniline, and 200 mM hydrogen peroxide. Data points represented six replicates.

Table 5-2. Comparison with other electrochemical detection methods for

miRNA

Method LOD

Selectivity for

one-base

mismatch

Detection

time Simplicity Repeatability Ref.

PNA probe+DNAzyme 0.5

fM 20:1 1.5 hrs + + + + + + + + +

This

work

Hairpin+DNAzyme+

AuNP

0.015

fM Not realized 3 hrs + + + + + + 256

DSN+alkaline

phosphatase

0.2

fM 15:1 40 min + + + + + + + 257

HCR+endonuclease+

DNAzyme

100

fM Not realized 5 hrs + + + + + + 258

HCR+ hairpin assembly

reaction

3.3

fM 40:1 4 hrs + + + + + + 259

The proposed bioassay for miRNA here was also compared with its

electrochemical counterparts. As summarized in Table 5-2, compared with the

previously published miRNA detection methods, the present work exhibited a

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good repeatability as well as selectivity toward one-base mismatched miRNA

family member. Some of the works have achieved ultralow LOD down to

several attomolar, however, they can not distinguish a specific miRNA among

its family members with high sequence similarity. And some of them achieved

comparable sensitivity, LOD and selectivity but sacrificed the simplicity.

5.4 Conclusion

The present study demonstrated a biosensor for miRNA expression profiling.

The combination of the charge neutral PNA CPs and the guided deposition of

PANI leads the unique amplified label-free EIS biosensor for miRNAs. Without

the need for miRNA labeling, the assay procedure is greatly simplified. The

adoption of the guided deposition of PANI provides a convenient means of

sensitivity improvement and LOD decreasement. Being capable of analyzing

miRNAs without thermal cycling and with minimal sample preparation, the

proposed biosensor is an attractive candidate for the development of a simple

and sensitive miRNA expression profiling tool for uses at point-of-care.

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Chapter 6. Conclusions and Suggestions for Future Research

In summary, the projects presented in this thesis were carried out successfully

to establish highly sensitive and selective bioassays for the detection of disease-

related nucleic acids, including DNA with SNPs and abnormally expressed

miRNAs.

In Chapter 2, a ferrofluid-based homogeneous bioassay for highly sensitive and

selective detection of disease-related SNPs was established. Excellent

selectivity and sensitivity as well as low LOD were accomplished in this assay

through the engagement of the FNPs and LCR. Firstly, colloidal MNPs with

PAA coating on their surface were modified with the two types of

oligonucleotides to prepare highly water soluble FNPs. Efficient homogeneous

hybridization between target DNA and the oligonucleotides on FNPs was

carried out and the first-step amplification was achieved by the incorporation of

LCR, in which a minute amount of target DNA crosslinks millions of the FNPs

together forming the aggregates of the FNPs. Separation of the aggregated FNPs

from the free ones was realized simply based on their drastic difference in

magnetic field strength under an external magnetic field. The intrinsic

peroxidase-like activity of the FNPs was then utilized to produce a detectable

colorimetric signal – the second step of amplification. The sensitivity was thus

greatly improved and LOD was successfully decreased by these two

amplification processes. The excellent sensitivity and low LOD were realized

through the judicious design of the two types of the FNPs with SNPs located at

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the ligation site as well as employing the allele-specific enzymatic LCR, the

latter is proved to be more specific than PCR regarding SNP genotyping.

Since it is a work of methodology development, most of the proof-of-concept

experiments were conducted with synthetic nucleic acid samples. In other words,

the proposed assay was only realized when the matrix effect was negligible and

has not yet been applied to real samples. More detailed investigations and real-

sample analysis can be done to further validate that the established assay can be

a prospective tool for uses in SNP detection at point-of-care. For example, the

colloidal MNPs prepared in Chapter 2 has a size of 6–8 nm and this is the only

material we employed to fabricate FNPs for SNP bioassays. Theoretically, all

the colloidal MNPs under 10 nm should have the properties we expected for this

bioassay. Considering that nanomaterials have dramatically different

performances when the size varied, colloidal MNPs with other sizes may also

be tried out in order to pursue the best performance when analyzing real samples.

As a whole view of SNP genotyping and quantification, rapid advances in SNP

research have offered an excellent opportunity to exploit SNPs for the purpose

of providing better diagnosis, prognosis and therapeutic assessment of human

disease. Robust, effective, and highly-specific SNP genotyping and

quantification techniques are therefore necessary for the study of SNPs and

diagnosis of disease. Despite impressive progress in research and development

of SNP genotyping and quantification techniques and multiplexing strategies,

no technique clearly represents a fundamental breakthrough. The most difficult

challenge may be the need to assess a large number of SNPs simultaneously.

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Judging from the speed with which new genotyping and quantification

techniques are being developed, there is great hope that an ideal SNP

genotyping and quantification platform will be developed in the near future.

Future efforts should therefore be devoted to development of SNP genotyping

and quantification techniques with enhanced sensitivity, selectivity,

multiplexing capability, decreased LOD, and greatly simplified protocols

without requiring any thermal amplifications. One possible approach is to

couple isothermal amplifications to allele-specific hybridization and a simple

signal-read-out mechanism, such as litmus test260 and colorimetry.261 Unlike the

thermal amplification strategies employed in many of the allele-specific

enzymatic reaction-based techniques, such as LCR and PCR, isothermal

protocols are more advantageous in the development of robust, easy-to-use SNP

genotyping and quantification platforms, which may eventually bring SNP

genotyping and quantification to point-of-care.

In Chapter 3, a simple and highly sensitive assay for disease-related miRNA let-

7a was developed by using MB-carried DNA detection probes coupled with

DSN-facilitated isothermal target cycling amplification. The proposed assay for

the detection of the target miRNA exhibited good sensitivity and selectivity as

well as low LOD, which should be attributed to the judicious design of the assay.

Firstly, the DNA detection probes were robustly attached to the MB carriers

through the strong interaction between the biotin moieties on the DNA detection

probes and the streptavidin moieties on the surface of the MBs. The dissociation

constant of biotin-streptavidin conjugates is seen to be small enough to warrant

that the release of fluorescent reporters (fluorescein) is only caused by the

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presence of target miRNAs. Secondly, the utilized DSN selectively cleaved the

DNA probes in the miRNA-DNA duplexes and released the target miRNA

strands back to solution, thereby establishing a target recycling amplification

mechanism. Such an accumulative nature of the target cycling amplification

offers a powerful means to significantly improve the sensitivity and decrease

the LOD of the assay. Moreover, the high specificity of the DSN to perfectly

matched duplexes endows this assay a good selectivity when analyzing target

miRNAs with high sequence similarities. Thirdly, the MBs ensure a complete

removal of possible interference from unreacted DNA detection probes before

fluorescence measurements, thus producing an ultralow fluorescence

background and an extended dynamic range.

The proposed assay was applied to analyze let-7a in three total RNA samples

extracted from cultured cells to evaluate its practicability. The results obtained

by the proposed assay agreed well with those obtained by the classic qRT-PCR

method, indicating that this assay is reliable for miRNA detection. Compared to

the qRT-PCR-based assays,262-264 the isothermal amplification via the DSN

facilitated target cycling together with its excellent selectivity is very valuable

since the freedom from thermal cycling would greatly improve the adaptability

of the assay for direct miRNA expression profiling with minimal requirement

of sample preparation. The simplicity, high sensitivity, low LOD, and freedom

from thermal amplification and miRNA tagging are some of the interesting

features for the development of a simple, robust, low cost, and highly sensitive

miRNA expression profiling tool used at point-of-care, which is of great

importance for early disease diagnosis and timely disease prevention. In

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addition, future work of real sample detection without sample pre-treatment

may be applied considering that without the denature step in isothermal assay,

pre-RNA with hairpin structure would not induce significant false signal and

thus would not affect the results of this assay. This is quite advantageous for

uses at point-of-care.

The final output fluorescent signals responding to target miRNAs at low

concentrations are relatively small. In the future work, fluorophores exhibiting

more intensive fluorescence such as Yakima yellow265 may be used to replace

fluorescein as the fluorescent reporter to modify the DNA detection probes. In

such a way, signals of different concentrations of target miRNAs at low

concentrations can differ from each other more distinctively, and therefore, the

assay could be more sensitive. Other fluorophores except the most popular

fluorescein were not been tried because that as long as the assay works and the

results are relatively good, the purpose of the project is achieved since we are

focused on methodology development.

The proposed assay demonstrated a detection method that only one miRNA

family member was analyzed each time. Multiplex detection using multiple

DNA capture probes on the MBs can be tried out in future research.

Theoretically, multiplex detection of more than one miRNA family member can

be achieved in a single tube undergone the same isothermal amplification

process provided that the emission spectra of different fluorophores are not

overlapped so that the output signals are not cross-interfered.

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In Chapter 4, a novel method for the synthesis of PANI in an acidic aqueous

medium, based on a G-quadruplex-hemin DNAzyme-catalyzed oxidation and

polymerization of aniline by hydrogen peroxide in the presence of a polyanionic

template, was demonstrated and well-studied for the first time. The kinetics of

the polymerization was found to be described by the classical Michaelis-Menten

mechanism. The linear correlation between the initial polymerization rate and

the concentrations of aniline and hydrogen peroxide were established, which

brings great benefit for its applications in bioassays. The synthesis is simple and

robust, and the experimental conditions are mild and environmentally friendly.

Due to these attractive features, this synthetic method was employed in Chapter

5 for disease-related miRNA detection.

In Chapter 5, a highly sensitive impedimetric miRNA bioassay was developed

on the basis of hybridized miRNA-guided deposition of PANI. A gold electrode

coated with charge neutral PNA CPs was first hybridized to target miRNAs.

The G-quadruplex-hemin DNAzyme catalyzed polymerization of aniline

demonstrated in Chapter 4 was then carried out, followed by the deposition of

PANI guided by the hybridized miRNA strands, thus resulting in the formation

of a thin PANI film on the surface of the gold electrode. The electro-transfer

impeding power of the PANI film was utilized to determine the concentration

of the target miRNA. Ultralow LOD down to lower femtomolar and superior

mismatch discrimination capability of this impedimetric bioassay was observed.

These should be largely due to the excellent hybridization selectivity of the PNA

CPs as well as the catalytic reaction-based signal amplification procedure.

128

When comparing Chapters 3 and 5, we can see that there is a similarity between

MBs and the gold electrode, i.e. both of them are used as carriers for CP

immobilization and can provide convenience when separating products from the

reaction mixture. Although MBs cannot replace the gold electrode mainly

because the gold electrode is essential for generating an electrochemical signal,

whereas the MBs could not even though they can be used in a way like the gold

electrode is used to some extent. Also, after some minor adjustments for signal

production, a series of new homogeneous bioassays could be constructed from

the heterogeneous ones, and they are expected to exhibit similar performance

but better repeatability.

All in all, the proposed bioassays for disease-related nucleic acids in this thesis

are still at proof-of-concept stage. Although one of them (project in Chapter 3)

has already obtained good sensitivity and selectivity as well as low LOD when

applied to real samples, it does not automatically ensure these assays a wide

spread applications or even commercialization. From application point of view,

the bioassay reported in Chapter 3 is the most possible one that can be widely

adopted and commercialized because it is one-step, isothermal, simple, fast,

reliable, and repeatable with relatively good selectivity and sensitivity as well

as low LOD. In addition, since most of the materials employed in the assay are

already commercially available, it implies that the assay are closer to

commercialization compared with other bioassays developed in this

thesis. Moreover, the possible property of free from pre-treatment is absolute

advantageous in point-of-care applications. These attractive features should be

129

born in mind when we are developing a reliable and practical bioassay for the

detection of disease-related nucleic acids.

130

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List of Publications

[1] W. Shen, C.L. Lim, Z. Gao, A ferrofluid-based homogeneous assay for

highly sensitive and selective detection of single-nucleotide polymorphisms,

Chem. Commun. 49 (2013) 8114-8116.

[2] W. Shen, H. Deng, Z. Gao, Synthesis of polyaniline via DNAzyme-

catalyzed polymerization of aniline, RSC Adv. 4 (2014) 53257-53264.

[3] W. Shen, Z. Gao, Quantum dots and duplex-specific nuclease enabled

ultrasensitive detection and serotyping of Dengue viruses in one step in a single

tube, Biosens. Bioelectron. 65 (2015) 327-332.

[4] W. Shen, K.H. Yeo, Z. Gao, A simple and highly sensitive fluorescence

assay for microRNAs, Analyst 140 (2015) 1932-1938.

[5] W. Shen, Y. Tian, T. Ran, Z. Gao, Genotyping and quantification

techniques for single-nucleotide polymorphisms, Trends Anal. Chem. 69 (2015)

1-13.

[6] H. Deng, W. Shen, Y. Peng, X. Chen, G. Yi, Z. Gao, Nanoparticulate

Peroxidase/ Catalase Mimetic and Its Application, Chem. - Eur. J. 18 (2012)

8906-8911.

[7] Z. Gao, W. Shen, H. Deng, Y. Ren, Detection of single-nucleotide

polymorphisms based on the formation of an electron-transfer impeding layer

on an electrode surface, Chem. Commun. 49 (2013) 370-372.

[8] H. Deng, W. Shen, Y. Ren, Z. Gao, A highly sensitive microRNA

biosensor based on hybridized microRNA-guided deposition of polyaniline,

Biosens. Bioelectron. 60 (2014) 195-200.

[9] S.Y. Lim, W. Shen, Z. Gao, Carbon quantum dots and their applications,

Chem. Soc. Rev. (2015) 362-381.