DEVELOPMENT OF ELECTROCHEMICAL DNA BIOSENSOR TO

DETECT SHORT DNA SEQUENCES

Dissertation submitted to the SASTRA University

in partial fulfillment of the requirements

for the award of the degree of

Bachelor of Technology in Bioengineering

By

G.S.SANTHOSHI SADHANAA (117011018)

V.VIJAYALAKSHMI (117011023)

SCHOOL OF CHEMICAL AND BIOTECHNOLOGY

SASTRA UNIVERSITY

(SHANMUGHA ARTS SCIENCE TECHNOLOGY & RESEARCH ACADEMY)

THANJAVUR – 613401, TAMILNADU, INDIA

April 2017

SASTRA UNIVERSITY

(SHANMUGHA ARTS SCIENCE TECHNOLOGY & RESEARCH ACADEMY)

(A University under section 3 of UGC Act 1956)

Tirumalaisamudram, Thanjavur-613 401, Tamil Nadu, India

BONAFIDE CERTIFICATE

This is to certify that the thesis, entitled “Development of electrochemical DNA biosensor to

detect short DNA sequences”, submitted to SASTRA University, in partial fulfillment of the

requirements for the award of the Degree of Bachelor of Technology in Bioengineering, is a

bonafide record of the work done by Ms. G.S. Santhoshi Sadhanaa (117011018) &

Ms. V. Vijayalakshmi (117011023) during the Final Semester of the academic year 2016-2017,

in the School of Chemical and Biotechnology, under my supervision and guidance. The

dissertation has not formed the basis for the award of any Degree / Diploma /Associateship /

Fellowship or other similar title to any candidate of any University.

Project Supervisor Associate Dean

Internal Examiner External Examiner

ACKNOWLEDGEMENTS

We take immense pleasure in thanking Prof. R. Sethuraman, our beloved Vice-Chancellor for

having permitted us to carry out this project work. We express our profound gratitude to Dr. S.

Swaminathan, Dean, (Sponsored Research) SASTRA for his overwhelming support throughout

our project.

We take it the rarest opportunity and proud privilege to express our profound sense of

immeasurable and debt of gratitude to Dr. R. John Bosco Balaguru, Associate Dean (Research),

SEEE, SASTRA University for his guidance, duly evaluating my progress and always

encouraging me by his invaluable suggestions and encouraging me during this project work. I also

thank all other members in the institute for providing me all the information, suggestions and

improvements at every stage of my project.

We owe deepest thanks to Dr. G. Arun Kumar, Professor SCBT and Dr. N. Noel, Professor,

SCBT, SASTRA University for their advice and support. We also extend our thanks for the lively

association and affection showered on us by Ph.D. scholars Ms. G. Manju Bhargavi and Ms.

Bhat Lakshmishri Ramachandra.

We wish to express my deep sense of gratitude to all the faculty members, my friends and others

who involved directly or indirectly for their encouragement during the project.

We sincerely thank Nanosensors laboratory, ASK-I, SASTRA University for their support in our

project work.

G.S. Santhoshi Sadhanaa (117011018)

V.Vijayalakshmi (117011023)

LIST OF CONTENTS LIST OF TABLES i

LIST OF FIGURES ii

LIST OF SYMBOLS iv

LIST OF ABBREVIATIONS v

ABSTRACT vi

CHAPTER 1: INTRODUCTION 1

1.1 Biosensors 1

1.2 Components of Biosensor 1

1.3 Classification of Biosensor 2

1.4 Methods Of Immobilization 7

1.4.1 Physical Immobilization 8

1.4.2 Chemical Immobilization 8

1.5 Applications of Biosensor

1.6 DNA Biosensor 8

1.6.1 Nucleic acid hybridization 9

1.7 Metal oxide and their sensing application 10

1.7.1 Significance of Zinc Oxide 11

1.7.1.1 Properties of Zinc Oxide 11

1.8 Scope of DNA Biosensor 11

1.8.1 HPV and its impacts 11

1.8.2 Importance of DNA Sensor for detection of HPV 12

CHAPTER 2: REVIEW OF LITERATURE 13

2.1 Review of HPV 13

2.2 Review of Electrochemical Sensing 13

2.3 Review of Zinc Oxide 14

2.4 Review of Microwave assisted synthesis 15

CHAPTER 3: AIMS AND OBJECTIVES 17

CHAPTER 4: MATERIALS AND METHODS 18

4.1 Materials and Reagents 18

4.1.1 Electrode System 18

4.2 Sample Preparation 18

4.3 Synthesis of Zinc Oxide Nanoparticles 19

4.4 Pre-treatment of working electrode 19

4.5 Fabrication of Electrode 20

4.6 Characterization Techniques 20

4.6.1 Scanning Electron Microscopy (SEM) 20

4.6.1.1 Experimental Setup 21

4.6.1.2 Advantages of SEM 21

4.6.2 X-Ray Diffraction (XRD) 21

4.6.2.1 Advantages 21

4.7 Electrochemical Sensing 22

4.7.1 Cyclic Voltammetry 22

4.7.2 Amperometry 22

4.7.3 Impedometry 23

CHAPTER 5: RESULTS AND DISCUSSIONS 24

5.1 Characterization Results

5.1.1 SEM Analysis for Zinc Oxide Nanoparticles 24

5.1.2 XRD data for Zinc Oxide Nanoparticles 25

5.2 Electrochemical Studies 26

5.2.1 Cyclic Voltammetry Studies

5.2.1.1 Effect of different electrode system 26

5.2.1.2 Effect of scan rates 28

5.2.1.3 Effect of Varying Concentrations 29

5.2.2 Amperometric Studies 31

5.2.3 Impedometric Studies 31

CHAPTER 6: CONCLUSION 32

CHAPTER 7: REFERENCES 33

ABSTRACT

In order to identify specific DNA sequences, an electrochemical DNA biosensor was developed

by modifying the electrode surface with zinc oxide nanoparticles. Here, the sensing was based on

the principle of nucleic acid hybridization, which involves base pairing between two

complementary sequences of nucleic acids. Zinc oxide nanoparticles were synthesized by

microwave method with zinc chloride and sodium hydroxide as precursors. These nanoparticles

were characterized using SEM and XRD. Fabrication of electrode was done by coating of zinc

oxide nanoparticles on the surface of working electrode (gold electrode) followed by

immobilization of the probe sequences specific to the target sequences. Zinc oxide nanoparticle

has good biocompatibility and high IEP (~ 9.5) to obtain strong physical adsorption.

Electrochemical detection of DNA hybridization will be done based on three parameters such as

potential, current and impedance based on cyclic voltammetric, amperometric and impedance

studies respectively. Electrochemical sensing of DNA sequence was done in the absence and

presence of zinc oxide nano-interface. 200 nucleotides and 20 nucleotides sequences were sensed.

Cyclic voltammograms of a probe of 200 nucleotides sequences and its respective target sequences

in the concentration range of 0.1 to 0.7 nM were studied with an optimized scan rate of 0.09 Vs-1.

Similarly a probe of 20 nucleotides and its target sequences of 96 pM, 100 pM and 108 pM were

also sensed in a similar way. Enhanced electron transfer offered by the ZnO nano-interface helped

to achieve better hybridization. As a result, rapid detection of DNA hybridization was observed.

Thus, sequence-selective DNA hybridization was examined in different cases by varying the

number of nucleotides and targets with different concentrations of target sequences. With the help

of these features, electrochemical DNA biosensor was tested for its efficacy to detect specific DNA

hybridization.

KEYWORDS: Zinc oxide nanoparticles, microwave, probe, target, electrochemical,

hybridization

i

LIST OF TABLES

Table 1: Comparison of different electrode system for 200 nucleotides and 20 nucleotides – 27

ii

LIST OF FIGURES

FIGURE NUMBER FIGURE NAME PAGE NUMBER

Figure 1 Components of a biosensor 1

Figure 2 Flowchart representation of

biosensors based on type of

transducer

2

Figure 3 Electrochemical cell setup 3

Figure 4 Schematic representation of an

optical biosensor

4

Figure 5 Classification of an optical

biosensor based on A) Optical geometry B) Transduction

5

Figure 6 Schematic representation of a

thermal biosensor

6

Figure 7 Schematic representation of an

acoustic biosensor

7

Figure 8 Methods of immobilization 7

Figure 9 Physical immobilization 8

Figure 10 Chemical immobilization 8

Figure 11 Schematic representation of a

DNA biosensor

9

iii

Figure 12 Microwave synthesis of ZnO

nanoparticles

19

Figure 13 Fabrication of working (gold)

electrode

20

Figure 14 Cyclic voltammogram 22

Figure 15 The FE-SEM micrograph of

the zinc oxide particle

24

Figure 16 X-ray diffraction pattern of

ZnO particle

25

Figure 17 CV for DNA sequences

showing the effect of different

electrode system

A) 200 nucleotides

B) 20 nucleotides

27

Figure 18 CV for DNA sequences

showing the effect of scan rate

A) 200 nucleotides

B) 20 nucleotides

28

Figure 19 CV for 200 nucleotide

sequences showing the A)

Effect of varying concentration

B) Bar graph representation

versus current

29

Figure 20 CV for 20 nucleotide

sequences showing the A)

Effect of varying concentration

B) Bar graph representation

versus current

30

Figure 21 Amperometric studies of 200

nucleotide sequences

31

Figure 22 Impedometric studies of 200

nucleotide sequences

31

iv

LIST OF SYMBOLS (If applicable)

pM Picomolar

µΩ Micro Molar

β Beta

nM Nano Molar

Ґ Surface coverage

Q Charge obtained by integration of anodic peak

n Number of electrons

F Faraday’s constant

A Area of the electrode

W Watt

C Coulomb

C Celsius

M Molar

mm Millimeter

λ Wavelength of the x-ray

θ Angle between the incident ray and the surface of the crystal

d Spacing between the layers of atoms

n Integer

v

LIST OF ABBREVIATIONS

DNA Deoxyribo Nucleic Acid

HPV Human Papilloma Virus

HIV Human Immunodeficiency Virus

IEP Iso Electric Point

ZnO– Zinc Oxide

ZnCl2 Zinc Cholride

NaOH Sodium Hydroxide

PCR Polymerase Chain Reaction

Au Gold

Ag Silver

AgCl Silver Chloride

CA Concentration of solution before dilution

CB Concentration of solution before dilution

VA Volume of solution after dilution.

VB Volume of solution before dilution

Rpm Revolutions per minute

SEM Scanning Electron Microscope

XRD X –ray diffraction

CV Cyclic Voltammetry

1

CHAPTER 1: INTRODUCTION

In this project, we have developed a nano-interface based biosensor for detection of short DNA

sequences. In order to perform this, the working electrode was fabricated with zinc oxide

nanoparticles along with probe DNA sequences and then electrochemical sensing was done.

We will discuss about the importance of biosensors in various fields, different types of

biosensors, various methods of immobilization and also the significance of zinc oxide

nanoparticles in electrochemical DNA biosensors.

1.1 BIOSENSORS:

Biosensors are analytical device which are used for the detection of an analyte by conversion

of biological response into electrical signal. Here the analyte or samples could be blood, urine,

saliva, cell cultures and food samples. The various types of biosensors are enzyme sensor,

immuno sensor, DNA sensor, thermal sensor and piezoelectric biosensor.

1.2 COMPONENTS OF A BIOSENSOR:

The three main components of a biosensor are as follows:

Bio receptor

Transducer

Signal processing unit

Data processing unit

Bio receptor is the region wherein the desired analyte is made to bind and then the biological

signal is converted into electrical signal by a transducer. This electrical signal is then amplified

with the help of signal processing unit and the obtained information or data are analysed using

data processing unit.

Fig.1: Components of a biosensor.

2

1.3 CLASSIFICATION OF BIOSENSORS:

Biosensors can be classified into different types based on various criteria’s as follows:

A) Based on the nature of interaction between biological material and the desirable analyte

B) Based on the types of transducer

A) BASED ON THE NATURE OF INTERACTION:

There are two types of biosensors based on the type of nature of interaction between the

biological molecules and the desired analyte. They are:

1) Catalytic biosensor

2) Affinity biosensor

1) CATALYTIC BIOSENSOR:

This type of biosensor employs the reaction between enzyme and substrate and the result is

analysed by the formed product. Here the enzyme will act as the bioreceptor with substrate

acting as the analyte.

2) AFFINITY SENSOR:

Here the bio receptor could be single stranded nucleic acids or antibodies. The possible analyte

could be either the other strand of nucleic acids or antigen respectively. For example, DNA

sensor is a type of affinity sensor. DNA sensor mainly employs binding between the two

strands of DNA which is based on electrostatic interaction between the nucleotides.

B) BASED ON THE TYPES OF TRANSDUCER:

BIOSENSORS BASED ON TYPES OF TRANSDUCER

ELECTROCHEMICAL

POTENTIOMETRIC

AMPEROMETRIC

CONDUCTOMETRICOPTICAL

THERMAL

RESONANT

Fig.2: Flowchart representation of biosensors based on type of transducer.

3

ELECTROCHEMICAL BIOSENSOR:

This sensor involves quantification of the analyte by means of redox reaction. Electrochemical

sensing is done based on the measurement of current or potential difference or conductance

obtained when the analyte makes contact with the bio receptor. The basic principle in

electrochemical sensing is the consumption or production of electrons as a result of chemical

reaction between the analyte and the bio receptor. Electrodes play a very important role in

electrochemical sensing. In most cases, three electrode system is preferred. Three electrode

system consists of working electrode, reference electrode and counter electrode. Working

electrode could be gold electrode, glassy carbon electrode, silver electrode, platinum electrode

and copper electrode. The function of working electrode is to transfer electrons to signal

processing unit. Bio receptor was placed on the surface of working electrode. There will be

production of ions and as a result, potential difference is created with respect to reference

electrode. The changes in the electron conduction may be due to pH changes, ionic strength

and redox reaction.

Fig.3: Electrochemical cell setup

A) POTENTIOMETRIC:

In this type of electrochemical sensing, the parameter taken into consideration will be

either oxidation potential or reduction potential. The principle behind electrochemical

sensing is there will be production of current as a result of an applied voltage. Based on

the applied voltage, detection is made.

B) AMPEROMETRIC:

At a constant potential, due to oxidation or reduction of electro active species, there will be

production of current. The magnitude of current is directly proportional to the concentration

of an analyte. In some cases, there will be a mediator which takes up the electrons and then

transfers it to the electrode. Amperometric sensors can work both in two or three electrode

configurations. Two electrode system consists of working electrode and reference electrode.

The drawback of two electrode configuration is maintaining the potential at the surface of

working electrode in the case of higher currents. In case of three electrode system, the third

electrode will be a counter electrode. Potential is applied between working and reference

4

electrodes and there will be current flow between the working electrode and counter

electrode.

C) CONDUCTOMETRIC:

In this type of electrochemical sensing, the parameter to be measured is resistance or

conductance of the solution. There will be change in resistivity or conductivity of the

solution when there is production of ions or electrons. Other way of measuring

conductance is by direct means of immobilization of the bio receptor onto the electrode

surface.

OPTICAL BIOSENSORS:

The important components of an optical biosensor are light source, transmission medium,

bioreceptor region and light detection system. Mostly, fibre optics are used as transducer in

optical sensing. When the incident light hits the sample, there will be change in optical

properties such as absorbance, reflection, fluorescence, refractive index, wavelength,

amplitude and also phase shift during transduction process.

Fig.4: Schematic representation of an optical biosensor

The features of optical biosensors are:

Selectivity and specificity

No electromagnetic interference

Biocompatibility

Remote sensing

CLASSIFICATION OF OPTICAL BIOSENSORS:

Optical biosensors can be classified based on the following characteristics:

1) Transduction

5

2) Optical geometry

B)

Fig.5: Classification of an optical biosensor based on A) Optical geometry and

B) Transduction

Disadvantages of optical biosensor:

Very expensive

Difficulty in immobilization

Loss in some of the optical properties

THERMAL BIOSENSOR:

Some biological materials can be detected and analysed based on the heat produced. It is

otherwise called as calorimetric biosensor. Temperature plays an important role in this

biosensor whose measurement is accomplished via the presence of a thermistor in the sensor

setup. The main feature of thermal biosensor is there is no need for frequent calibration of the

instrument. Here the bioreceptor is attached in such a way that it makes contact with the

thermistor so that, in the presence of an analyte, there will be a heat reaction. Based on this, the

concentration of that analyte could be estimated. The main disadvantage faced with thermal

biosensor is the maintenance of temperature.

A)

OPTICAL GEOMETRY

SURFACE PLASMON RESONANCE (SPR)

BIO - OPTRODEFIBRE GRATING BASED SENSOR

TRANSDUCTION

ABSORPTION FLUORESCENCE LUMINESCENCE

6

RESONANT BIOSENSOR:

Resonant biosensor is otherwise called as acoustic biosensor. Since it employs the principle of

piezoelectric effect, it is also called as piezoelectric sensor. In the presence of an analyte, the

mass of bio receptor changes which in turn results in change in frequency. In other words, with

the help of piezoelectric crystals, the sound waves are excited at an input transducer and

detected with an output transducer.

1.4 METHODS OF IMMOBILIZATION:

There are two methods of immobilization – physical and chemical immobilization.

Fig.6: Schematic representation of a thermal biosensor [Source: Google images]

Fig.7: Schematic representation of an acoustic biosensor. [Source: Google images]

7

1.4.1 PHYSICAL IMMOBILIZATION:

It is the most common method of surface immobilization because it does not require any

reagents during immobilization process. Entrapment type of immobilization results in very less

damage to the enzyme during its catalytic activity in the case of enzyme biosensor.

1.4.2 CHEMICAL IMMOBILIZATION:

In the case of crosslinking, there will be presence of functional reagents which cross links with

the biomolecule. The main advantage of crosslinking is the strong binding of biomolecule with

the functional reagents. But the disadvantage faced with crosslinking is the activity loss. In the

case of covalent coupling, there will be change in the conformation of the biomolecule.

IMMOBILIZATION

PHYSICAL

ADSORPTION

ENTRAPMENT

CHEMICAL

COVALENT COUPLING

CROSS LINKING

Fig.8: Methods of immobilization.

Fig.9: Physical immobilization.

8

1.5 APPLICATIONS OF BIOSENSORS:

Biosensors has numerous applications owing to its small size, sensitivity, selectivity, low cost

and quicker detection. The main applications of biosensors are as follows:

1. Biosensors play an important role in maintaining the environment (i.e.,) in controlling

the environmental pollution. Pesticide sensor is an example for environmental type of

sensor. The need for oxygen is also measured by this type of biosensors. Biosensors are

helpful to evaluate the amount of environmental pollutants.

2. Biosensors is helpful in estimating various ions and also fermentation products. To

obtain a better yield. In order to measure the odour of food samples, biosensors are

highly beneficial.

3. They are highly advantageous in detection of hazardous gases especially for military.

4. One of the most important application of biosensor is healthcare related. They play a

very crucial role in estimating different biological ssubstances present inside the body.

Glucose biosensor is one of the best-known biosensor in the field of medicine and

healthcare. These biosensors are highly useful in diagnosis of numerous diseases.

1.6 DNA BIOSENSORS:

DNA biosensor is a type of genosensor and the sensing depends on nucleic acid hybridization.

DNA sensing has gained importance owing to its major application in the field of medicine and

healthcare.

Fig.10: Chemical immobilization.

9

1.6.1 NUCLEIC ACID HYBRIDIZATION:

Nucleic acid hybridization is a process where the single strand nucleic acid molecule is made

to interact with their synthesized complementary sequences. The principle of nucleic acid

hybridization is the formation of hydrogen bond due to affinity between the two strands. It is

the measure of sequence similarity and from that process we can also detect the specific type

of sequences. Earlier, nucleic acids were detected by visualizing under the electron microscope.

Here, the process can be carried out in a liquid buffer or some type of gels or nitrocellulose

paper where one strand of nucleic acid (probe which are radio labelled or with fluorescent dye)

are immobilized.

Three types of nucleic acid hybridization are possible:

DNA-DNA hybrid

DNA-RNA hybrid

RNA-RNA hybrid

1.7 METAL OXIDES AND THEIR SENSING APPLICATION:

In recent years, nanomaterials play a crucial role in the development of biosensor. Among

different nanomaterials, metal oxides have its own characteristics which can be listed as

follows:

• Good biocompatibility

• Morphological structure in nano level

• Better sensing characteristics

• Strong physical adsorption properties

• Enhanced electron transfer

• Non – toxicity

• Highly suitable for immobilization of biomolecules

Fig.11: Schematic representation of a DNA biosensor

10

Following are the various methods by which these metal oxides can be synthesized in nano

scale:

• Sol-gel methods

• Sputtering technology

• Hydrothermal technique

From either of these techniques, metal oxide nanoparticles can be synthesized with uniform

size and appropriate morphology.

The main challenge in the development of a biosensor is the fabrication of electrode. An

excellent fabrication is possible with the help of the metal oxide nanoparticles.

DIFFERENT METAL OXIDES FOR BIOSENSING APPLICATION:

Cerium oxide

Cupric oxide

Manganese oxide

Magnesium oxide

Zinc oxide

Iron oxide

Titanium oxide

Tin oxide

Zirconia

SALIENT FEATURES OF THE METAL OXIDES:

1. BIOCOMPATIBILITY: This is the most important feature of most of the

nanoparticles based on metal oxides. Biocompatibility is the foremost property

required for a metal oxide to be used in a biosensing application with metal oxides

as the interface between the electrochemical and biological parts.

2. ISOELECTRIC POINT: It is defined as the pH exhibited by a molecule at which it

does not possess any electric charge.

3. ELECTRON TRANSFER: The main benefit of metal oxides is they help to promote

the electron transfer

1.7.1 SIGNIFICANCE OF ZINC OXIDE:

Many researchers proved that, zinc oxide is the best among the various metal oxides which is

suitable for the biosensing application.

1.7.1.1 PROPERTIES OF ZINC OXIDE:

Nanostructured zinc oxide is a semiconductor based nanomaterial

11

Researchers identified a strong electrostatic interaction between the zinc oxide which

is positively charged and the DNA sequences which is negatively charged.

Very low detection limit for biological samples and offers higher sensitivity

They possess larger excitation energy of around 60 meV

Zinc oxides has wider band gap which is almost equal to 3.37 eV

Very good biocompatibility which is highly necessary for biosensors

They offer high isoelectric point of about 9.5 and this is perfect in the case of DNA

biosensors

Increased surface area for DNA immobilization and hybridization

1.8 SCOPE OF DNA BIOSENSORS:

1.8.1 HPV AND ITS IMPACTS:

Human papilloma virus is the most common cause of sexually transmitted disease affecting

both men and women throughout the world. It is found to be a very significant topic due to its

high rate of morbidity and mortality and is found to be rapidly increasing nowadays. More than

200 different types of HPV types are recognized based on DNA sequence data. Among these

types, 85 HPV genotypes are found to be very well characterized and 120 other HPV types are

partially characterized. In general, HPV mainly infects basal epithelial cells of skin and

anogenital epithelium. It also targets the inner lining of tissues. To be more specific, it infects

the cutaneous (hands and feet) and also the mucosal types (lining of mouth, throat and

respiratory tract).

Sexually transmitted HPV types are categorized into two types: Low risk HPV genotypes and

high risk HPV genotypes. Low risk HPV genotypes are as follows:

HPV -6, 11, 42, 43, 44

High risk HPV genotypes are as follows:

HPV – 16,18,31,33,34,35,39 etc.

Though low risk HPV genotypes does not cause cancer but it can cause Condylomata

acuminata (skin warts) around the genitals and anus. It also targets the respiratory tract causing

benign tumors. High risk HPV genotypes are highly known for causing cancer. Among various

high risk HPV genotypes, HPV -16 and HPV -18 causes cervical cancer.

1.8.2 IMPORTANCE OF DNA SENSOR FOR DETECTION OF HPV:

Though many conventional techniques are used for the detection of HPV and cervical cancer,

the level of effectiveness is not assured. Some of the common conventional techniques

available for the detection of HPV are Pap smear test (cytological test), analysis of protein

expression, VIA test and DNA test. These above mentioned techniques suffer from many

disadvantages. Cytological test does not provide clear information at molecular level and hence

fails for the diagnosis in molecular level. VIA test is less accurate and also shows moderate

specificity and the quality is not assured. DNA test is highly time consuming. Pap test also has

a drawback that some of the cells are lost during fixation step of histological analysis and other

12

disadvantage is the accurate collection of cells from the infected area. Owing to these

disadvantages, biosensors is the best alternative for the detection of HPV sequences since they

offer good sensitivity and specificity.

Therefore, the main aim of this project work is to develop an electrochemical DNA sensor

based on ZnO platform for the detection of DNA sequences by means of hybridization principle

and in future, this sensor can be used for the detection of HPV sequences, thereby cervical

cancer can be diagnosed at a very early stage itself [8].

13

CHAPTER 2: REVIEW OF LITERATURE

2.1 REVIEW ON HPV:

1) Elaine VM Souza et al., [16] explained about the prevailing risk for the development

of cervical cancer which is mainly due to HPV type 16 sequences. This paper has

described about the electrochemical sensing for the sequence specific detection of E1

HPV 1 (type 16) in the presence of methylene blue as the indicator. They concluded

that the detection limit of the HPV sequences respective to its complementary

sequences was found to be 1.49 pM.

2) Isaac A.M. Frias et al., [13] stated about the conventional strategies available for the

detection of HPV sequences. Since these techniques are very complex, sensing is

preferred. It involves the interaction between the biological molecule and its transducer

for detection of the HPV sequences. Different methodologies have been adopted to

study the detection which are as follows: electrochemical sensors, optical sensors and

thermal biosensors. They concluded that future researches need to be conducted in order

to increase the rates of immobilization and also to increase the stability of probe

sequences.

3) Danielly S.Campos-Ferreira et al.,[7] developed an electrochemical sensor for

detection of HPV type 6 sequences in real samples. Here, the electrochemical sensing

was studied by differential pulse voltammetry in the presence of methylene blue

indicator. Conventional techniques for detection of HPV sequences has many

disadvantages owing to their very low specificity. Few advantages of electrochemical

DNA sensor are cheaper, simpler and effective sensing. Due to difficulties faced with

conventional techniques, diagnosis of HPV type 16 sequences should be done using

sensing and nano technology.

2.2 REVIEW ON ELECTROCHEMICAL SENSING:

1) Ana Maria Olveira Brett [2] stated in a review article that the major advantage of

choosing electrochemical method for sensing of DNA sequences is because of the rapid

response time, quantitative analysis, sensitivity, less expensive. The behaviour of DNA

sequences upon electrochemical technique paves way for detection of damaged DNA

sequences. This enables us to study the interaction of DNA immobilized on the surface

of electrode with the target sequences in solution.

2) T. Gregory Drummond et al., [10] proposed that electrochemical based sensors has

features such as sensitivity, selectivity and also cheaper for the detection of DNA

sequences. This review article focused on three properties as follows: Direct DNA

electrochemistry, indirect DNA electrochemistry, enhanced electrochemical response

depending upon the presence of specific nanoparticles, detection of DNA sequences

based on redox indicators. Some of the most important challenges faced with

electrochemical sensing is the fabrication of electrode and complexity with the

14

biological samples. But it offers a huge advantage of having inherent sensitivity for the

corresponding DNA sequences.

3) Eric Bakker et al., [8] explained about different types of electrochemical sensors and

their applications in biological perspective. Researchers prefer electrochemical sensing

due to their quick, simple and inexpensive characteristics for detection of biological

molecules. Several detection approaches were recognized as follows: direct detection

technique, use of some mediators, labelling with enzymes and other labelling

compounds.

4) Lam Dai Tran et al., [16] carried out their research based on HIV sequences by means

of electrochemical detection based on iron oxide nanoparticles. The main feature of this

study is their micro environment which results in enhanced electron transfer and good

sensitivity which was confirmed in the presence of methylene blue indicator. Though

iron oxide nanoparticles have certain advantages such as highly sensitive and enhancing

electron transfer, there could be biocompatibility issues.

2.3 REVIEW ON ZINC OXIDE:

ZINC OXIDES:

Zinc oxides are classified under the category of metal oxides which have the most stunning

unique property for the areas of the sensing application especially in bio sensing for nano level

sensing. From the literature survey, the properties of the zinc oxides are listed as having high

ratio of surface to volume, high IEP, nontoxicity, good biocompatibility, faster electron transfer

between the analyte and the working electrode and also provide the nano structure stability.

1. Shafiquzzaman Siddique et al., [15] Zinc oxide nanoparticles were taken to be one of the

most famous as well as important nanoparticle, because of the unique characteristics such as

electronic, metallic and structural properties. Most of the research work in sensing have

concerned about the high electron transfer property of the zinc oxide nanoparticle to promote

the conductance between the electrically active species and the working electrode at nano

interface. The zinc oxide nanoparticle also has the great advantage to increase the surface area

to promote the more binding of the receptor and the target in order to enhance the sensitivity

and the rapid occurrence of the process.

2. Maumita Das et al., [5] zinc oxide particle does have property of compatible with biological

environment in case of sensing with the nucleic acids, antibodies and other biological elements.

It also have the isoelectric point in the range of (~9.5) which gives good and strong physical

adsorption between the nucleic acids (DNA) (~4.5) by the high electrostatic interaction. Other

than those properties, it has the high electron transport with no toxicity, chemically stable

which also enhance the good immobilization of the receptor on to the surface of the electrode

and it can be recruited for most of the bio sensing techniques.

3. Tugrul Yumak et al., [18] Recent works are more eager about the some unique character

of the zinc oxide nanoparticle such as broad band gap and high excitation energy. Several types

of nanostructure of zinc oxide particle such as nanorods, nanosphere, nanoflowers, nanosheets,

15

nanospikes can also be easily modified, because it provides good and stable nanomorphology.

It also has several advantages in the application such as biosensor, batteries, nanolasers etc.

2.4 REVIEW ON MICROWAVE ASSISTED SYNTHESIS:

MICROWAVE SYNTHESIS:

This technique is one of the simple, faster and easy method of hydrothermal synthesis of

nanoparticle. It was considered to be more eco-friendly and effective in consumption of energy.

Furthermore, it promotes the material quality and uniform structure, and then it also enhance

the uniform temperature distribution and yield of the particle. By this method, different types

of nano structure are produce by varying different parameters.

1. M. Hasanpoor et al., [3] Nowadays, currently undergoing research shows interest on

microwave assisted hydrothermal method to produce the particle such as hydroxide, sulphide

and oxides. This type of microwave assisted technique prevents the problem related to thermal

gradient factor. In comparison with the other ancient technique for hydrothermal synthesis, it

provides simple and easily controlling environment. It makes the reaction to be fast and within

that simple environment suitable temperature and pressure for the reaction was easily attained

with short time. It also makes the uniform structure and size distribution of the preferred

nanoparticle in controlled manner.

2. G. Barreto et al., [4] In order to overcome the disadvantage of the ancient method of

hydrothermal techniques, microwave assisted technique was employed in current studies and

research. Here, by the use of the microwave radiation the difficulty created by the thermal

factors are easily eliminated. In the microwave assisted method, microwave heating generally

employs mainly two types of mechanism such as dipole-dipole vibration and conduction

between the ionic species. The process of the reaction linked directly to the chemical mixture

of the preferred particles, where the reaction happened because of the conversion of

electromagnetic microwave radiation to the thermal radiation as energy.

3. Ashok K. Singh et al., [3] Among the different types of nanomaterial synthesis, microwave

method of preparation provides effectiveness in the reaction process and also have simple

reaction mechanism, where the thermal energy by electromagnetic radiation are directly

applied to the samples, where in case of convectional hydrothermal nanoparticle synthesis there

is loss of heat due to the enlarged medium for reaction process. By this technique, the heat

energy was penetrated over the particle, results in uniform supply of heat energy throughout

the surface and volume of the particle.

16

CHAPTER 3: AIMS AND OBJECTIVES

The overall objective of this project is to detect hybridization of short DNA sequences at nano

interface with ZnO as nano particle by electrochemical sensing technique. In order to fulfil this

objective, the following aims have been identified:

SPECIFIC AIM # 1: Preparation of ZnO nanoparticles by microwave synthesis method

followed by characterization of this zinc oxide nanoparticles.

SPECIFIC AIM # 2: Fabrication of the working electrode through immobilization method

(physical adsorption) on zinc oxide nanoparticles over gold electrode.

SPECIFIC AIM # 3.1: Detection of DNA – DNA hybridization with 200 nucleotide

sequences (probe and its respective complementary sequences).

SPECIFIC AIM # 3.2: Detection of DNA – DNA hybridization with 20 nucleotide sequences

(probe and its respective complementary sequences).

19

CHAPTER 4: MATERIALS AND METHODS

4.1 MATERIALS AND REAGENTS:

Zinc chloride powder (ZnCl2) and sodium hydroxide pellets (NaOH) were purchased from

Merck Specialities Private Limited, Mumbai, India. The purity of zinc chloride and sodium

hydroxide are 95% and 97% respectively. Distilled water (obtained from Nano fluids

laboratory, SASTRA University) was used for dilution purpose during preparation of zinc

oxide solution. Deionised water was purchased from Aqua purification system, Salem, India.

The electrical conductivity of deionised water is <1µΩ. 20 µL of oligonucleotides (DNA- PCR

products) were extracted from β- globin which was obtained from Genomics laboratory,

SASTRA University. Alumina powder was used for micro polishing.

4.1.1 ELECTRODE SYSTEM:

In this biosensor, three electrode system was used. The working electrode, reference electrode

and counter electrode used for electrochemical sensing were gold (Au), Ag/AgCl and platinum

wire respectively. The surface area of the working electrode used was 0.0314 cm2.

4.2 SAMPLE PREPARATION:

DNA forward PCR product was used as immobilizing DNA probe. This solution was prepared

by dilution of 20 µL forward PCR product in 5 mL of deionised water. DNA reverse PCR

product was used as target (analyte). This solution was prepared by dilution of 20 µL reverse

PCR product in 5 mL of deionised water. Seven different concentrations such as 0.1, 0.2, 0.3,

0.4, 0.5, 0.6, 0.7 nM of target were prepared using volumetric equation as follows:

CBVB = CAVA

where,

CB & VB – Concentration and volume of solution before dilution.

CA & VA – Concentration and volume of solution after dilution.

These probe and target consisted of approximately 200 nucleotides. Similarly, these steps were

repeated for 20 nucleotides. The concentration of the electro active species DNA probe and

target on the electrode surface were calculated with the help of surface coverage analysis

technique and thereby the molecular weight was also calculated using the following equation:

Surface coverage analysis:

Ґ = 𝑄

𝑛𝐹𝐴

where,

Ґ – Surface coverage

Q – Charge obtained by integration of anodic peak

n – Number of electrons.

F – Faraday’s constant (96485 sA/mol).

A – Area of the electrode.

20

4.3 SYNTHESIS OF ZINC OXIDE NANOPARTICLES:

Zinc oxide nanoparticles was synthesized by microwave technique. Zinc oxide solution was

prepared by mixing the zinc chloride solution with sodium hydroxide (0.5 M), under the

magnetic stirrer for 30 min, where the chemicals are purchased from MERCK chemical private

ltd. After that, it was kept in microwave oven under 800W for 10 min. Nanoparticles get

separated by centrifuge method (Conditions: 1200 rpm for 15 min- repeated for 3 times). And

then it was dried in a hot air oven for 24 hours under 80˚C. It was annealed in muffle furnace

under 200˚C for 4 hours.

4.4 PRETREATMENT OF WORKING ELECTRODE:

The working electrode used here is gold electrode of 3mm diameter. First, the electrode surface

was cleaned and then it was polished with alumina powder using wet pad until a mirror glossy

appearance is seen on the surface of this electrode. After sometime, the electrode was gently

washed in running water. It is then dried in order to prevent crystallization of the particles at

the electrode surface.

4.5 FABRICATION OF ELECTRODE:

First, the working electrode is pretreated and then zinc oxide nanoparticles are coated over the

surface of it. Then the 200 nucleotide sequences are attached to the nano interface.

Hybridization event is facilitated when the probe sequence binds with its respective target

Fig.12: Microwave synthesis of ZnO nanoparticles

21

sequences. Similar steps are followed in the case of 20 nucleotide sequences also and

hybridization is analysed.

4.6 CHARACTERIZATION TECHNIQUES:

4.6.1 SCANNING ELECTRON MICROSCOPY (SEM):

Scanning electron microscopy is the most versatile microscopy which is used for the

exploration of surface morphology, grain size and also many micro level features. In

conventional microscope, light waves are used for imaging. On the other hand, SEM uses

electrons for imaging purpose. So, this type of electron microscope produces highly magnified

image of the surface of the respective material. First, the electrons are produced from electron

gun and then passed onto the corresponding sample to be analysed. These accelerated electrons

will produce kinetic energy. There will be many types of electrons coming out of the electron

gun. When these electrons hit the sample, there will be dissipation of kinetic energy in the form

of various signals. The various signals could be back scattered electrons and also in some cases,

diffracted back scattered electrons for determination of the crystal structure. From this,

information about the surface topography, composition and distribution of the particle is

revealed. In this type of microscopy, sample should have the conductive surface for the electron

transfer to occur for imaging the surface topography. In the case of non-conductive material,

the material should have been coated with conductive material before imaging by low vacuum

sputter coating or high vacuum evaporation. This is mainly done to prevent the accumulation

of the static charges on the surface of the sample during electron irradiation. This can produce

the high resolution image up to x300000 with great clarity.

Fig.13: Fabrication of working (gold) electrode.

22

4.6.1.1 EXPERIMENTAL SETUP:

The very basic components of scanning electron microscope are as follows:

• Electron gun

• Lenses

• Stage for samples

• Detectors (secondary electron detector)

• Output unit

4.6.1.2 ADVANTAGES OF SEM:

High resolution image of the samples

Three-dimensional morphology of samples

Very high and powerful magnification

4.6.2 X-RAY DIFFRACTION (XRD):

X-ray diffraction pattern of the sample contain the information about the crystalline nature,

size and orientation of the crystallites and also the phase composition of the samples. When the

incident beam of x-ray (electromagnetic radiation) hits the sample, the process of scattering

occurs. The diffraction of the x-ray occurs when the scattered rays are in phase with other

scattered rays from other atomic planes. The diffraction of x-ray was based on the principle of

Bragg’s equation.

According to Bragg’s law,

n λ= 2d sin(θ)

where,

λ is the wavelength of the x-ray

θ is the angle between the incident ray and the surface of the crystal

d is the spacing between the layers of atoms

n is the integer

Crystalline materials such as semi crystalline polymer, metal and metal oxide nanoparticle,

layered silicate nanoclay has unique x-ray diffraction characteristics patterns.

4.6.2.1 ADVANTAGES:

Effective tool for identification of unknown material

Very low quantity of samples for determination

Easier data interpretation

23

4.7 ELECTROCHEMICAL SENSING TECHNIQUES:

4.7.1 CYCLIC VOLTAMMETRY:

This technique shows the information about the thermodynamics of the charges released or

absorbed during the redox reaction, adsorption process and the kinetics of the charge transfer.

In this technique, the graph was plotted against the potential linearly versus time, which gives

the information about chemical reaction happened at the electrode interface as redox peaks.

Depend upon the requirements of the certain analysis or the specific experiment control, single

or several scan can be done. During this type of technique, under the different potential and

current, the analyte or the substance in the electrolyte solution or at the electrode interface

undergo electron communication, this may be directly proportional to the concentration of the

substance which acts as the analyte.

4.7.2 AMPEROMETRY:

The method of amperometry was as same as the cyclic voltammetry where the current was

produced when the constant potential was applied between the two electrodes such as reference

and the counter electrode. The current was measured between the counter electrode and the

working electrode. This produced current was proportional to the change in the density of the

species involved in the chemical reaction. The maintaining of the potential was achieved by

varying the potentiostat.

4. 4.7.3 IMPEDOMETRY:

The impedometric technique of the electrochemical sensing comprises the both resistance and

capacitive nature of the substance at the electrode interface. In this technique, the constant

potential was maintained to determine the impedance of the system, where the current can be

varied. It is the effective tool for interrogating the electrode and the electrolyte kinetics

Fig.14: Cyclic voltammogram

24

CHAPTER 5: RESULTS AND DISCUSSION

5.1 SEM ANALYSIS FOR ZINC OXIDE NANOPARTICLES:

Fig.5.1 represents the FE-SEM micrograph of the ZnO particles. From the image, the presence

of the zinc nanoparticle was observed. From the SEM micrograph, it can be inferred that

agglomeration has taken place. This agglomeration was due to the high surface energy

exhibited by ZnO particles. So, the ZnO nanoparticles tends to form cluster by the dipole –

dipole interaction. Morphology of ZnO particles can also be studied using SEM analysis and it

almost appears to be hexagonal in nature.

Fig.15: The FE-SEM micrograph of the zinc oxide particle.

25

5.2 XRD DATA FOR ZnO NANOPARTICLES:

The X-ray diffraction pattern of ZnO particles were synthesized at 800 W under microwave

synthesis were represented in the fig.16. There were number of Bragg’s reflections with two-

theta values of 33°, 35°, 37°, 48°, 57°, 64°, 66°, 69°, 70°, 77° corresponding to Miller indices

plane such as (110), (002), (101), (102), (110), (103), (112), (201), (202) which confirmed the

crystalline nature of the zinc oxide particles and was found to be similar with [JCPDS 36-

1451]. The hexagonal structure of ZnO was further confirmed by means of diffraction peaks

obtained in XRD analysis. It was also found that, the broader the peak, the smaller the size of

the particle.

0 10 20 30 40 50 60 70 80 90

0

100

200

300

400

500

600

700

ZnO

A

ZnO

Fig 16: X-ray diffraction pattern of ZnO particle.

26

5.3 ELECTROCHEMICAL STUDIES:

5.3.1 CYCLIC VOLTAMMETRY STUDIES:

5.3.1.1 EFFECT OF DIFFERENT ELECTRODE SYSTEM:

From figure 17, the effect of different electrode system was studied. The CV was run in three

cases as follows: i) with the bare electrode in target sequences solution, ii) bare electrode with

immobilized probe in the target sequences solution and iii) bare electrode with coated ZnO

interface immobilized with probe in the target solution.

A) 200 NUCLEOTIDES:

Figure 17 A) represents the CV plot for probe and target sequences consisting of 200

nucleotides. From this graph, it can be seen that, when compared with bare electrode system

and also with bare – probe electrode system, working electrode with nano interface (ZnO

particles) showed a higher peak. This is because of enhanced electron transfer in the presence

of ZnO particles.

-0.8 0.0 0.8

0.000000

0.000002

0.000004

Cu

rren

t(A

)

Potential(V)

BARE

BARE+PROBE

BARE+PROBE+NPS

0

B) 20 NUCLEOTIDES:

Figure 17 B) represents the CV plot for probe and target sequences consisting of 20

nucleotides. From this graph, it can be seen that, when compared with bare electrode system

and also with bare – probe electrode system, working electrode with nano interface (ZnO

particles) showed a higher peak. This is because of enhanced electron transfer in the

presence of ZnO particles.

27

-0.7 0.0 0.7

-0.000002

0.000000

0.000002

0.000004

Cu

rren

t(A

)

Potential(V)

BARE

BARE+PROBE+NPS

BARE+PROBE

COMPARISION

BARE ELECTRODE

BARE + PROBE

BARE + PROBE +

NANOPARTICLE

NUMBER OF BASES

200

BASES

20 BASES 200

BASES

20 BASES 200

BASES

20 BASES

FWHM 2.6856 2.6856 3.3380 3.2142 3.0323 3.3581

KS 2.4982 2.4982 0.08828 4.3552 0.08509 0.07695

Ґ (µmol/cm2) 177 177 191 121 744 509

EP (V) -0.4405 -0.4405 0.44 -0.2432 -0.571 -0.704

IP (µA) 2.6856 2.6856 0.1025 3.2142 0.3838 0.2378

TABLE 1: Comparison of different electrode system for 200 bases and 20 bases.

Table 1 represents the comparison of different electrode system for both 200 nucleotide

sequences and 20 nucleotide sequences with various electrochemical parameters such as

FWHM (full width –half maximum), KS (Reaction rate), Ґ (surface coverage), Ep (peak

potential) and Ip (peak current).

5.3.1.2 EFFECT OF SCAN RATES:

From figure 18, it can be observed that a linearity was obtained in terms of scan rates in the

range of 0.01 to 0.09 V/S. It was found that 0.09 V/S was found to be the optimized scan rate.

Fig.17: CV for DNA sequences showing the effect of different

electrode system A) 200 nucleotides B) 20 nucleotides

28

A) 200 NUCLEOTIDES:

B) 20 NUCLEOTIDES:

0.00 0.04 0.08 0.12

0

10

20

30

Cu

rre

nt

(µA

)

Scan rate(V/s)

Slope= 32.004

Fig.18: CV for DNA sequences showing the effect of scan

rates A) 200 nucleotides B) 20 nucleotides

29

5.3.1.3 EFFECT OF VARYING CONCENTRATIONS:

I) 200 NUCLEOTIDES:

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0

-0.000002

0.000000

0.000002

0.000004

0.000006

0.000008

0.000010

curr

ent

(A)

Potential (v)

0.1 nM

0.2 nM

0.3 nM

0.4 nM

0.5 nM

0.7 nM

0.0 0.2 0.4 0.6 0.80

1

2

3

4

5

Cu

rre

nt

(A)

Concentration (nM)

concentration

A)

Fig.19: CV for 200 nucleotide sequences showing the A) effect of

varying concentration B) Bar graph for concentration versus current

30

II) 20 NUCLEOTIDES:

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0

-0.000002

0.000000

0.000002

0.000004

0.000006

0.000008

0.000010

0.000012

0.000014

Cu

rren

t (A

)

Potential (V)

96 pM

108 pM

100 pM

B)

96 102 108

0

1

2

3

4

5

Cu

rren

t (A

)

Potential (V)

Concentration

Fig.20: CV for 20 nucleotide sequences showing the A) effect of varying

concentration B) Bar graph for concentration versus current

A)

31

5.3.2 AMPEROMETRIC STUDIES:

0.2 0.3 0.4 0.5 0.6 0.70.8

1.0

1.2

1.4

1.6

1.8

Cu

rre

nt

(A

)

Target (nM)

5.3.3 IMPEDOMETRIC STUDIES:

0.2 0.3 0.4 0.5 0.6 0.7

5500

6000

6500

7000

7500

8000

8500

Re

t (o

hm

)

Target (nM)

From figures 19, 20 and 21, it can be concluded that when the concentration parameter was

varied for both 200 and 20 nucleotides, we obtained a non- linear graph. For further

confirmation, Amperometric and impedance studies were carried out both in the case of 200

nucleotides and 20 nucleotides sequences. From these studies, it can be inferred that further

optimization have to be performed by means of varying the concentration range over a larger

and smaller scale.

FIG.21: Amperometric studies of 200 nucleotide sequences

FIG.22: Impedance studies of 200 nucleotide sequences

32

CHAPTER 6: CONCLUSION

In this project work, ZnO particles were synthesized in a nano scale by microwave synthesis.

The morphology, size and distribution of zinc oxide nanoparticles were studied by SEM and

XRD analysis. From this characterization results, it was confirmed that the zinc oxide powder

had a tetragonal zincite structure.

This approach combines the influence of both nanotechnology and electrochemistry. From this,

it can be concluded that, after inclusion of zinc oxide nanoparticles, electrochemical sensing of

nucleotide sequences increased significantly. Zinc oxide nanoparticles play a very crucial role

in such a way that it helps to enhance the transfer of electrons for hybridization of the probe

and its corresponding complementary target sequences. Zinc oxide nanoparticles also offers

good biocompatibility and has high isoelectric point (IEP ~9.5) in order to obtain strong

physical adsorption of DNA sequences. Electrochemical sensing was performed using cyclic

voltammetric, amperometric and impedance studies. This has resulted in the development of

very simple and inexpensive electrochemical DNA biosensor with nano-interface and hence it

can be used for rapid detection of various defective sequences and this, in turn, is highly useful

for diagnostic applications. For example, by checking for hybridization of HpV type 16

sequences, cervical cancer can be detected at an earlier stage.

33

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3) Microwave synthesis, characterization and photocatalytic properties of SnO2

nanoparticles Ashok K singh, Umesh T. Nakate, Advances in particles 2 (2013) 66-70

4) Microwave assisted synthesis of ZnO tridimensional nanostructures

G. Barreto et al., Procedia material and science 8 (2015) 535-540

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