Summary of Doctoral thesis in Materials science: Research on fabrication of the electrochemical miocrosensor based on modified conductive polymer for application in biomedical and

Study on modification/functionalization of conductive polymers using nanostructured materials (CNTs, Fe3O4 nanoparticles and Graphene) to develop the electrochemical biosensor and apply this biosensor in biomedical and environmental analysis.

MINISTRY OF EDUCATION AND TRAINING

VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY

GRADATE UNIVERSIY OF SCIENCE AND TECHNOLOGY



Nguyen Hai Binh

RESEARCH ON FABRICATION OF THE ELECTROCHEMICAL MIOCROSENSOR BASED ON MODIFIED CONDUCTIVE POLYMER FOR APPLICATION IN BIOMEDICAL AND

This thesis was done at:

Laboratory of Biomedical Nanomaterials, Institute of Materials and Sciene, Vietnam Academy of Science and Technology

Supervisor: Prof Ph.D Tran Dai Lam

INTRODUCTION

Currently, biosensors are considered as a potential device for application in many fields such as biology, pharmaceuticals, agriculture, food safety and hygiene, enviromental protection and industrial safety, etc Biosensor is a device that uses specific biological components in combination with a signal converter to detect, measure or analyze chemical agents

Electrochemical microsensor has a simple structure, easy to design and develop structure, easy to integrate with micro-elements of the system, bactch fabrication The working electrodes, counter electrodes and reference electrodes are integrated on one chip, which reduces the volume and mass of the sample to be analyzed due to reduces electrode size The elements of the electrochemical sensor are all employed on planar technology so it is easy to pack, increase stability and repeatability

Around the world, many research groups have developed micro-biosensor based on microelectromechanical components with different physical-chemical effects such as mass, presure, electrochemical Comparison with micro-sensor using mass/pressure effect, the electrochemical micro-sensor has more advantages such as designing and manufacturing on the MEMS technology so small size, easy to batch fabricate to reduce the price, more simple structure, easier integrate with microchannel- microvalve – micropump system, easier package, easy to use the electrochemical methods to testing the properties of the device In Vietnam, some initial results on fabrication and development of biosensor has been published by domestic research groups The research on the develop electrochemical microsystem applied in biomedical diagnosis and environmental monitoring is being paid attetion and strongly invested in many countries around the world Vietnam is a country with a strong developing economy with o population of nearly 90 million people, prospects for develeping electrochemical microsystems and devices based on nanostructured materials would push science and technology and has profound socio-ecomonic signification Based on the science and

practical requirements, I choose to carry out the thesis “Research on fabrication of the

electrochemical miocrosensor based on modified conductive polymer for application

in biomedical and environmental”

The issue of this thesis is to fabricate, develop and test the electrochemical microbiosensor (as platform devices) with simply operation mode, fast response time, high accuracy, easy to customize the structure, easy to integrate with other components With the aim of manufacturing some electrochemical microbiosenor based on conductive polymers in which are modified by nanostrutured materials in existing technological conditions in Vietnam, the thesis sets out the necessary problem have to solve: designing an electrochemical miocrosensor suitable to the existing technological conditions, conducting experiment to employe sensors, surveying the properties of the fabricated microsensor, applying to analyze some indicators in biomedical, environmental pollutants and food safety substances On the obtained results, we would concluse about the ability to fabricate, develop and apply the microsensor sytem in the

current technological conditions in the country

Objectives of the thesis:

The electrochemical microbiosensors based on conductive polymer (PANi và DAN)) are modified/functionalized with nanostructured materials (CNTs, Fe3O4 nanoparticles and Graphene)

P(1,5-Goals of the thesis:

Research on fabrication of the electrochemical microbiosensors based on conductive polymer (PANi và P(1,5-DAN)) are modified/functionalized with nanostructured materials (CNTs, hạt nano Fe3O4 nanoparticles and Graphene)

Applying the fabricated electrochemical micro-biosensor in biomedical and environmental analysis

Scientific and application of the thesis:

Study on modification/functionalization of conductive polymers using nanostructured materials (CNTs, Fe3O4 nanoparticles and Graphene) to develop the electrochemical biosensor and apply this biosensor in biomedical and environmental analysis

Research methods:

The thesis is conducted by experimental method The integrated electrochemical microelectrodes was fabricated by CMOS/MEMS technology The surface morphology of composite membrane based on modified/functionalized conductive

polymer with nanostructured materials was investigated by some techniques: FTIR, Raman spectrum, FESEM, AFM The electrochemical properties of the composite film was evaluated by electrochemical analysis techniques: CV, Square Wave Voltammetry and Electrochemical Impendance spectra The biomedical and environmental testing

of electrochemical biosensor was performed by electrochemical techniques: CV, Chronoamperometric and Square Wave Voltammetry on the Autolab PGS/TAT 30A system (EcoChimie, Netherlands)

Contents of the thesis:

Research on electropolymerize the composite films based on conductive polymer (PANi, P(1,5-DAN)) modified/functionalized by nanostructured materials (CNTs, Fe3O4 nanoparticles, Graphene)

Study the surface morphology and electrochemical properties of composite films on the surface of the integrated electrochemical microelectrodes

Evaluate the characteristics of electrochemical biosensor based on composite membrane (PANi, P(1,5-DAN)) and apply on the biomedical and environmental analysis

Structure of the thesis:

The main content of thesis is presented in 4 chapters Chapter 1 is an overview of electrochemical biosensors, conductive polymer materials (PANi, P(1,5-DAN)), nanostructured materials and applications of electrochemical biosensors Chapter 2 presents the technological and experimental processes to manufacture an integrated electrochemical miocroelectrode system, electrochemical polymerization of composite film, analytical techniques Chapter 3 gives the results of the properties of employed composite films based on conductive polymers (PANi và P(1,5-DAN)) Chapter 4 describes the results of apply the electrochemical microbiosensor in biomedical and environmental analysis

The research results of thesis was published in 10 scientific paper, including 04 articles published in ISI journal, 02 articles published in International Scopus journal and 04 articles published in national journal

Main results of the thesis:

Successfully fabricated the integrated electrochemical microelectrodes with CMOS/MEMS technology

Successfully electropolymerized the composite film based on conductive polymer

in which has been modified/functionalized by nanostructured materials The structural

and electrochemical properties of composite films on the surface of electrochemical microelectrodes have been studied

Successfully developed the electrochemical biosensor based on conductive polymer (PANi, P(1,5-DAN)) and applied in biomedical and environmental analysis

Chapter I: OVERVIEW

I Introduction to electrochemical biosensor

An electrochemical biosensor is a type of biosensor in which the working principle

based on electrochemical phenomenons that occur when an electric current through electrolyte flask or by oxidation – reduction on the electrodes, the above phenomena depend on the properties of the electrode, the nature and concentration of the solutions Electrochemical microsensor is an electrochemical sensor system with working electrode has dimension smaller than 1mm (similar to the definition of Micro ElectroMechanical System – MEMS) Electrochemical micro-biosensor allows directly converting the biochemical signals as a results of interaction of protein-protein, antigen-antibody, DNA-DNA, enzyme-subtrate into electrical signals

II Conducting polymer in electrochemical biosensor

Two types of electronic conductive polymers (PANi and PDAN) have been polymerized and modified to develop the electrochemical biosensors thanks to their advantages: good conductivity, easy processing, low cost, functional group – NH2 in the polymer structure to create bonding with biological element, good stability and durability In addition, to enhance their conductivity, electrochemical activity, some nanostructure materials (such as Carbon nanotubes, Graphene, Fe3O4 magnetic nanopartices) will also be used for doping/denaturing with conductive polymers in manufacturing – development the elctrochemical micro-biosensors

III Applications of electrochemical biosensor

The electrochemical biosesnsor has many applications in different fields such as: in the field of health care (monitoring of blood glucose/cholesterol levels, determination

of DNA of HPV virus), in environmental monitoring (determination of the residues of Atrazine), in food safety control (detection of mycotoxin Aflatoxin M1 in milk, determination of concentration of lactose in milk)

Chpater 2 THE FABRICATION OF ELECTROCHEMICAL BIOSENSOR

In this chapter, the experimental processes in fabrication - development and testing

of electrochemical biochemical sensors based on doped/modified conductive polymer with nanostructured materials (Fe3O4 nanoparticles, carbon nanotubes, graphene materials ) are presented in detail The diagram of experimental steps is shown in Figure II.1 below

Figure II.1 Diagram of experimental steps for manufacturing - testing electrochemical

biosensor based on conductive polymer

I Fabrication of the electrochemical microelectrodes

In the experimental framework of this thesis, we implement integrated electrochemical microelectrode system on 1 chip including: working electrode (Pt), counter electrode (Pt) and reference electrode (Ag/AgCl) on Si /SiO2 wafer (purchased from Wafernet Inc, USA) (where Si p <100> wafer has a thickness of ~ 50 m and a thickness of 1m SiO2) with thin Chromium (Cr) layer to increase the adhesion of layers on the substrate

Integrated electrochemical microelectrodes are fabricated based on microelectronic technology by UV-photolithography, PVD-Physical Vapor Deposition, lift-off at the Institute of Materials Science (IMS), Vietnam Academy of Science and Technology (VAST) and at some abroad laboratories (Institute of Fundamental Electronics, University of Paris 11, France and Department of Engineering and Science Systems, National Tsinghua University, Taiwan) Integrated electrochemical microelectrodes have dimensions: diameter of working electrode is 100m/200m or 500m, the width

of counter electrode/reference electrode is 100m/200m, the distance between the electrodes is 100m/200m with the contact pad designed according to the USB configuration

II Electropolymerization of the conductive polymer membrane

II.1 Electropolymerization of the polyaniline membrane

POLYME DẪN CHỨC NĂNG HÓA

CỐ ĐỊNH PHÂN TỬ ĐẦU DÒ SINH

HỌC

ĐO ĐẠC, PHÂN TÍCH, THỬ NGHIỆM

Electrolytic conducting solution consists of ANi 0.1M monomer in 0.5M H2SO4 containing MWCNTs-COOH (or Fe3O4-COOH) 1% w.t (compared to Aniline) The polymerization process uses the Cyclic Voltammetry (CV) method in the potential range of 0.0 - 0.9V (vs Ag/AgCl), the scan rate of 50mV/s with a step of 10mV in 20 cycles The synthesis process of pure PANi films in the same condition is also conducted for comparison

II.2 Electropolymerization of the polydiaminonaphthalen membrane

The P(1,5-DAN)-doped Fe3O4 films coated on working electrode (Pt) were polymerized in 1,5-diaminonapthalene (DAN) solution of 5mM in 1M HClO4 and Fe3O4 solution (10mg/ml) 0.5% w.t (compared to DAN) by electrochemical polymerization CV method in the range of -0.02V to + 0.95V, scan rete of 50mV/s, step of 10mV in 10 cycles Pure PDAN films are also synthesized in the same conditions to compare properties

III Immobilization of the biorecognition on the electrochemical miocroelectrodes

After the composite films on the basis of a multifunctional conductive polymer membrane (denatured by nanostructured materials) was electropolymerized on the surface of the working electrode (of the integrated microelectrode system), the biological elements (biological probes such as enzymes, aptamers, DNA chains or monoclonal antibodies ) should be immobilized to the surface of the composite membrane to develop electrochemical biosensors Biological probes are immobilized

on the surface of composite membrane through chemical linkage (-NH-COO-) by biological engineering The biorecognition elements used in this thesis are biological probes with high specificity such as enzymes (Glucose oxidase, Cholesterol oxidase ), monoclonal antibodies, DNA sequences, aptamer sequences

IV Electrochemical analytical methods

In this thesis, we have used many different electrochemical analysis methods to investigate the properties of composite films (based on PANi and PDAN) and determine the concentration of analytes in solutions such as: CV, SWV, Chronoamperometric, EIS Electrochemical experiments were performed on the multifunction electrochemical device Autolab PGS/TAT 30 (EcoChimie, Netherlands)

at the Institute of Materials Science (VAST), Institute for Tropical Technology (VAST), CETASD (Hanoi University of Science, Hanoi - Vietnam National University)

V The analytical methods for surface and structure of thin films

The surface and strutural analysis techniques such as FESEM, HRTEM, AFM, FTIR, Raman spectrum are used in the study of the surface morphology of employed membanes in the elctrochemical microbiosensors

Chapter III DEVELOPMENT OF THE ELECTROCHEMICAL

MICRO-BIOSENSOR BASED ON CONDUCTING POLYMER

I Development of the electrochemical micro-biosensor based on polyaniline

I.1 Functionalization the PANi film by using CNTs

CV spectra obtained in both cases are presented in Figure III.1 with similar shape, this is the typical CV spectrum of PANi membrane electropolymerization However, it

is very interesting that the intensity of electric current obtained in the case of composite

is about 10 times larger than the case of PANi Thus with CNT doping in the membrane may have increased: (i) the conductivity of the film and / or (ii) the contact surface between the membrane and the solution containing the monomer

0.0 0.2 0.4 0.6 0.8 1.0 -600

-400 -200 0 200 400 600 800

E (V)

PANi/CNTs PANi

Figure III.1 Spectrum polymerization by CV method of PANi film (a) and PANi / CNTs

membrane (b) at the 20th cycle on integrated microelectrodes

The electrochemical synthesis spectra of Fe3O4 doped PANi films are shown in Figure III.2 We observed an increase in the electrochemical current density of the

Fe3O4-doped PANi membrane (solid line) when compared to the PANi membrane (dashed line) (as shown in Figure III.3); This means that Fe3O4 nanoparticles may have increased the current density of PANi films in the same experimental conditions (design of electrode and PANi membrane properties equally), demonstrating the doping of Fe3O4 nanoparticles into PANi membrane increase the electrochemical activity or the contact surface between the membrane and the monomer solution; that leads to an increase in the ability of electron transfer in the configuration of electrochemical sensors

-600 -400 -200 0 200 400 600 800 1000

Figure III.2: Electropolymerization

spectrum of Fe 3 O 4 doping PANi films

Figure III.3 Comparison of

electrochemical polymerization spectra of PANi / Fe3O4 and PANi films

I.3 Development of the electrochemical micro-biosensor based on PANi/Grpahene layer-by-layer structure

The thickness and structure and the functional group of PANi/Graphene films are evaluated by Raman spectra (as shown in Figure III.4) The structural variation of Graphene films before and after transferring to the working electrode surface Pt/PANi

is clearly observed in the Raman spectrum through comparison with Raman spectra of PANi films and Graphene films Raman spectrum of PANi/Graphene films (black lines) shows the bands attributed to the PANi and Graphene (Gr), confirming the occurrence

of both of these components in the film The question here is if the Gr has firmly bonded

by chemical bonding to PANi film or the Gr has only been mounted on this film temporarily In the thesis, it was found that the band situated at 1507 cm-1 (N-H bonding, bipolaron) was collapsed, and in the same time, the band located at 1612 cm-1 (C-C

bonding, benzenoid) red shifts to 1597 cm-1 These results clearly demonstrated the increase in concentration of benzenoid units; or on other hand, the chemical bonding between PANi and Gr occurred It was believed that those bondings are π-π bonding between quinoid rings of PANi and Gr Such bondings can facilitate charge transfer between Gr and PANi, therefore influence the charge-carrier transport properties of the material

Figure III.4 Raman spectra of the films: Graphen, PANi và PANi/Graphen

The influence of glutaraldehyde (GA) on electrochemical behavior of PANi/Graphene films is shown in Fig III.5

Figure III.5 Electrochemical behavior of PANi/Gr film before and after GA imomobilization

-0,2 0,0 0,2 0,4 0,6 0,8 -100

-80 -60 -40 -20 0 20 40

The shape of CV curves did not change but the current intensity was decreased slightly, suggesting the assembly of non-conductive organic compounds on the membrane The fact is that the GA was successfully immobilized on the surface of micorosensor and influence on the electrochemical behavior of biosensor

I.4 Development of the electrochemical micro-biosensor based on

The surface morphology of composite PANi-Fe3O4/Graphen was examined by FESEM (S-4800, Hitachi) at Institute of Materials Science (as show in Fig III.6)

Figure III.6 FESEM image of PANi-Fe 3 O 4 /Graphen film

Some observations can be made from FE-SEM image of graphene/Fe3O4/PANi films (Figure III.6) First, it shows a spongy and porous structure of PANi, which in turn can

be very helpful for enzyme entrapment Second, doped core-shell Fe3O4 NPs (with the

diameter core of ca 30 nm) could also contribute to further immobilization of

biomolecule, owing to their carboxylated shell Furthermore, a thin and opaque graphene layer was distinguishably seen on the top of the electrode surface

The electrochemical activity of PANi/Fe3O4/graphene film increased about 8 times compared with PANi film (Figure III.7) on the CV spectrum The Fe3O4 nanoparticle plays the role of electrolyte in the composite films From Fig.III.7 4 it is clear that the

conductivity of composite was strongly enhanced with the presence of graphene film

Graphene

Fe 3 O 4 NPs

-0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 -300

-200 -100 0 100 200 300 400

Figure III.7 The electrochemical behavior of composite film PANi-Fe 3 O 4 /Graphen

II DEVELOPMENT OF THE ELECTROCHEMICAL MICRO-BIOSENSOR BASED ON P(1,5-DAN) MEMBRANCE

When doping Fe3O4 nanoparticles into PDAN films during in-situ electropolymerization process, the Fe3O4 magnetic nanoparticles were linked to DAN monomers via the bonding [Fe3O4]-COO-NH-[DAN] and increasing the electroactivity

of the membrane material After 20 cycles, the current intensity of the PDAN/Fe3O4 film reaches ~ 120 A while the current intensity of the PDAN film is only ~ 8A, so the current intensity of the PDAN/Fe3O4 film has increased greatly compared to the with conventional PDAN film

The electrochemical activity of PDAN/Fe3O4 films was investigated and compared with PDAN films by CV spectrum (Figure III.8) Electrochemical spectrum of PDAN/Fe3O4 composite has no change in shape but the signal strength increases clearly, the spectral area is also increased (expressing the increase in electrochemical conductivity of the film) about 10 times Due to the electrical conductivity of PDAN/Fe3O4 film increase, the output of electrochemical sensor also increased accordingly, so which the sensitivity of sensor also increased