MINISTRY OF VIETNAM ACADEMY OF EDUCATION AND TRAINING 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 ENVIRONMENTAL FIELDS Major: Electronic materials Code: 9.44.01.23 SUMMARY OF DOCTORAL THESIS IN MATERIALS SCIENCE Ha Noi - 2018 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 Reviewer 1: Reviewer 2: Reviewer 3: The dissertation will be defended at Graduate University of Science and Technology, 18 Hoang Quoc Viet street, Hanoi Time: ., , 2018 This thesis could be found at National Library of Vietnam, Library of Graduate University of Science and Technology, Library of Institute of Materials and Science, Library of Vietnam Academy of Science and Technology 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 P(1,5DAN)) are modified/functionalized with nanostructured materials (CNTs, Fe3O4 nanoparticles and Graphene) Goals of the thesis: Research on fabrication of the electrochemical microbiosensors based on conductive polymer (PANi 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 chapters Chapter is an overview of electrochemical biosensors, conductive polymer materials (PANi, P(1,5-DAN)), nanostructured materials and applications of electrochemical biosensors Chapter presents the technological and experimental processes to manufacture an integrated electrochemical miocroelectrode system, electrochemical polymerization of composite film, analytical techniques Chapter gives the results of the properties of employed composite films based on conductive polymers (PANi P(1,5-DAN)) Chapter 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 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 CHẾ TẠO HỆ VI ĐIỆN CỰC TÍCH HỢP CỐ ĐỊNH PHÂN TỬ ĐẦU DÒ SINH HỌC TỔNG HỢP MÀNG POLYME DẪN CHỨC NĂNG HĨA ĐO ĐẠC, PHÂN TÍCH, THỬ NGHIỆM 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 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 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 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 MICROBIOSENSOR 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 800 PANi/CNTs PANi 600 I (A) 400 200 -200 -400 -600 0.0 0.2 0.4 0.6 0.8 1.0 E (V) Figure III.1 Spectrum polymerization by CV method of PANi film (a) and PANi / CNTs membrane (b) at the 20th cycle on integrated microelectrodes I.2 Functionalization the PANi film by using Fe3O4 nanoparticles 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 1000 1000 800 800 600 600 400 400 I /A I /A Fe3O4/PANi 200 0 -200 -200 -400 -400 -600 -600 -800 -0,2 0,0 0,2 0,4 0,6 0,8 PANi 200 -0,2 1,0 0,0 0,2 0,4 0,6 0,8 1,0 E /V vs Ag/AgCl E /V vs Ag/AgCl Figure III.2: Electropolymerization Figure III.3 Comparison of spectrum of Fe3O4 doping PANi films 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 400 (1) PANi/Fe3O4/Graphene films (1) 300 (2) PANi films I / A 200 100 (2) -100 -200 -300 -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 E /V vs Ag/AgCl Figure III.7 The electrochemical behavior of composite film PANi-Fe3O4/Graphen II DEVELOPMENT OF THE ELECTROCHEMICAL MICRO-BIOSENSOR BASED ON P(1,5-DAN) MEMBRANCE II.1 Electropolymerization of the Fe3O4 nanoparticles-dopped 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 11 60 40 I /A 20 -20 -40 P1,5-DAN P1,5-DAN/Fe3O4 -60 0,0 0,2 0,4 0,6 0,8 1,0 E /V vs Ag/AgCl Hình III.8 Electrochemical behavior of fimls: PDAN and PDAN/Fe3O4 II.2 Fabrication of the electrochemical micro-biosensor based on Graphen/PDAN membrance Electrochemical behavior of Graphen/PDAN was studied and compared with PDAN membrane by CV spectrum (Fig III.9 below) 150 100 I / A 50 -50 Pt/Gr/P(1,5-DAN) Pt/P(1,5-DAN) -100 -150 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 E /V vs Ag/AgCl Hình III.9 Electrochemical behavior of fimls: Pt/PDAN Pt/Graphen/PDAN Compared to the pure PDAN membrane, the electrochemical spectrum of the Graphen / PDAN polymer film has no change in shape but the signal strength increases markedly, the spectral area is also increased (demonstrating the enhancement of electrochemical conductivity) of membrane) about 15 times Due to the 12 electrochemical conductivity of the Gr/PDAN membrane, the output current of the electrochemical sensor also increased accordingly, from which the sensor's sensitivity increased The increase of electrochemical conductivity of PDAN film on Graphene material may be due to the interaction of NH2-Graphene group, which has changed the band gap of the material, leading to an increase in the electronic conductivity of the material Chương APPLYING THE ELECTROCHEMICAL MICROBIOSENSOR ON THE ANALYTICAL I APPLYING ON THE BIOMEDICAL DIAGNOSTICS I.1 Determination of the concentration of glucose I.1.1 Determination of the concentration of glucose by using PANi/CNTs microbiosensor The real-time response current of PANi/CNTs/GOx microsensor (with the percentage of doping CNTs doped is 1.0% by weight) is shown in Figure IV.1 below Đường chuẩn vi cảm biến sở màng composite PANi/CNTs có pha tạp 1,0%CNTs 0.7 9mM 8mM 0,4 7mM 0.6 6mM 0,3 4mM 0.5 3mM ∆i (μA) I (A) 5mM 2mM 0,2 1mM 0.4 y = 0,0371x + 0,0074 R² = 0,9962 0,1 0.3 150 200 250 300 350 400 t (s) 0 Nồng độ (mM) 10 Figure IV.1 The real-time response Figure IV.2 The response curve of current of PANi/CNTs/GOx microsensor PANi/CNTs/GOx microsensor in range 1-9 mM It can be seen that the current intensity when measured in PBS solution (10mM, pH = 7) is stable after about 200 seconds When adding glucose solution, the current intensity increases rapidly and reaches stability after about 30-40 seconds However, when the concentration of glucose exceeds 9mM, the increase in flow intensity is very weak, even reduced This may be due to the immense amount of GOx enzymes on the electrode and the low activity (20kU) 13 The calibration curve describes the relationship between the difference in the response current intensity ΔI (A) and the glucose concentration C (mM) added to the electrolyte as shown in Figure IV.2 The regression equation has the form ΔI (A) = 0.0074 + 0.0371 * C (mM) The correlation coefficient of the regression equation reaches 0.9962 I.1.2 Determination of the concentration of glucose by using PANi-Fe3O4 microbiosensor The current intensity of the oxidation process of glucose on the PANi/Fe3O4/GOx sensor increases with the concentration of glucose in the solution shown in Figure IV.3 1.2 3.5mM 1,4 PANi with Fe3O4 3.0mM PANi 1,2 1.0 2.0mM 0,8 0.8 I (A) Current (A) 2.5mM 1,0 1.5mM 1.0mM 0,6 0.6 0.5mM 0,4 0.4 0,2 0.2 0,0 200 400 600 800 1000 0.5 Time (s) 1.0 1.5 2.0 2.5 3.0 3.5 Concentration (mM) Figure IV.3 The current response of the Figure IV.4 The calibaration curve of PANi/Fe3O4/GOx microbiosensor PANi/Fe3O4/GOx sensor From the results in Figure IV.3, we determine the sensitivity of the micro sensor to 10 A.mM-1.cm-2 and the response time is less than 10s From the calibration curve of the sensor (Figure IV.4), the linear range of the PANi/Fe3O4/GOx micro-biosensor is determined to be 0.5 to 3.5mM with R2 = 0.9992, LOD = 0.25mM The regression equation has the form: I (A) = 0.33021 * C (mM) + 0.04503 I.1.3 Determination of the concentration of glucose by using PANiFe3O4/Graphen/Gox micro-biosensor Figure IV.5 shows a typical current–time plot for the sensor at +0.7 V during successive injections of glucose (3 mM increased injection, at room temperature, without stirring, air saturated, in 50 mM PBS solution) 14 45 21,26 13,04 8,26 5,66 10,71 Iresponse /A I /A 25 20 15 2,91mM 10 400 40 I (A) = 1,484*Cglucose + 6,764 35 R = 99,69 17,36 15,25 35 30 45 23,08 40 30 25 20 15 10 PA-Fe-Gr Glucose sensor PANi-Fe3O4/co(St-AA)-Graphene films 600 800 1000 1200 1400 1600 Time /s 10 15 20 25 Glucose concentration C/mM Hình IV.5 Amperometric responses of Hình IV.6 Glucose calibration line and PANi-Fe3O4/Graphen/GOx microsensor to respective regression equation of PANi- different added glucose concentrations Fe3O4/Graphen/GOx microsensor We found that the sensor has a short response time for changing glucose concentrations in the solution, tresponse ~ 10-15s, the response current intenstity has good stability at the various concentrations of glucose The calibration plot indicates a good and linear amperometric response to glucose within the concentration range from 2.9 to 23 mM (with regression equation of I (A) = 1.484 * C (mM) + 6.764, R2= 0.9969) (as Fig IV.6) Thus, with a miniaturized dimension (500 µm) the above graphene patterned sensor has shown much improved sensitivity to glucose, as high as 47 AmM-1cm-2 compared to non-graphene one (10-30 AmM-1cm-2) I.2 Determination of the concentration of cholesterol I.2.1 Determination of the concentration of cholesterol by using PANi/CNTs microbiosensor The current response curve of PANi/CNTs/ChOx micro-biosensors with the presence of mediator K3[Fe(CN)6] at voltage E = -0.3V given in Figure IV.7 The concentration of cholesterol is the diluted concentration (considering the change in volume is negligible) 15 0.6 3.4 0,12mM 0.5 0,10mM 3.3 0,08mM 3.2 0.4 I (A) I (A) 0,06mM 3.1 0,04mM 0.3 Y=A+B*X Parameter Value Error -A 0.01740 0.00926 B 4.30143 0.11885 3.0 0.2 0,02mM 2.9 R Sy N P -0.99848 0.00994