Research on fabrication of the electrochemical miocrosensor based on modified conductive polymer for application in biomedical and environmental fields

lactose in milk). Chpater 2. THE FABRICATION OF ELECTROCHEMICAL BIOSENSOR 5 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 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 CHẾ TẠO HỆ VI ĐIỆN CỰC TÍCH HỢP TỔNG HỢP MÀNG POLYME DẪN CHỨC NĂNG HÓA CỐ ĐỊNH PHÂN TỬ ĐẦU DÒ SINH HỌC ĐO ĐẠC, PHÂN TÍCH, THỬ NGHIỆM 6 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). 7 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) I ( A ) 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 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 8 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. -0,2 0,0 0,2 0,4 0,6 0,8 1,0 -600 -400 -200 0 200 400 600 800 1000 I / A E /V vs. Ag/AgCl -0,2 0,0 0,2 0,4 0,6 0,8 1,0 -800 -600 -400 -200 0 200 400 600 800 1000 I / A E /V vs. Ag/AgCl Fe3O4/PANi PANi Figure III.2: Electropolymerization spectrum of Fe3O4 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 9 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 I / A E /V vs. Ag/AgCl PANi/Graphen PANi/Graphen/Glutaraldehyde 10 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 PANi- Fe3O4/Graphene structure 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-Fe3O4/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 Fe3O4 NPs 11 -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 -300 -200 -100 0 100 200 300 400 (2)I / A E /V vs. Ag/AgCl (1) PANi/Fe3O4/Graphene films (2) PANi films (1) 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. 12 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). Hình III.9. Electrochemical behavior of fimls: Pt/PDAN và 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 0,0 0,2 0,4 0,6 0,8 1,0 -60 -40 -20 0 20 40 60 I / A E /V vs. Ag/AgCl P1,5-DAN P1,5-DAN/Fe3O4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 -150 -100 -50 0 50 100 150 I / A E /V vs. Ag/AgCl Pt/Gr/P(1,5-DAN) Pt/P(1,5-DAN) 13 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 4. APPLYING THE ELECTROCHEMICAL MICRO- BIOSENSOR 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 micro- biosensor 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. 150 200 250 300 350 400 0.3 0.4 0.5 0.6 0.7 9mM 8mM 7mM 6mM 5mM 3mM 4mM 2mM 1mM I (A ) t (s) Figure IV.1. The real-time response current of PANi/CNTs/GOx microsensor Figure IV.2. The response curve of 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). y = 0,0371x + 0,0074 R² = 0,9962 0 0,1 0,2 0,3 0,4 0 2 4 6 8 10 ∆i (μ A ) Nồng độ (mM) Đường chuẩn của vi cảm biến trên cơ sở màng composite PANi/CNTs có pha tạp 1,0%CNTs 14 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 micro- biosensor 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.. 200 400 600 800 1000 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 3.5mM 2.5mM 3.0mM 2.0mM 1.5mM 1.0mM C ur re nt ( A ) Time (s) PANi with Fe3O4 PANi 0.5mM 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.2 0.4 0.6 0.8 1.0 1.2 I ( A ) Concentration (mM) Figure IV.3. The current response of the PANi/Fe3O4/GOx microbiosensor Figure IV.4. The calibaration curve of 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 PANi- Fe3O4/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). 15 400 600 800 1000 1200 1400 1600 5 10 15 20 25 30 35 40 45 23,08 21,26 17,36 15,25 13,04 10,71 8,26 5,66 I / A Time /s PA-Fe-Gr Glucose sensor 2,91mM 0 5 10 15 20 25 5 10 15 20 25 30 35 40 45 I re sp on se / A Glucose concentration C/mM I (A) = 1,484*Cglucose + 6,764 R2 = 99,69 PANi-Fe3O4/co(St-AA)-Graphene films Hình IV.5. Amperometric responses of PANi-Fe3O4/Graphen/GOx microsensor to different added glucose concentrations Hình IV.6. Glucose calibration line and respective regression equation of PANi- 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 micro- biosensor 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). 16 400 500 600 700 800 2.8 2.9 3.0 3.1 3.2 3.3 3.4 0,10mM 0,12mM 0,04mM 0,06mM 0,08mM 0,02mM I ( A ) Thêi gian (gi©y) 0.02 0.04 0.06 0.08 0.10 0.12 0.1 0.2 0.3 0.4 0.5 0.6 Y = A + B * X Parameter Value Error ------------------------------------------------------------ A 0.01740 0.00926 B 4.30143 0.11885 ------------------------------------------------------------ R Sy N P ------------------------------------------------------------ 0.99848 0.00994 6 <0.0001 ------------------------------------------------------------ I (A ) Nång ®é (mM) Figure IV.7. The real-time response curve of PANi/CNTs/ChOx microsensor Figure IV.8. The calibration curve of PANi/CNTs/ChOx microsensor The PANi/CNTs/ChOx microsensor reachs a stable current (-2,8A) in PBS buffer solution of 50mM (pH = 7) after about 400 seconds. Based on the difference in the response current intensity of the PANi/CNTs/ChOx microsensor and the total added amount of cholesterol, a calibration curve for determining cholesterol at -0.3V (compared with Ag/AgCl ) in the presence of K3[Fe(CN)6]. The regression equation has the form ΔI (A) = 0.0174 + 4,3014*C (mM). Correlation coefficient of the regression equation: R2 = 0.9985. I.2.2 Determination of the concentration of cholesterol by using PANi/Fe3O4 The response current spectra of PANi/Fe3O4/ChOx-Fe3O4 microsensors is shown in Figure IV.9 below. The results showed that the sensor gave good response (linear) in the range of cholesterol concentrations from 0.196mM ÷ 1,803mM. At higher cholesterol concentrations, when added to the electrolyte, the signal is noisy, the current is poor. This is due to ChOx when catalyzing hydrolytic reactions of choleterol that do not keep up with the added rate of substrate. For the results in the calibration graph of the sensor (Figure IV.10), the regression equation of the calibration curve will take the form: I (µA) = (21.45±1,271)×C (mM) + (-0,8352±1,1474), the correlation coefficient of the regression equation reached R2 = 0.9929. The average sensitivity of PANi/Fe3O4/ChOx-Fe3O4 micro sensors is S = 21.44 A.mM-1.cm-2. 17 0 200 400 600 800 1000 0 10 20 30 40 I ( A ) t (s) cholesterol 0.0 0.3 0.6 0.9 1.2 1.5 1.8 2.1 0 5 10 15 20 25 30 35 40 45 I (A ) cholesterol (mM) Equation y = a + b*x Adj. R-Square 0.99297 Value Standard Error -- Intercept -0.8352 0.66165 -- Slope 21.44897 0.57052 Figure IV.9. The real-time response curve of PANi/Fe3O4/ChOx-Fe3O4 microsensor Figure IV.10. The calibration curve of PANi/Fe3O4/ChOx-Fe3O4 microsensor I.2.3 Determination of the concentration of cholesterol by using PANi- Fe3O4/Graphen micro-biosensor Figure IV.11 shows a typical current–time plot for the sensor at +0.7 V during successive injections of cholesterol (2 mM increased injection, at room temperature, without stirring, air saturated, in 50 mM phosphate buffered solution). The response time of cholesterol sensor was smaller than 5s with cholesterol concentration change. The calibration plot indicates a good and linear amperometric response to cholesterol within the concentration range from 2 to 20 mM (with regression equation of I (µA) = (2.15 ± 0.13) * C (mM), R2= 0.9986) (the inset in Figure IV.11). Thus, with a miniaturized dimension (250 µm) the above graphene patterned sensor has shown much improved sensitivity to cholesterol, as high as 1095.54 AmM-1cm-2. The sensitivity of graphene cholesterol sensor was 2 times higher than that of CNT- cholesterol sensor. 18 0 200 400 600 800 1000 0 10 20 30 40 0 2 4 6 8 10 12 14 16 18 20 0 5 10 15 20 25 30 35 40 C ur re nt (m A ) Concentration (mM) C ur re nt / A Time (s) Hình IV.11. Amperometric responses of PANi-Fe3O4/Graphen/ChOx microsensor to different added cholesterol concentrations (inset: the calibration curve of fabricated cholesterol sensor) I.3. Testing of DNA of HPV virus The SWV graph is recorded after each process (activated with EDC/NHS, before and after immobilized aptamer, HPV antigen), as shown in Figure IV.12. -0,4 -0,2 0,0 0,2 0,4 0,6 -1,0x10-4 -5,0x10-5 0,0 5,0x10-5 1,0x10-4 1,5x10-4 2,0x10-4 2,5x10-4 3,0x10-4 3,5x10-4 (6) (5)(4) (3) I / A E /V vs. Ag/AgCl (1) + EDC/NHS (2) + HPV-16-L1 (3) + 10nM anti-HPV (4) + 20nM anti-HPV (5) + 30nM anti-HPV (6) + 40nM anti-HPV (7) + 50nM anti-HPV (1) (7) (2) 0 20 40 60 80 1.4x10-4 1.6x10-4 1.8x10-4 2.0x10-4 2.2x10-4 2.4x10-4 2.6x10-4 I / A Anti-HPV-16 concentration /nM Figure IV.12. SWV of PANi/CNTs microsensor recorded after treatment with EDC/NHS (curve 1), after grafting HPV- 16-L1 (curve 2) and after complexation with anti-HPV-16 (curve 3-7) Figure IV.13. The response curve of PANi/CNTs microsensor with anti-HPV-16 concentration range from 10-80 nM The spectrum of SWV analysis proved very clearly the formation of the complex of aptamer HPV-16-L1 and its specific HPV antibody, through the linearly attenuating of SWV peak current intensity. The calibration curve was constructed with a range of 19 different HPV concentrations in the range of 10-80 nM (shown in Figure IV.13). The PANi/CNTs biosensor has a sensitivity response of 1.75 ± 0.2 (A.nM-1) (R2 = 0.997) in a concentration range of 10–50 nM with limit of detection (LOD ) is 490pM. It can be seen that the signal tends to be saturated with a concentration value above 80nM II. APPLYING IN FOOD SAFETY CONTROL II.1. Determination of the concentration of Aflatoxin M1 in milk The ability to recognize the concentration of AFM1 of microsensors is determined by a calibration curve with a range of different concentrations (from 6ng/L to 78ng/L relative to a concentration of 18-240pM of AFM1) of AFM1 (molecular weight is ~ 328Da). The analytical results of AFM1 concentration of microsensor by SWV method shown in Figure IV.14 is quite similar to the electrochemical CV signal of micro sensor. -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 0 1 2 3 4 5 6 7 0 10 20 30 40 50 60 70 80 2,0 2,5 3,0 3,5 4,0 4,5 5,0 R2 = 0,9986 I / A AFM1 concentration /ngL-1 I (A) = -4,77*CAFM1 + 5,17 (A) LOD = 1,98 ngL-1 LOQ = 6,62 ngL-1 I / A E /V vs. Ag/AgCl (1) Fe3O4/PANi (2) Fe3O4/PANi/Glu (3) Fe3O4/PANi/Glu/APT (4) + AFM1 06ngL-1 (5) + AFM1 18ngL-1 (6) + AFM1 30ngL-1 (7) + AFM1 60ngL-1 SIGNAL OFF Figure IV.14. SWV response of PANi/Fe3O4 microsensor with various AFM1 concentration The results of microsensors are: sensitivity of 4.77 ± 0.2 (A/ ngL-1) (R2 = 0.9986) in the range of 6 - 60 ngL-1 (approximate 18 to 240 pM) with LOD reaching 1.98ng/L (the inset of Figure IV.14: the calibration line of the sensor). II.2 Determination of the concentration of lactose in milk II.2.1 Determination of the concentration of lactose by using P(1,5-DAN)/Fe3O4 mic