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MINISTRY OF EDUCATION AND TRAINING HANOI UNOVERSITY OF TECHNOLOGY AND SCIENCE INTERNATIONAL TRAINING INSTITUTE FOR MATERIALS SCIENCE - TRIEU VAN VU QUAN DEVELOPMENT OF ELECTROCHEMICAL MICRO-SYSTEM TOWARDS THE APPLICATION IN BIOMEDICAL ANALYSIS MASTER THESIS OF MATERIALS SCIENCE Batch ITIMS-2014B SUPERVISOR Assoc Prof Mai Anh Tuan Hanoi – 2016 CONTENTS LIST OF ABBREVIATIONS LIST OF TABLES LIST OF FIGURES INTRODUCTION Chapter - REVIEW ON METHOD FOR DNA HYBRIDIZATION DETECTION AND HEAVY METAL DETECTION DNA sensor 10 1.1 Optical Method: 12 1.2 Piezoelectric method: 15 1.3 Magnetic methods 17 1.4 Electrochemical methods 17 Heavy metal detection in Food Safety analysis 26 2.1 Inductively Coupled Plasma – Mass Spectroscopy (ICP-MS) 27 2.2 Inductively Coupled Plasma – Atom Emission Spectroscopy 27 2.3 UV-VIS 27 2.4 X-ray Fluorescence 28 2.5 Atomic Absorption Spectroscopy 28 Chapter - DEVELOPMENT OF DATA ACQUISITION AND PROCESSING DEVICE FOR ELECTROCHEMICAL SENSOR 30 Triple electrode configuration 30 Potentiostat Circuit Operating Principle 32 Electronics Circuit Design 36 2.1 Power Source 36 2.2 Micro-controller Section 37 2.3 Analog Section 40 LabView Software in Communication with Computer 45 Chapter - RESULTS AND DISCUSSION 48 Device Specifications 48 Evaluation of the Entire Data Acquisition and Processing Device (DAP) 50 2.1 Evaluation of the Linear Sweep Mode 50 2.2 Evaluation of the Cyclic Voltammetry Mode 51 Application of the DAP in DNA sensor 55 Electrochemical synthesis of Poly-pyrrole nanowire 55 DNA Probe Immobilization 56 Detection of the DNA target using DAP and DNA sensor 57 Detection of heavy metal ion for food safety application 61 Future Work 66 General Conclusion 68 REFERENCES 69 LIST OF ABBREVIATIONS IUPAC: International Union of Pure and Applied Chemistry DNA: Deoxyribonucleic Acid RNA: Ribonucleic Acid SPR: Surface Plasmon Resonance MOSFET: Metal Oxide Semiconductor Field Effect Transistor UV-VIS: Ultraviolet – Visible Spectroscopy WE: Working Electrode RE: Reference Electrode CE: Counter Electrode AC: Alternating Current DC: Direct Current VI: Virtual Instrument DAP: Data Acquisition and Processing Device USB: Universal Serial Bus DAC: Digital Analog Converter ADC: Analog Digital Converter CV: Cyclic Voltammetry Op-amp: Operational Amplifier EIS: Electro-Impedance Spectroscopy LIST OF TABLES Table 1.1 - Toxic heavy metals and their effects on daily lives 26 Table 3.1- Aptamer Sequence 56 LIST OF FIGURES Figure 1.1 - Structure of a biosensor Figure 1.2 - Nucleic Acid Hybridization 11 Figure 1.3 - Scheme of ssDNA labelled with fluorescent agent detection 12 Figure 1.4- Scheme of interposed intercalators at the hybridization event 13 Figure 1.5 - SPR DNA sensor 14 Figure 1.6 - A QCM – based DNA biosensor 16 Figure 1.7 - Direct DNA detection 19 Figure 1.8 - Hybridization signal from ISFET 19 Figure 1.9 - Electrochemical assay for mismatches through DNA – mediated charge transport 20 Figure 1.10- Nyquist Plot of EIS 22 Figure 1.11 - DNA structure 24 Figure 2.1 - Potentiostat Principle 33 Figure 2.2 - Double Layer Model 34 Figure 2.3 - Impedance model of electrochemical cell 35 Figure 2.4 - Power Source Transformation 36 Figure 2.5 - PSoC Architecture 37 Figure 2.6 - MCU Firmware Flowchart 39 Figure 2.7 - General Analog Diagram 40 Figure 2.8 - Potentiostat Core 41 Figure 2.9 - Trans-impedance Amp using Instrumentation Amplifier 41 Figure 2.10 - Simulation Result of TIA circuit 42 Figure 2.11 - Low-pass Filter at 0.1 Hz 43 Figure 2.12 - Filter Circuit Simulation Result 44 Figure 2.13 - Voltage Level Shifter Circuit 44 Figure 2.14 - Simulation Result of Voltage Level Shifter 45 Figure 2.15 - LabView Program Flowchart 47 Figure 3.1 - Data Acquisition and Processing Device Board 48 Figure 3.2 - LabView Computer User Interface 49 Figure 3.3 - Resistor Test Setup 50 Figure 3.4 - Result of Resistor Test 50 Figure 3.5 - Triple Electrode Sensor draw and real one 52 Figure 3.6 - Measurement Setup with EC301 52 Figure 3.7 - CV Voltammogram by both devices 53 Figure 3.8 - The peak current values obtained by the DAP and the EC301 54 Figure 3.9 - SEM Image of working electrode with PPy-NWs 56 Figure 3.10 - CV Voltammogram after each steps at Target C = 10-6 by EC301 57 Figure 3.11 - CV Voltammogram after each steps at C = 10-6 by the DAP 58 Figure 3.12 - Relation between ∆Ip to concentrations of DNA target when measured with EC301 and PSoC circuit 60 Figure 3.13 - Voltage form for ASV measurement for Arsenic Detection 62 Figure 3.14 - ASV Measurement at As3+ 50ppb with the DAP 63 Figure 3.15 - ASV Measurement at As3+ 50ppb with EC301 63 Figure 3.16 - ASV Voltammogram for different As3+ concentrations by the DAP 64 Figure 3.17 - ASV Voltammogram for different As3+ concentrations by EC301 64 Figure 3.18 - Regression lines for both devices 65 INTRODUCTION Today, as the living standard of people increases, healthcare section receives huge attention People come to periodic medical examination and check for their health status From the data of Biomedical Network in Vietnam, almost all the analyses in hospital now focus on liquid sample such as blood, urine and endothelial cell analysis Traditionally, those analyses are conducted by cumbersome, complicated instruments and skilled technicians in laboratories With the development of micro- and nanotechnology, biosensors have proved a boon to analysts, for they allow the miniaturization of those instruments into hand-held devices while still being able to produce fast and accurate results Biosensors include two main parts: a biological components and a transducer The working mechanism of sensors is based on the reactions between biological elements as well as physics/biology/chemistry effects, and those signal detected from the reactions will reveal the information we need through a test Recently, electrochemical biosensors are receiving interest in biomedical analysis field The main transducing elements include support electrodes of noble metals and carbon derivatives These electrodes can be modified to improve the connection with the recognizing agents, thus enhance the charge transfer process and signal intensity, which is helpful to the signal acquisition stage For signal acquisition stage, a device should be developed alongside with the sensor, because it is a replacement for the laboratory instrument The users will prefer a complete kit of analysis, which will give them understandable figures rather than just some sensors and they have no idea how to interpret the signal obtained from them It helps reduce the cost of the analyses and direct to on-site measurement In addition, electrochemical measurements are not only applied in medical analysis but can also be applicable in environment analysis and food safety Understanding the principle of such circuits and sensors will be very helpful for the developers in the development of the common analysis platform, because both hardware and software can be used for different purposes Within the scope of this thesis, we will focus on the development of a measurement circuit which will be able to obtain and display signals from electrochemical biosensors The thesis will be divided into three chapters: The first chapter one will review on the current method for DNA detection and heavy metal detection in food safety analysis This will summarize the advantages/disadvantages of each method In addition, the working principle of electrochemical sensor is presented The second chapter will present the impedance model for electrochemical sensor and the operating principle of the circuit The design process for Data Acquisition and Processing Device will be shown In the third chapter, functional blocks and entire device will be evaluated via characteristics test After that, the device’s application in DNA sensor and heavy metal detection for food safety analysis will be presented The results will be compared with that of EC301 – a commercial device for electrochemical analysis Chapter - REVIEW ON METHOD FOR DNA HYBRIDIZATION DETECTION AND HEAVY METAL DETECTION According to IUPAC, Biosensor is defined as the device uses specific biochemical reactions mediated by isolated enzymes, immune-systems, tissues, organelles or whole cells to detect chemical compound by electrical, thermal or optical signals [39] Today, biosensors are finding its application in almost all the fields such as agricultural sensing and control, pharmaceutical process and biomedical signal processing [15] Biosensors provide a method to record signal generated from biological and biochemical processes, which is important for researchers to understand medicine, biology and biotechnology phenomena The structure of a biosensor is shown below: Figure 1.1 - Structure of a biosensor As we can see on Figure 1.1, a biosensor is composed of three components: bioreceptors, transducer and processing parts Bio-receptors are biological substances that can involve in characteristic biological reactions For example, enzyme glucose oxidase accelerates reaction between glucose and oxygen Bio-receptors can be divided into some categories including enzymes, anti-genes/antibodies, DNAs, microbials (microorganism) and molecular structures (cells) The next component, transducer is used to convert the bio-chemical signal resulting from the interaction of the sample and bioreceptors to other signal types (eg electric, optic and mechanic signal) The intensity of signal produced by the transducer is directly proportional to the sample concentration A few types of transducers can be mentioned such as electrochemical, optical and piezoelectric transducers The signal produced from transducer will be then amplified in processing part In the next part, the mechanism of DNA sensor will be presented REFERENCES [1] Bard, A J., L R Faulkner (2001), "Electrochemical Instrumentation" in “ELECTROCHEMICAL METHODS Fundamentals and Applications,” Elsevier, pp 632–658 [2] 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Zhang, C D Kodira, X H Zheng, L Chen, M Skupski, G Subramanian, P D Thomas, J Zhang, G L G Miklos, C Nelson, S Broder, A G Clark, J Nadeau, V A Mckusick, N Zinder, A J Levine, R J Roberts, M Simon, C Slayman, M Hunkapiller, R Bolanos, A Delcher, I Dew, D Fasulo, M Flanigan, L Florea, A Halpern, S Hannenhalli, S Kravitz, S Levy, C Mobarry, K Reinert, K Remington, J Abuthreideh, E Beasley, K Biddick, V Bonazzi, R Brandon, M Cargill, I Chandramouliswaran, R Charlab, K Chaturvedi, Z Deng, V Di Francesco, P Dunn, K Eilbeck, C Evangelista, A E Gabrielian, W Gan, W Ge, F Gong, Z Gu, P Guan, T J Heiman, M E Higgins, R Ji, Z Ke, K A Ketchum, Z Lai, Y Lei, Z Li, J Li, Y Liang, X Lin, F Lu, G V Merkulov, N Milshina, H M Moore, A K Naik, V A Narayan, B Neelam, D Nusskern, D B Rusch, S Salzberg, W Shao, B Shue, J Sun, Z Y Wang, A Wang, X Wang, J Wang, M Wei, R Wides, C Xiao, C Yan, A Yao, J Ye, M Zhan, W Zhang, H Zhang, Q Zhao, L Zheng, F Zhong, W Zhong, S C Zhu, S Zhao, D Gilbert, S Baumhueter, G Spier, C Carter, A Cravchik, T Woodage, F Ali, H An, A Awe, D Baldwin, H Baden, M Barnstead, I Barrow, K Beeson, D Busam, A Carver, M L Cheng, L Curry, S Danaher, L Davenport, R Desilets, S Dietz, K Dodson, L Doup, S Ferriera, N Garg, A Gluecksmann, B Hart, J Haynes, C Haynes, C Heiner, S Hladun, D Hostin, J Houck, T Howland, C Ibegwam, J Johnson, F Kalush, L Kline, S Koduru, A Love, F Mann, D May, S Mccawley, T Mcintosh, I Mcmullen, M Moy, L Moy, B Murphy, K Nelson, C Pfannkoch, 72 E Pratts, V Puri, H Qureshi, M Reardon, R Rodriguez, Y Rogers, D Romblad, B Ruhfel, R Scott, C Sitter, M Smallwood, E Stewart, R Strong, E Suh, R Thomas, N N Tint, S Tse, C Vech, G Wang, J Wetter, S Williams, M Williams, S Windsor, E Winn-deen, K Wolfe, J Zaveri, K Zaveri, J F Abril, R Guigo, A Kejariwal, H Mi, B Lazareva, T Hatton, A Narechania, K Diemer, A Muruganujan, N Guo, S Sato, V Bafna, S Istrail, R Lippert, R Schwartz, B Walenz, S Yooseph, D Allen, A Basu, J Baxendale, L Blick, M Caminha, J 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13, pp 4446–4452 [36] Wei, Y S., Q Q Jin, and T Z Ren (2011), “Expanded graphite/pencil-lead as counter electrode for dye-sensitized solar cells” Solid State Electron., vol 63, no 1, pp 76–82 [37] Yang, I V and H H Thorp (2001), “Modification of indium tin oxide electrodes 73 with repeat polynucleotides: electrochemical detection of trinucleotide repeat expansion” Anal Chem, vol 73, no 21, pp 5316–5322 [38] Zhang, Q and V Subramanian (2007), “DNA hybridization detection with organic thin film transistors: Toward fast and disposable DNA microarray chips” Biosens Bioelectron., vol 22, no 12, pp 3182–3187 [39] McNaught, A D and A Wilkinson, Eds (1997), IUPAC Compendium of Chemical Terminology, 2nd Blackwell Scientific Publications, Oxford 74 PUBLICATIONS Trieu V V Q., Nguyen H N., Chu T X and Mai A T (2016), Simple Anodic Stripping Voltametry Method For the Determination of As3+, ICAMN2016, p 191 - 194, ISBN: 978-604-95-0010-7 Trieu V Q., Tran Th L., Vu D H Tong D Hien, Mai A T (2014), Development of a portable data acquisition device for electrochemical biosensor application, ICAMN2014, p 71-75, ISBN: 978-604-911-946-0 Tran Th L., Chu T X , P Q Do, D Th Pham, Trieu V Q., , D C Huynh and Mai A T (2015), In-Channel-Grown Polypyrrole Nanowire for the Detection of DNA Hybridization in an Electrochemical Microfluidic Biosensor, Journal of Nanomaterials, vol 2015, Article ID 458629, pages doi:10.1155/2015/458629 Luu T H T., Tran Th L D Th Pham, Trieu V Q, Pham V T., Ta T N A., Chu T X., Mai A T (2015), Fabrication of PDMS-based Microfluidic Devices Toward Biomedical Applications, Tạp chí Khoa học và Công nghệ các Trường Đại học Kỹ thuật, 105A, p 38 - 42, ISSN: 2354 - 1083 75 APPENDIX // // C main line // #include #include #include #include #include #include #include #include "PSoCAPI.h" "RefMux_1.h" "stdlib.h" "delay.h" "string.h" "math.h" // part specific constants and macros // PSoC API definitions for all User Modules volatile int tick2ms = 0; volatile char flag2ms = 0; volatile int tick1s = 0; int max_val = 0; char flag_range; BYTE Port0_shadow_register = #define button_1 0x01 #define button_2 0x10 #define button_3 0x04 0b00000001; // 0b00000001 - P0.0 //choose 1, 2, up, down // 0b00010000 - P0.4 //write button // 0b00000100 - P0.2 #define MINVAL #define MAXVAL 510 void delay1ms(void) { Delay50uTimes(20); } void delay_ms(WORD factor) { int i; for (i=0; i scan rate 25mV/s void Timer24_1_ISR(void){ DAC9_1_WriteBlind(CV_0_3_0_6_vs_Ag[Pointer]); Pointer++; if(Pointer >= 292) Pointer = 0; } void Start(void){ BYTE bRefSignal; bRefSignal = RefMux_1_AGND; RefMux_1_RefSelect(bRefSignal); RefMux_1_Start(RefMux_1_HIGHPOWER); //reference voltage 2.390V LCD_1_Start(); UART_1_IntCntl(UART_1_ENABLE_RX_INT); UART_1_EnableInt(); UART_1_Start(UART_1_PARITY_NONE); //UART_1_PutChar(41); } char buffer[5]; void send_ADC(int Data) { buffer[0] = Data/1000+ '0'; 77 buffer[1] = (Data%1000)/100 + '0'; buffer[2] = ((Data%100)/10) + '0'; buffer[3] = ((Data%100)%10) + '0'; buffer[4] = '\0'; UART_1_PutString(buffer); //UART_1_PutChar(10); } int readadc(void) { return ADCINC_1_iClearFlagGetData(); } void Display_int(int value){ form of ten base to LCD char str[10]; LCD_1_PrString(itoa(str, value, 10)); } void Display_float_range_10_100(int val){ float division; int *status; division = ((val*3.11E-4)-1.233)*10; LCD_1_PrString(ftoa(division, status)); } void Display_float(float val){ int *status; LCD_1_PrString(ftoa(val, status)); } char check_ASV_button(void){ if(!(PRT0DR&button_1)) { return 1; } else return 0; } char check_CV_button(void){ //if (!(PRT0DR&button_3)) if ((PRT0DR&button_3)) { return 1; } else return 0; } int check_button(void){ PRT0DR = Port0_shadow_register; if (check_ASV_button()) { Delay10msTimes(8); 78 //display int type value in if (check_ASV_button()==1) return 1; } //task = 1; else if (check_CV_button()) { Delay10msTimes(8); if (check_CV_button()==1) return 2; } return 0; } void ASV_out_ADC_read(void) //function to output DAC value and read ADC value { static int DAC_value 0_45V = 165; // 1.998V static int DAC_value 0_3V = 190; //2.148V static int DAC_value_0_6V = 380; //3.048V if (tick1s 100) //DAC output at 2.2V in the period 10 to 20s { LCD_1_Position(0,0); LCD_1_PrCString("Phase 2"); DAC9_1_WriteStall(DAC_value 0_3V); } if (tick1s == 110) // DAC output increase to 3.048V with rate of change 1V/s { if(tick2ms%4==0 && flag2ms) //check increment condition { flag2ms=0; 79 DAC_value 0_3V +=1; DAC9_1_WriteStall(DAC_value 0_3V); } } if (tick1s > 110 && tick1s < 200) { LCD_1_Position(0,0); LCD_1_PrCString("Phase 3"); DAC9_1_WriteStall(DAC_value_0_6V); } } void Enable_scan_range10_100(void) { int result; LCD_1_Control(LCD_1_DISP_CLEAR_HOME); //clear LCD Timer8_1_EnableInt(); //enable timer Timer8_1_Start(); DAC9_1_Start(DAC9_1_HIGHPOWER); PGA_1_SetGain(PGA_1_G1_00); PGA_1_Start(PGA_1_HIGHPOWER); ADCINC_1_Start(ADCINC_1_HIGHPOWER); ADCINC_1_GetSamples(0); while (1) { ASV_out_ADC_read(); if (ADCINC_1_fIsDataAvailable()) { result = readadc(); if (max_val < result) max_val = result; UART_1_PutChar(flag_range); UART_1_PutChar(13); send_ADC(result); UART_1_PutChar(32); asm ("nop"); } if (tick1s > 150) { LCD_1_Position(1,0); LCD_1_PrCString("Peak A:"); //write to LCD Display_float_range_10_100(max_val); LCD_1_Position(1,17); LCD_1_PrCString("uA"); } } } void Enable_scan_range100_1000(void) { int result; LCD_1_Control(LCD_1_DISP_CLEAR_HOME); Timer8_1_EnableInt(); 80 Timer8_1_Start(); DAC9_1_Start(DAC9_1_HIGHPOWER); PGA_1_SetGain(PGA_1_G1_00); PGA_1_Start(PGA_1_HIGHPOWER); ADCINC_1_Start(ADCINC_1_HIGHPOWER); ADCINC_1_GetSamples(0); while (1) { ASV_out_ADC_read(); if (ADCINC_1_fIsDataAvailable()) { result = readadc(); if (max_val < result) max_val = result; UART_1_PutChar(flag_range); UART_1_PutChar(13); send_ADC(result); UART_1_PutChar(32); asm ("nop"); if (tick1s > 150) { LCD_1_Position(1,0); LCD_1_PrCString("Peak A:"); //write to LCD Display_float_range_10_100(max_val); LCD_1_Position(1,17); LCD_1_PrCString("uA"); } } } } void Test_DAC_val(void) { DAC9_1_Start(DAC9_1_HIGHPOWER); LCD_1_Control(LCD_1_DISP_CLEAR_HOME); LCD_1_PrCString("test"); while (1) { DAC9_1_WriteBlind(300); } } void CV_Get_Results (void){ int result_CV; LCD_1_Control(LCD_1_DISP_CLEAR_HOME); LCD_1_PrCString("Scanning "); Timer24_1_EnableInt(); Timer24_1_Start(); DAC9_1_Start(DAC9_1_HIGHPOWER); PGA_1_SetGain(PGA_1_G4_00); PGA_1_Start(PGA_1_HIGHPOWER); ADCINC_1_Start(ADCINC_1_HIGHPOWER); 81 ADCINC_1_GetSamples(0); while (1) { //CV_scan(); if (ADCINC_1_fIsDataAvailable()) { result_CV = readadc(); //UART_1_PutChar(13); send_ADC(CV_0_3_0_6_vs_Ag[Pointer]); //send_ADC(CV_0_1[Pointer]); UART_1_PutChar(32); send_ADC(result_CV); UART_1_PutChar(13); //asm ("nop"); } } } void Start_ASV_scan(void){ int choose_range; LCD_1_Control(LCD_1_DISP_CLEAR_HOME); //Timer8_1_EnableInt(); //Timer8_1_Start(); PRT0DR = Port0_shadow_register; LCD_1_Position(0,0); LCD_1_PrCString("1.10-100ppb"); LCD_1_Position(1,0); LCD_1_PrCString("2.100-1000ppb"); while (1) { choose_range = check_button(); switch (choose_range) { case 1: flag_range = 48; Enable_scan_range10_100(); break; case 2: flag_range = 57; Enable_scan_range100_1000(); break; } } } void main1(void){ int adc_val; int action; LCD_1_Position(0,0); LCD_1_PrCString(">CV"); LCD_1_Position(1,0); LCD_1_PrCString(">ASV"); 82 PRT0DR = Port0_shadow_register; //button polling for PORT0 while (1) { action = check_button(); switch (action) { case 1: Start_ASV_scan(); break; case 2: //Start_CV_scan(); CV_Get_Results(); //Test_DAC_val(); //test_timer_LCD(); break; } } } void main(void) { M8C_EnableGInt ; M8C_EnableIntMask(INT_MSK1, INT_MSK1_DBB01); M8C_EnableIntMask(INT_MSK1, INT_MSK1_DCB12); Start(); main1(); //main2(); } 83 ... complementary bases Adenine (A) – Thymine (T) and Cytosine (C) – Guanine (G) In the 1990s, DNA sequencing method, which determines the order of a specific DNA strand by making use of an array of informed... onto the surface of piezoelectric crystal The crystal will then interact with the analyte in the solution The change in mass, associated with the hybridization process, results in a decrease of the. .. Evaluation of the Linear Sweep Mode 50 2.2 Evaluation of the Cyclic Voltammetry Mode 51 Application of the DAP in DNA sensor 55 Electrochemical synthesis of Poly-pyrrole nanowire
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