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. Mai Anh Tuan Hanoi – 2016 17051113936051000000 CONTENTS LIST OF ABBREVIATIONS. 3 LIST OF TABLES. 4 LIST OF FIGURES.
7 Chapter 1 - REVIEW ON METHOD FOR DNA HYBRIDIZATION DETECTION AND HEAVY METAL DETECTION. Heavy metal detection in Food Safety analysis. Inductively Coupled Plasma – Mass Spectroscopy (ICP-MS). Inductively Coupled Plasma – Atom Emission Spectroscopy.
Atomic Absorption Spectroscopy. 28 Chapter 2 - DEVELOPMENT OF DATA ACQUISITION AND PROCESSING DEVICE FOR ELECTROCHEMICAL SENSOR. Triple electrode configuration. 30 Potentiostat Circuit Operating Principle.
Electronics Circuit Design. Micro-controller Section. LabView Software in Communication with Computer. 45 Chapter 3 - RESULTS AND DISCUSSION.
Evaluation of the Entire Data Acquisition and Processing Device (DAP). Evaluation of the Linear Sweep Mode. Evaluation of the Cyclic Voltammetry Mode. 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. Detection of heavy metal ion for food safety application.
69 2 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 3 LIST OF TABLES Table 1.1 - Toxic heavy metals and their effects on daily lives. 56 4 LIST OF FIGURES Figure 1.1 - Structure of a biosensor .2 - Nucleic Acid Hybridization .3 - Scheme of ssDNA labelled with fluorescent agent detection .4- Scheme of interposed intercalators at the hybridization event .5 - SPR DNA sensor .6 - A QCM – based DNA biosensor .7 - Direct DNA detection .8 - Hybridization signal from ISFET .9 - Electrochemical assay for mismatches through DNA – mediated charge transport .10- Nyquist Plot of EIS.2 - Double Layer Model .3 - Impedance model of electrochemical cell .4 - Power Source Transformation .6 - MCU Firmware Flowchart .7 - General Analog Diagram .9 - Trans-impedance Amp using Instrumentation Amplifier .10 - Simulation Result of TIA circuit .11 - Low-pass Filter at 0.12 - Filter Circuit Simulation Result .13 - Voltage Level Shifter Circuit .14 - Simulation Result of Voltage Level Shifter .15 - LabView Program Flowchart .1 - Data Acquisition and Processing Device Board .2 - LabView Computer User Interface .3 - Resistor Test Setup .4 - Result of Resistor Test .5 - Triple Electrode Sensor draw and real one .6 - Measurement Setup with EC301 .7 - CV Voltammogram by both devices .8 - The peak current values obtained by the DAP and the EC301 .9 - SEM Image of working electrode with PPy-NWs .10 - CV Voltammogram after each steps at Target C = 10 -6 by EC301 .11 - CV Voltammogram after each steps at C = 10-6 by the DAP .12 - Relation between ∆Ip to concentrations of DNA target when measured with EC301 and PSoC circuit .13 - Voltage form for ASV measurement for Arsenic Detection .14 - ASV Measurement at As 3+ 50ppb with the DAP .15 - ASV Measurement at As 3+ 50ppb with EC301 .16 - ASV Voltammogram for different As3+ concentrations by the DAP .17 - ASV Voltammogram for different As3+ concentrations by EC301 .18 - Regression lines for both devices. 65 6 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. 7 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. 8 Chapter 1 - 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: bio- receptors, 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 (micro- organism) and molecular structures (cells). The next component, transducer is used to convert the bio-chemical signal resulting from the interaction of the sample and bio- receptors 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. DNA sensor For detection of DNA sequences, the techniques are based on the nature hydrogen linkage between 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 DNA sequences, were used intensively in DNA mapping [33]. Famous methods used in DNA sequencing are Maxam – Gilbert sequencing and chain – termination method. Oligonucleotides probes are attached to a solid surface, and then a sample DNA or RNA section (called target) can be hybridized under stringent conditions. The hybridization results are detected and quantified by fluorescence labelling method.
These methods are reliable and easy to use but requires complex sample preparation and large amounts of purified DNA [2]. They are more suitable in genome – wide genetic mapping, physical mapping, proteomics and gene expression studies [25]. Recently, Polymerase Chain Reaction (PCR) and Enzyme - Linked Immuno Sorbent Assay (ELISA) are the two most widely used methods in biology, medicine and food technology for the detection of DNA. PCR perceives the change of temperature when the hybridization occurs while ELISA method relies on the antibodies and color change to identify a substance.
Those methods are powerful and well-known for accuracy, strong specificity and high specificity. However, they are costly, time-consuming and requires complicated sample preparation. DNA biosensor is a type of biosensors which takes advantage of direct hybridization of aptamers or single-stranded DNA (ssDNA), as shown in Figure 1. The recognition process of nucleic acid will give rise to a signal which then will be detected.
Based on the intensity of signal and the method we use, specific characteristics of biological substances can be recognized. More matches in double stranded DNA will result in stronger hybridization which causes signal to be more obvious. DNA biosensors are receiving a great interest due to its profound potential in obtaining specific gene 10 information in a faster, cheaper and more precise manner compared to traditional analysis. With the development of biological and chemical methods, nucleic acids can now be easily synthesized and regenerated by chemical methods or molecular biology [20].
Moreover, they are highly stable and readily reusable after thermal heating [30]. DNA biosensors are now widely used in medical diagnostics, agriculture and analytical application.2 - Nucleic Acid Hybridization [11] DNA biosensors are categorized based on transduction methods, namely optics, electrochemistry, piezoelectricity and magnetism [2][13]. We will discuss these techniques briefly to view at the advantages and disadvantages of each method. To be more specific, we can divide the analysis methods for hybridization into label- based detection and label – free detection.
Label-based detection method includes redox intercalators to recognize dsDNA, DNA mediators supporting in electron transfer and enzyme labelling to enhance the sensitivity and intensity of signal. Label-based detection method utilizes some indicators, such as ruthenium bipyridine, methylene blue or redox chemicals. Label – free detection, as its name suggests, can detect the 11 hybridization of single – stranded DNAs directly. Each transduction method combining with detection methods will create a new approach to the hybridization phenomenon.
The next part will show some common approach used in today analyses. Optical Method: Each transduction technique comprises of label (fluorescence) method and label – free method. In optical technique we can mention some methods such as optical fibers, surface plasmon resonance and reflection interference contrast microscopy. Fluorescence Methods: Figure 1.3 - Scheme of ssDNA labelled with fluorescent agent detection Fluorescence methods are also known as labelling methods.
Fiber optics DNA sensors are now receiving enormous attention from scientists as nanotechnology develops in recent years [30]. Fiber optics DNA sensors can be divided into single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) labelling.