Nghiên cứu chế tạo hệ vi điện hóa định hướng ứng dụng trong phân tích y sinh Development of electrochemical micro system towards the application in biomedical analysis

Nghiên cứu chế tạo hệ vi điện hóa định hướng ứng dụng trong phân tích y sinh Development of electrochemical micro system towards the application in biomedical analysis Nghiên cứu chế tạo hệ vi điện hóa định hướng ứng dụng trong phân tích y sinh Development of electrochemical micro system towards the application in biomedical analysis luận văn tốt nghiệp thạc sĩ

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 3

LIST OF TABLES 4

LIST OF FIGURES 5

INTRODUCTION 7

Chapter 1 - REVIEW ON METHOD FOR DNA HYBRIDIZATION DETECTION AND HEAVY METAL DETECTION 9

1 DNA sensor 10

1.1 Optical Method: 12

1.2 Piezoelectric method: 15

1.3 Magnetic methods 17

1.4 Electrochemical methods 17

2 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 2 - DEVELOPMENT OF DATA ACQUISITION AND PROCESSING DEVICE FOR ELECTROCHEMICAL SENSOR 30

1 Triple electrode configuration 30

Potentiostat Circuit Operating Principle 32

2 Electronics Circuit Design 36

2.1 Power Source 36

2.2 Micro-controller Section 37

2.3 Analog Section 40

3 LabView Software in Communication with Computer 45

Chapter 3 - RESULTS AND DISCUSSION 48

1 Device Specifications 48

2 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

3 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

4 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 9

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 1 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 3.7 - 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

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 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:

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

Figure 1.1 - Structure of a biosensor

1 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 1.2 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

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

Figure 1.2 - 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 based detection and label – free detection Label-based detection method includes

label-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

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

1.1 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

1.1.1 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 Optical fibers play the role of transmission path for fluorescent signal emitted from hybridization event In ssDNA labelling, fluorescent chemicals can bind covalently to ssDNA The fluorescent intensity

will be improved when the hybridization occurs between ssDNAs (Figure 1.3) Another

approach in this method is the use of fluorescent intercalators After dsDNAs are formed, the fluorescent indicator, such as ethidium bromide (EB) and thiazole orange (TO), will

be selectively tied to DNA hybrids by noncovalent bonds Once these intercalators interpose between base pairs, excitation will occur which leads to fluorescence

enhancement or quenching [34], as shown in Figure 1.4

Figure 1.4- Scheme of interposed intercalators at the hybridization event

Many methods are based on labelling using radioisotopes, fluorophores and absorbing molecules [2] With the development of nanotechnology, today quantum dots and nanowires are integrated into hybridization labelling method Quantum dots is a nanostructure generated by bottom-up approach which can be used in fluorescence tagging of biomolecules The most important characteristic of quantum dots is color change due to the replacement of particle sizes, which is also known as confinement effect ssDNAs can be tagged with quantum dots, each of them will emit a different color, so that the hybridization can be discerned directly by naked eyes or a further process called fluorescence resonance energy transfer (FRET)

UV-1.1.2 Label free method:

Surface Plasmon Resonance (SPR) takes advantage of a quantum phenomenon in which the conduction electrons go into oscillation state when stimulated by incident light [34] The resonance condition is established when the frequency of incident photons matches the natural frequency of surface electrons In SPR DNA sensor, ssDNA was immobilized onto the surface of thin metal film while another aqueous solution flows over the thin film surface Light is transmitted to the surface through a prism, then it will be reflected back with characterized optical reflectivity If the hybridization of dsDNA occurs, the optical characteristics including reflectivity, refractive index and intensity will change

accordingly [6] When used with optic fiber, the system is setup as in the Figure 1.5

The metal layer part is kept in contact with the sample aqueous solution As in based technique, this one is deemed costly and complex, making them more suitable with research rather than reality application

labelled-Figure 1.5 - SPR DNA sensor

Labelled-free methods also include other methods such as Reflection Interference Contrast Microscopy (RICM) or Raman Spectroscopy to detect the DNA hybridization process [4]

With the development of nanotechnology, fiber-optic DNA biosensors have got great progresses because fiber-optic can be easily miniaturized to the nanometer scale size by chemical etching [2] or tube etching [30] and mechanically pulled with CO2 laser heating

setup [25], they are immuned to electromagnet [34], disposability and long-distance transmission [6]

1.2 Piezoelectric method:

Piezoelectric materials such as quartz when properly cut and applied a certain AC voltage will oscillate at defined frequency This frequency is sensitive to the mass of the crystal, which is the rudimentary idea of Quartz Crystal Microbalance The change in mass can relate to the change in frequency by Sauerbrey equation [28]:

∆𝑓 = − 2𝑓02

𝐴√𝑝𝑞𝜇𝑞∆𝑚 With

𝑓0: Resonant frequency

∆𝑓: Frequency change

∆𝑚: Mass change

𝐴: Piezoelectrically active crystal area

𝑝𝑞: Density of quartz 𝜇𝑞: Shear modulus of quartz for crystal

Figure 1.6 shows the configuration of the QCM-based DNA biosensor It consists of a

piezoelectric crystal, an oscillator to apply AC voltage the crystal and a frequency counter device to measure the change of frequency due to the change of the mass ssDNAs will be immobilized 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 oscillating frequency [27]

Figure 1.6 - A QCM – based DNA biosensor [12]

Besides QCM, other methods can be utilized for mass detection with a piezoelectric crystal, such as surface acoustic wave Inter-digital transducers are attached on the piezoelectric substrate, and it also takes the role of electrodes Biology materials are coated on the system, which includes targets and probes The mechanic wave generated

by the piezoelectric will be transmitted periodically The process of generation and reception of signal will be affected by the hybridization of probes and targets Due to the alteration of the intensity and frequency of mechanical wave, we can detect the happened phenomenon [10], [17] This method is often used with microfluidic system where analytical solution flows over the surface of immobilized biology materials, which can

be presented as a complete DNA sensor Another approach is the use of micro-fabricated cantilever to detect mass change The mechanism of this method is the same as Atomic

Force Microscopy (AFM), where the movement of cantilever is detected by the deflection of a laser beam from the cantilever surface This technique lends itself to array assay and mass detection However, this method is constrained by high cost equipment and often applied in laboratory [6]

1.3 Magnetic methods

Magnetic method is one kind of labelling methods, but they are often disregarded as true label due to their micro-scale However, they are famous for two important advantages including the easiness of detecting a small amount of magnetic beads and application of using micro-fluidic setup for better assay performance [30] Magnetic beads are often used for target pre-concentration purpose rather than detection Probe-modified magnetic beads are hybridized with target DNA, after that they are separated magnetically from the pool of analytes for further identification In fact, the magnetic methods are often used in conjunction with other methods such as optics, electronics and electrochemistry to produce the desired results [3]

1.4 Electrochemical methods

In electrochemical sensors, ssDNAs are immobilized onto an electrically active metal electrode surface The hybridization of ssDNAs will cause changes in electronic characteristics such as current, potential, impedance, thus implying the happening process Electrochemical sensors can be divided into sub-categories based on their transducing methods or types of detection Electrochemical methods is of paramount importance in the thesis so it will be focused and analyzed comprehensively Electrochemical methods can be looked at on two different aspects

In terms of detection techniques, electrochemical sensor can be divided due to their change in physio-chemistry properties, including Amperometry, Potentiometry, Conductometry and Impedimetry In terms of sensing platforms, DNA sensor can be

functionality to detect probe in analyte but also discern the mismatch in pre-hybridization dsDNA strands, and this application is in favor of enzymatic – based DNA detection Research attempt in human gene has arisen for a few decades now, and people have just succeeded in deciphering the DNA map In this long and arduous quest, a myriad of methods have been adopted to identify the order of DNA sequences, which comprise of only four nucleotides The sensing platform is divided into direct detection and indirect detection

1.4.1 Direct (Label – free) Platform:

The seminal electrochemical DNA was based on reduction and oxidation reaction at a mercury electrode The amount of oxidized or reduced DNA will reflect the concentration of DNA captured Palecek and his colleagues [22] developed methods to distinguish between single and double-stranded DNA through direct DNA reduction The feasibility of the assay is due to the fact that when two single-stranded DNA hybridized, physiochemical changes will occur on the surface of the transducer The direct detection of DNA requires high potentials, which cause high background noise current As a result, even though the platform is really simple, application in real life require careful treatments and modifications to improve signal-to-noise ratio of the measurement Gold electrodes have been modified with thiolated and SAM – immobilized probes to detect short oligonucleotides [14] Label – free DNA sensor used polyaniline has also been received much attention from scientists [9][23]

Ion-selective Field effect transistor (ISFET) has been used in detection of DNA (Figure 1.7) ISFET has the same working mechanism as Metal Oxide Semiconductor Field

Effect Transistor (MOSFET), except the gate structure In normal MOSFET, the current flowing from drain to source is controlled by the voltage applied on the gate In ISFET, the gate is replaced with a chemical sensitive thin film and an electrode In application

of DNA sensor, ssDNAs are immobilized directly on the gate surface The hybridization

occurred at the gate will produce a change in transistor current, and this allows us to

perceive the concentration of DNA hybridized, as shown in Figure 1.8 [38]

1.4.2 Indirect (Label – based) Platform:

Indirect methods was utilized to increase signal to noise ratio of the measurement Label – based processes take advantage of chemical mediators such as methylene blue (MB),

Figure 1.7 - Direct DNA detection [9]

Figure 1.8 - Hybridization signal from ISFET [21]

Ruthenium Bypiridine, Co(phen)33+ (phen = phenanthroline) [18], [19], Ru(NH3)6+ and Ferrocene Yang and Thorp [37] used Ruthenium complexes to directly oxidize guanine

in DNA Another approach is to label the target DNA with redox indicator This method

is somehow similar to fluorescence labelling, but instead of optical signal, the redox reporter will unveil itself through electrochemical reaction In some situations, DNA serves as mediator for charge transportation Electrochemical substances in solution are oxidized or reduced and exchange electrons with the electrode via DNA The hybridization will increase the surface concentration of those substances, therefore signal the hybridization process, for example methylene blue was used as intercalative probe

is referred to as amperometry A different variation is voltammetry, where the current is recorded while the voltage is varied Due to different electron transfer processes, three

“generations” of amperometric sensor have been presented In the first generation, the product of reaction diffuses to the transducer and causes the electric current In the second type, biosensors involve a specific “mediator” between the reaction and the transducer to improve response while in the third one, biosensors measure directly the signal generated from the reaction One example of amperometric first generation sensor

is glucose sensor, in which the current measured is the electron flow from H2O2, a product of reaction between glucose with oxygen

b) Potentiometric sensor

Potentiometry method determine the amount of charge accumulated on the surface of electrodes Those pile up electrons will generate a potential compared to the reference electrode, where no current is allowed to pass through This can be referred to as a static method, as there is no flow existed in the analyte Therefore, the method is often used in quantitative measurement The amount of charge can be calculated by Nernst equation [24]:

𝐸 = 𝐸0−𝑅𝑇

𝑛𝐹ln 𝑎 With

𝐸: Electrode Potential

𝐸0: Standard Potential of the electrode

𝑅: Universal gas constant (8.314 𝐽

𝐾.𝑚𝑜𝑙) 𝐹: Faraday Constant(96485𝑚𝑜𝑙𝐶 ):

𝑇: Temperature in Kelvin

𝑁: Charge of the ion or number of electrons participating in reaction

𝐴: Activity of the ions

One important factor in this method is the reference electrode There are many kind of reference electrode, for example silver/silver chloride is a popular one which is independent of concentration of analytes and temperature This method was used in several DNA sensor platforms to detect DNA hybridization [8], [21]

c) Impedimetry sensor

This type of sensor is based on the change of impedance (including both real and imaginary parts) of the interface between analyte and the immobilized substances on the electrode surface In the measurement, an AC voltage of the form 𝐸 = 𝐸𝐷𝐶+

𝐸0sin(𝑤𝑡 + ∅) with varying frequency is applied to the electrode The measured impedance was then displayed in Nyquist Plot which represents the relationship between real and imaginary impedance

Figure 1.10- Nyquist Plot of EIS [27]

In detection of DNA hybridization, the impedance of the interface is measured before and after the immobilized probe DNA come into contact with target DNA The Nyquist plot contains two parts: the semi – circle part represents the high – frequency

impedance and linear part represents the low – frequency impedance of the interface

As indicated on Figure 1.10, Nyquist plot are different before and after the

hybridization takes place

The nature of electrochemical processes which happen at the site of reaction will be thoroughly examined, the answer to the question “What happens when we apply a voltage to the solution?” will be presented The structure of DNA is double helix form with four bases, which are bound together by two strands called back-bone of the DNA The backbone of DNA is based on a repeated pattern of a sugar group and phosphate group As indicated in the name, the sugar group is deoxyribose (C5H10O4), and the other group which are also part of the backbone is the phosphate group (H3PO4) The combination of these two groups makes the entire molecule heavily charged with negative potential [35] A group of deoxyribose, phosphate group and a base (Adenine, Guanine, Cytosine and Thymine) will produce a so-called nucleotide The nucleotides

will then join together to form a DNA strand As shown on Figure 1.11, two DNA

strands can link together through the hydrogen linkage between bases: Adenine (A) links with Thymine (T) with two hydrogen bonds while Guanine (G) links with Cytosine (C) with three hydrogen bonds

The nucleotides must overcome the strong repulsion force between each other in hybridization process due to negative charge of each bodies In conventional detection methods, temperature and chemicals are used to reduce the repulsion force of these molecules However, they proved to be not so much effective because very high temperature or ion buffer concentrations are required to overcome the energy barrier, and such conditions are likely to damage the bio-system The nature of electrochemical

process is to apply an electrostatic field to the surface, and this electrical field will help

to overcome the energy barrier [35][4]

Figure 1.11 - DNA structure (U.S National Library of Medicine)

Each method mentioned above has its own advantages and disadvantages Optical methods are highly praised for its accuracy, rapidity, non-destructiveness and comparatively inexpensive analysis Additionally, these techniques can allow real time detection of hybridization However, it requires careful control in the operation as well

as the operator’s skills The integration of optical sensor into complete systems is also a

problem encountered by many scientists [5] On the one hand, the piezoelectric methods are very sensitive but its minus mark is the selectivity Any change in mass (about nanograms) can trigger the signal variation On the other hand, electrochemical methods offer advantages of simple operation, high sensitivity and relatively inexpensive instrumentation that can be easily miniaturized, making this method very attractive for the development of portable devices or in situ monitoring Also, they requires many pretreatment steps and different chemicals which can cause damage to the DNA However, these drawbacks can be overcome if we can build a standard and careful procedure for sensor fabrication This gives an opportunity for mass production of high quality DNA sensor, which have been applied in reality in the case of glucose sensor

2 Heavy metal detection in Food Safety analysis

Heavy metals are found naturally in the earth’s crust Through inhalation, diet and manual handling, heavy metals are accumulated to plants, animals and human tissues Heavy metals are important in biological processes but the over uptake of heavy metals ions can have deleterious effects on human’s health There are many kinds of heavy metals, but only 7 elements are considered to be toxic [Table 1]

Heavy

metal Toxicities

MCL (mg/L) Arsenic Skin manifestations, visceral cancers, vascular disease 0.050

Cadmium Kidney damage, renal disorder, human carcinogen 0.01

Chromium Headache, diarrhea, nausea, vomiting, human

carcinogen

0.05

Copper Liver damage, Wilson disease, insomnia 0.25

Nickel Dermatitis, nausea, chronic asthma, coughing, human

Lead Damage the fetal brain, diseases of the kidneys,

circulatory system, and nervous system

0.006

Mercury Rheumatoid arthritis, and diseases of the kidneys,

circulatory system, and nervous system

0.00003

Table 1.1 - Toxic heavy metals and their effects on daily lives

Long term exposure to those heavy metals can cause serious health problems, as indicated in Table 1 Today, through the use of industrial and agricultural processes, heavy metal doses in food exceeded the safety standard and became a menace to population Therefore, we need a routine to detect and treat the heavy metals sources, and ameliorate the situation for indigenous people A few methods have been applied in laboratory to detect the heavy metal components, including Inductively Coupled Plasma – Mass Spectroscopy (ICP-MS), Inductively Coupled Plasma-Atom Emission

Spectroscopy (ICP-AES), UV-VIS, X-ray Fluorescence (XRF) and Atomic Absorption Spectroscopy (AAS)

ICP-MS is an analytical tool for elements determination ICP-MS includes a high temperature ICP (Inductively Coupled Plasma) and a mass spectrometer Firstly, the sample is introduced to the chamber to and converted into gas or liquid forms The inductively coupled Plasma is a type of plasma source produced by electromagnetic induction Argon atoms will become argon ions when enter electric field, then they collide with each other to form plasma Plasma will strip off electrons of heavy metals elements and turn them into ions These ions are then separated and directed to the mass spectrometer Based on the relation between mass and charge of the elements, these ions will be filtered and counted by an ion deflector As a result, the spectrum will represent the components of the sample

ICP-AES also contains one Inductively Coupled Plasma (ICP) source and an optical spectrometer The Argon atoms are transformed into ions by an intense electromagnetic field, which is created by high power radio frequency signal running through a working coil Argon plasma can produce excited atoms and ions that emit characteristic wavelength which defines a particular element

UV-VIS is an analytical quantitative method It’s often based on the bonding of heavy metals ions with ligands – sol particles or emulsion Through the absorption or reflectance of certain wavelength in UV and near infrared range when interact with the particles in solution, the concentration and composition of sample can be analyzed

2.4 X-ray Fluorescence

X-ray Fluorescence is an analytical method used to determine the elemental composition

of materials A high energy beam is directed to the samples, where it will interact with electrons at the inner shells of atoms The electrons at inner shells are ejected from its orbits and another electron from the outer shells fill in The difference in energy binding between the inner electron orbital and outer electron orbital gives rise to a radiation Because the energy of emitted photon is characteristic of a transition between specific electron orbitals in a particular element, the resulting fluorescent X-ray can be used to detect the traces of elements that present in the sample

Atomic Absorption Spectroscopy is an analytical method for quantitative determination

of chemical elements The principle of the method is based on the specific absorption ability of atoms in gaseous state When an optical light is exposed to the sample in gaseous state, the atoms will absorb optical light at suitable energy level and turn into excited state Calculating the proportion between the remaining and initial light intensity,

we can infer the concentration of the analyzed element This method has one disadvantage is that it requires an atomizer to convert sample from liquid to gaseous state

Regarding sensitivity, accuracy and limit of detection, those aforementioned techniques are of high quality, however they require complicated instrumentation and high-skilled technicians Moreover, they are not easily transformed into portable analytical tools for screening, detecting, identifying and quantifying metal ions Recently, electrochemical methods have been optimized intensively in heavy metal detection It can employ direct volt – ampere characteristics between the analyte and electrode surface It includes two main steps: ion absorption and ion stripping, which occur on the surface of electrodes The principle of the method will be detailed in chapter 3

In Chapter 1, we have already demonstrated the mechanism of DNA detection and heavy metal detection and the diverse methods in obtaining the signal produced by the sensor Among these, electrochemical methods are of favor and we also introduced electrochemical – based techniques

In Chapter 2, we will focus on the design and development of a device which will be able to acquire the signal produced by the sensor

Chapter 2 – DESIGN AND IMPLEMENTATION OF DATA ACQUISITION AND PROCESSING DEVICE FOR

ELECTROCHEMICAL SENSOR

This Chapter will discuss the design of the data acquisition and processing device which can be applicable for electrochemical analysis In the scope of the thesis, only cyclic voltammetry and linear voltage sweeping are available for the device The triple electrode configuration will be used for electrochemical implementation

1 Triple electrode configuration

Today, the most common electrochemical analysis cell is the triple electrode configuration (or three electrode system) As its name indicates, it comprises three electrodes, which are Working Electrode (WE), Reference Electrode (RE) and Counter Electrode (CE) They are used mainly in analytical chemistry, where we need to control the voltage over a surface and measure the current produced by the chemical reaction occurred due to the applied potential

An electrochemical cell generally consists of two half-cells, each containing an electrode

in contact with an electrolyte A typical example of two electrode cell is the battery, where the redox reaction occurs and causes an electric current flowing through the circuit It consists of anode and cathode, in which oxidation occurs at anode and reduction occurs at cathode However, we can only achieve the relative voltage between the two terminals, while in analytical electrochemistry we need to control the accurate potential applied to each terminal

To solve the problem, the third electrode is introduced to the system: the reference electrode A fundamental thing one has to bear in mind is that the potential of a single electrode or half-cell by itself cannot be measured There must be a “reference”, it is the same in electronics where the measuring voltage of a node needs two points, one of them commonly referred as ground In the triple electrode configuration, the voltage between

working electrode and reference electrode is kept under control, and the counter (or auxiliary) electrode is added to complete the electrochemistry circuit

The working electrode is the electrode in an electrochemical system on which the reaction of interest is occurring The working electrodes are of various geometries and materials, ranging from noble metals such as gold, platinum to glassy carbon [7] Materials selected for working electrode must show favorable redox behavior with the analyte, have high potential window and they must be inert as not to participate in electron transfer process It acts as the cathode in the two-electrode system In this thesis, gold has been chosen for its stability, availability and wide potential range Also, gold is famous for its linking ability with biology substances [26]

The reference electrode should provide a reversible half-reaction with Nernstian behavior, be constant over time, and be easy to assemble and maintain Two most commonly used reference electrodes for aqueous solutions are the calomel electrode and silver/silver chloride electrode [16] Basically, the saturated calomel electrode composes

of an inner tube, packed with a paste of Hg, HgCl2 and saturated KCl, situated within a second tube filled with a saturated solution of KCl Electrode potential is determined by the reaction in the equation 2.1:

𝐻𝑔2+ → 𝐻𝑔 − 2𝑒 | 𝐶𝑙2+ 2𝑒− → 2𝐶𝑙− (2.1) The experiments showed that electrode potential at calomel electrode is very stable and ease of use However, the electrode may cause hazards if crashed

The other type is the silver/silver chloride electrode (Ag/AgCl), it includes an Ag wire with AgCl at the tip of the wire in saturated KCl solution Its potential is determined by the reaction in the equation 2.2:

𝐴𝑔𝐶𝑙 + 𝑒− → 𝐴𝑔 + 𝐶𝑙− (2.2) For Saturated calomel Electrode (SCE), the reference potential is +0.241V while this value is +0.197V for silver/silver chloride electrode Except for the redox potential, these

integrating the reference electrode onto electrode structure and employing electrode as on-site biosensor, silver/silver chloride is more feasible Therefore, Ag/AgCl will be used in this thesis

micro-Counter electrode allows the electrons produced at the working electrode surface to pass

by, as the reference electrode is designed to block current Most often the counter electrode made of a noble metal like Platinum or Gold The graphite is also used in some cases [36] In this thesis, gold is chosen to be counter electrode for its stability Besides,

to simplify the fabrication process, counter electrode material is chosen to be the same with working electrode

The aforementioned configuration will be implemented in both cyclic voltammetry and anodic stripping voltammetry (Linear sweep mode)

Potentiostat Circuit Operating Principle

As the electrode configuration for measurement was presented, the function of the instrument used to control three-electrode system will be discussed

According to Bard and Faulkner [1], a potentiostat, an electronic instrument, is used to control a three electrode cell and operate most electro-analytical experiment The system functions by maintaining the potential of the working electrode at a constant level with respect to reference electrode by adjusting the current passing through counter electrode

(Figure 2.1)

In regards to electronic circuit, an impedance (resistors/capacitors/inductors) model should be built for easier understanding of operation principle and functions implementation of the circuit In this case, the electrode – solution behaviors which impede the charge transfer process will be analyzed and working mechanism of potentiostat circuit will be derived from that

Figure 2.1 - Potentiostat Principle

Several theories have been developed to explain the nature of solution - electrode interface [29], according to which there is a layer structure that impedes the charge transfer process between the solution and the electrode In this thesis, the Stern model seems to be suitable to describe the electrochemical behavior when applying a voltage

at the interface

Figure 2.2 - Double Layer Model

As shown in Figure 2.2, when we apply a positive voltage to the electrode, negative

charge (ions) in the solution will be attracted to the surface However, those negative ions cannot directly come into contact with the electrode They are separated by solvent molecules, which have smaller size and contribute to the polarization process Positive charges and negative charges separated by a distance d will create a capacitor The second layer is made of free ions loosely associated with the object, so that this diffuse layer is not fixed Those two double layer capacitors combining with electrode resistance create impedance between the electrode and the solution, which will show its role while being put into electronic model used to explain potentiostat operation principle

Figure 2.3 - Impedance model of electrochemical cell

Figure 2.3 shows the most basic model of a potentiostat, which only utilizes a voltage

source and a control amplifier (CA) In the model, Z1 equals to Relectrode in series with the interfacial impedance of the counter electrode and the solution resistance between the counter and the reference Z2 represents the interfacial impedance of the working electrode in series with the solution resistance between the working electrode and the reference electrode

The role of the CA is to amplify the potential difference between the positive inverting) input and the negative (inverting) input This can be translated mathematically into equation 2.3:

(non-𝐸𝑜𝑢𝑡 = 𝐴 (𝐸+− 𝐸−) = 𝐴 (𝐸𝑖− 𝐸𝑟) (2.3) where A is the amplification factor of the CA

At this point, it is assumed that no or only insignificant current is flowing through the reference electrode This corresponds to the real situation since the reference electrode

is connected to a high impedance electrometer Thus the cell current can be put in two ways:

𝐼𝑐 = 𝐸𝑜𝑢𝑡

𝑍1+𝑍2𝑜𝑟 𝐼𝑐 =𝐸𝑟

𝑍2 (2.4)

Combining two equations yields a new one:

𝐸𝑟 = 𝑍2

𝑍1+ 𝑍2 𝐸𝑜𝑢𝑡 = 𝛽 𝐸𝑜𝑢𝑡 (2.5) Where 𝛽 is the fraction of the output voltage of the control amplifier returned to its negative input or the feedback factor

𝛽 = 𝑍2

𝑍1+ 𝑍2 (2.6) Combining equation (2.3) and (2.5), we get equation (2.7)

𝐸𝑟

𝐸𝑖 =

𝛽 𝐴

𝛽 𝐴 + 1(2.7) When the quantity 𝛽 𝐴 becomes very large with respect to (2.3), equation (2.7) reduces

to equation (2.8), which is one case of negative feedback equation:

𝐸𝑟 = 𝐸𝑖 (2.8) Equation (2.8) proves that the control amplifier works to keep the voltage between the reference and the working close to the input source voltage

2 Electronics Circuit Design

In the previous section, the working principle of a potentiostat has been presented The implementation requires some modules as would be introduced below:

The device is powered at 5V DC, as CY8C27443 microcontroller and op-amps operate

at 5V.Normally, the city-line voltage needs to go through a few steps to shrink down to the desired value

Figure 2.4 - Power Source Transformation

AC Voltage Transformer Rectifier Voltage Regulator

In power source section, the AC voltage at 220V 50 Hz must go through the transformer

to scale down to AC voltage at a lower amplitude The rectifier is used to “smooth” the remaining ripple After that, the voltage regulator will stabilize the voltage at a fixed value, namely +5V DC and -5V DC In our application, we will utilize a 9V-1A adapter for the first three stages, the voltage regulators are AMS1117 and ICL7660 to create a stable +5V DC and -5V DC

Figure 2.5 - PSoC 1 Architecture

The PSoC 1 architecture consists of four main areas as in Figure 2.5: (1) PSoC Core,

(2) Digital System, (3) Analog System, and (4) System Resources Configurable global busing allows all the device resources to be combined into a complete custom system Cypress offers PSoC 1 devices that have up to 16 digital blocks and 12 analog blocks Digital blocks are programmable digital blocks that the designer can use to implement glue logic, UART, PWMs, counters, LUTs, etc These blocks contain a programmable data path and status and control registers, and includes both basic blocks (BB) and communications blocks (CB) Analog blocks are programmable analog blocks that the designer can use to implement Op-Amps, PGAs, comparators, etc, and include continuous time (CT) blocks and switched capacitor (SC) blocks Both the digital and analog blocks are configurable using user modules (UMs) in PSoC Designer

PSoC Designer

PSoC Designer provides a friendly platform which utilizes the drag-and-drop design environment for programming The parameters setup implemented via user interface combining with written code bring a quick and easy way to put modules into work Application code was written in C In our application, the microcontroller needs to fulfill some requirements:

- Create a desired voltage waveform

- Acquire the analog-processed signal and transform to digital signal

- Transmit the converted data to the computer for further processing

The flow chart of programming model is shown in Figure 2.6:

Stop

Start

RESET

True False

Figure 2.6 - MCU Firmware Flowchart