68 Trang 4 LIST OF ABBREVIATIONS IUPAC: International Union of Pure and Applied Chemistry DNA: Deoxyribonucleic Acid RNA: Ribonucleic Acid SPR: Surface Plasmon Resonance MOSFET: Metal
REVIEW ON METHOD FOR DNA HYBRIDIZATION DETECTION
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 methods, such as Maxam-Gilbert sequencing and the chain-termination method, became essential for DNA mapping by determining the order of specific DNA strands using informed sequences These techniques involve hybridizing oligonucleotide probes to a solid surface with target DNA or RNA under stringent conditions, followed by detection through fluorescence labeling While reliable and user-friendly, they demand complex sample preparation and large quantities of purified DNA, making them ideal for genome-wide genetic mapping, physical mapping, proteomics, and gene expression studies Recently, Polymerase Chain Reaction (PCR) and Enzyme-Linked Immunosorbent Assay (ELISA) have emerged as the most prevalent methods in biology, medicine, and food technology for DNA detection PCR detects temperature changes during hybridization, while ELISA utilizes antibodies and color changes for substance identification Although both methods are known for their accuracy and specificity, they can be costly, time-consuming, and require intricate sample preparation.
DNA biosensors utilize the direct hybridization of aptamers or single-stranded DNA (ssDNA) to detect nucleic acids, generating a measurable signal The strength of this signal correlates with the degree of hybridization, where more matches in double-stranded DNA lead to enhanced signal intensity This technology is gaining significant attention for its potential in identifying specific genes and biological substances.
Recent advancements in biological and chemical techniques have enabled the rapid, cost-effective, and precise synthesis and regeneration of nucleic acids, surpassing traditional analytical methods.
[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
DNA biosensors can be classified according to their transduction methods, which include optical, electrochemical, piezoelectric, and magnetic techniques This article will provide a brief overview of each method, highlighting their respective advantages and disadvantages.
The analysis methods for hybridization can be categorized into label-based and label-free detection Label-based detection involves the use of redox intercalators for recognizing double-stranded DNA (dsDNA), DNA mediators that facilitate electron transfer, and enzyme labeling to improve signal sensitivity and intensity Common indicators in this method include ruthenium bipyridine, methylene blue, and various redox chemicals In contrast, label-free detection, as the name implies, identifies targets without the use of labels.
The hybridization of single-stranded DNAs can be achieved through various transduction methods combined with detection techniques, leading to innovative approaches for studying this phenomenon The following section will explore some commonly used methods in contemporary analyses.
Transduction techniques include both label-based methods, such as fluorescence, and label-free methods Notable optical techniques encompass optical fibers, surface plasmon resonance, and reflection interference contrast microscopy.
Figure 1.3 - Scheme of ssDNA labelled with fluorescent agent detection
Fluorescence methods, often referred to as labelling methods, have gained significant attention in recent years, particularly in the context of fiber optics DNA sensors These sensors are categorized into single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) labelling techniques Optical fibers serve as the transmission pathway for fluorescent signals emitted during hybridization events In ssDNA labelling, fluorescent chemicals can covalently bind to ssDNA, enhancing the fluorescent intensity and enabling more effective detection.
The hybridization of ssDNAs significantly enhances the process, as illustrated in Figure 1.3 A key method involves using fluorescent intercalators, such as ethidium bromide (EB) and thiazole orange (TO), which bind selectively to DNA hybrids through noncovalent interactions after dsDNAs are formed When these intercalators insert between base pairs, they trigger excitation, resulting in either fluorescence enhancement or quenching, as demonstrated in Figure 1.4.
Figure 1.4- Scheme of interposed intercalators at the hybridization event
Here is a rewritten paragraph that complies with SEO rules:Various labelling methods rely on radioisotopes, fluorophores, and UV-absorbing molecules However, the integration of nanotechnology has led to the development of hybridization labelling methods using quantum dots and nanowires Quantum dots, generated through a bottom-up approach, are nanostructures that can be used for fluorescence tagging of biomolecules Notably, their colour changes in response to particle size variations, a phenomenon known as the confinement effect, allowing ssDNAs to be tagged with distinct colours This enables direct visual detection of hybridization by the naked eye or through fluorescence resonance energy transfer (FRET).
Surface Plasmon Resonance (SPR) utilizes a quantum phenomenon where conduction electrons oscillate in response to incident light The resonance condition occurs when the frequency of incoming photons aligns with the natural frequency of surface electrons In SPR DNA sensors, single-stranded DNA (ssDNA) is immobilized on a thin metal film while an aqueous solution flows over it Light is directed to the surface via a prism and reflected back with specific optical reflectivity Upon hybridization of double-stranded DNA (dsDNA), changes in optical characteristics such as reflectivity, refractive index, and intensity are observed When integrated with optical fiber, the system maintains contact between the metal layer and the sample solution However, this labeled-based technique is often considered expensive and complex, making it more suitable for research applications than practical use.
Labelled-free methods also include other methods such as Reflection Interference Contrast Microscopy (RICM) or Raman Spectroscopy to detect the DNA hybridization process [4]
The advancement of nanotechnology has significantly enhanced fiber-optic DNA biosensors, as these sensors can be efficiently miniaturized to the nanometer scale through techniques like chemical and tube etching, as well as mechanical pulling using CO2 laser heating.
15 setup [25], they are immuned to electromagnet [34], disposability and long-distance transmission [6]
Piezoelectric materials, like quartz, oscillate at a specific frequency when cut correctly and subjected to an alternating current (AC) voltage This frequency is directly influenced by the mass of the crystal, forming the fundamental principle behind Quartz Crystal Microbalance (QCM) According to the Sauerbrey equation, variations in mass correspond to changes in frequency, highlighting the sensitivity of this technology.
Density of quartz Shear modulus of quartz for crystal
The QCM-based DNA biosensor configuration includes a piezoelectric crystal, an oscillator for applying AC voltage, and a frequency counter to measure frequency changes caused by mass variations Single-stranded DNAs (ssDNAs) are immobilized on the crystal's surface, enabling interaction with analytes in solution The hybridization process leads to a mass change that decreases the oscillating frequency.
Figure 1.6 - A QCM based DNA biosensor [12] –
In addition to Quartz Crystal Microbalance (QCM), surface acoustic wave technology can be employed for mass detection using piezoelectric crystals This method involves inter-digital transducers that function as electrodes on the piezoelectric substrate, where biological materials, including targets and probes, are coated The mechanical wave generated by the piezoelectric element is transmitted periodically, and the signal generation and reception are influenced by the hybridization of the probes and targets Changes in the intensity and frequency of the mechanical wave allow for the detection of these interactions This technique is commonly integrated with microfluidic systems, facilitating the flow of analytical solutions over immobilized biological materials, effectively serving as a complete DNA sensor Another effective method for mass change detection is the use of micro-fabricated cantilevers, which operates on a principle similar to that of atomic force microscopy.
Heavy metal detection in Food Safety analysis
Heavy metals naturally occur in the earth's crust and can accumulate in plants, animals, and human tissues through inhalation, diet, and manual handling While these metals play a role in biological processes, excessive uptake of heavy metal ions can negatively impact human health Among the various heavy metals, only seven are classified as toxic.
(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 carcinogen 0.20
Zinc Depression, lethargy, neurological signs and increased thirst 0.80
Toxic heavy metals, such as lead and mercury, pose significant health risks, particularly affecting vulnerable populations Lead exposure can damage the fetal brain and lead to diseases of the kidneys, circulatory system, and nervous system, with a notable impact at levels as low as 0.006 Similarly, mercury is linked to rheumatoid arthritis and can also harm the kidneys, circulatory system, and nervous system, even at minimal exposure levels of 0.00003 Understanding the effects of these heavy metals is crucial for safeguarding public health and well-being.
Long-term exposure to heavy metals poses serious health risks, as highlighted in Table 1 Industrial and agricultural practices have led to elevated levels of heavy metals in food, surpassing safety standards and threatening public health It is crucial to establish a routine for detecting and mitigating heavy metal sources to improve conditions for indigenous populations Various laboratory methods, such as Inductively Coupled Plasma – Mass Spectroscopy (ICP-MS) and Inductively Coupled Plasma-Atom Emission, have been implemented to identify heavy metal components effectively.
Spectroscopy (ICP-AES), UV-VIS, X-ray Fluorescence (XRF) and Atomic Absorption Spectroscopy (AAS)
2.1 Inductively Coupled Plasma – Mass Spectroscopy (ICP-MS)
ICP-MS (Inductively Coupled Plasma Mass Spectrometry) is a powerful analytical tool used for the determination of elemental composition It combines a high-temperature inductively coupled plasma with a mass spectrometer to analyze samples The process begins with the introduction of a sample, which is converted into gas or liquid form The inductively coupled plasma is generated through electromagnetic induction, where argon atoms become ions in an electric field, leading to plasma formation This plasma strips electrons from heavy metal elements, converting them into ions These ions are then separated and directed to the mass spectrometer, where they are filtered and counted based on their mass-to-charge ratio The resulting spectrum provides a detailed representation of the sample's elemental components, making ICP-MS an essential tool for elemental analysis.
2.2 Inductively Coupled Plasma Atom Emission Spectroscopy –
Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) utilizes an ICP source and an optical spectrometer to analyze elements In this process, Argon atoms are ionized by a strong electromagnetic field generated by a high-power radio frequency signal passing through a coil The resulting Argon plasma produces excited atoms and ions that emit specific wavelengths, allowing for the identification of distinct elements.
UV-VIS is a quantitative analytical method that analyzes the concentration and composition of samples by measuring the absorption or reflectance of specific wavelengths in the UV and near-infrared range This technique often involves the bonding of heavy metal ions with ligand sol particles or emulsions, allowing for precise interactions with the particles in solution.
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 (AAS) is a precise analytical technique used for the quantitative analysis of chemical elements This method operates on the principle that atoms in a gaseous state exhibit specific absorption characteristics when exposed to optical light As the light interacts with the sample, atoms absorb it at particular energy levels, leading to their excitation By measuring the ratio of the initial light intensity to the remaining light intensity, we can determine the concentration of the element being analyzed However, a notable drawback of AAS is its requirement for an atomizer to convert liquid samples into a gaseous state.
Recent advancements in electrochemical methods have significantly improved the sensitivity, accuracy, and detection limits for heavy metal detection, despite the complexity of traditional techniques that require sophisticated instrumentation and highly skilled technicians These methods utilize direct volt ampere characteristics between the analyte and the electrode surface, involving two main steps: ion absorption and ion stripping on the electrode surface Further details on the principles of this method will be provided in Chapter 3.
In Chapter 1, we explored the mechanisms for detecting DNA and heavy metals, highlighting various methods for obtaining sensor signals Among these techniques, electrochemical methods are particularly favored, and we introduced several electrochemical-based approaches.
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
This chapter focuses on the design of a data acquisition and processing device tailored for electrochemical analysis, specifically utilizing cyclic voltammetry and linear voltage sweeping The device will employ a triple electrode configuration to facilitate effective electrochemical implementation.
The triple electrode configuration, also known as the three-electrode system, is the most prevalent electrochemical analysis cell used in analytical chemistry This system consists of a Working Electrode (WE), a Reference Electrode (RE), and a Counter Electrode (CE) It allows for precise control of voltage across a surface while measuring the current generated by chemical reactions resulting from the applied potential.
An electrochemical cell comprises two half-cells, each featuring an electrode immersed in an electrolyte A common example is a battery, where redox reactions generate electric current in the circuit The cell includes an anode, where oxidation occurs, and a cathode, where reduction takes place While we can measure the voltage between the terminals, analytical electrochemistry requires precise control over the potential applied to each terminal for accurate measurements.
To address the issue, a reference electrode is added to the system, which is crucial for accurate measurements It's important to understand that the potential of a single electrode or half-cell cannot be assessed in isolation; a reference point is necessary This concept parallels electronics, where measuring voltage at a node requires two points, with one typically designated as ground In a triple electrode configuration, the voltage is measured between the working electrode and the reference electrode, ensuring precise readings.
31 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 in an electrochemical system is crucial as it is where the reaction of interest occurs These electrodes can vary in geometry and materials, including noble metals like gold and platinum, as well as glassy carbon When selecting materials for the working electrode, it is essential that they exhibit favorable redox behavior with the analyte, possess a high potential window, and remain inert to avoid participating in the electron transfer process In a two-electrode system, the working electrode functions as the cathode This thesis focuses on gold due to its stability, availability, and extensive potential range, along with its renowned ability to link with biological substances.
DEVELOPMENT OF DATA ACQUISITION AND PROCESSING
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 operates on a 5V DC power supply, as both the CY8C27443 microcontroller and op-amps require this voltage Typically, the city-line voltage must undergo several steps to be reduced to the necessary level.
AC Voltage Transformer Rectifier Voltage Regulator
The power source section begins with a 220V AC voltage at 50 Hz, which is transformed to a lower AC voltage using a transformer A rectifier is then employed to smooth out the remaining ripple, followed by a voltage regulator that stabilizes the output to fixed values of +5V DC and -5V DC For our application, a 9V-1A adapter is utilized in the initial stages, with AMS1117 and ICL7660 voltage regulators ensuring a stable +5V DC and -5V DC output.
The Cypress Programmable System on Chip CY8C27443, a PSoC 1 device, is the world's first Programmable Embedded System on Chip that integrates configurable analog and digital peripheral functions, memory, and a microcontroller into a single chip Featuring Cypress’s proprietary 8-bit MCU core, PSoC 1 devices offer system analog integration, flexible I/O routing, and configurable peripherals, enabling designers to rapidly create systems and implement changes efficiently, ultimately reducing BOM costs and development time.
The PSoC 1 architecture consists of four main areas as in Figure 2.5: (1) PSoC Core,
Cypress PSoC 1 devices feature configurable global busing that integrates digital and analog resources into a custom system, offering up to 16 programmable digital blocks and 12 analog blocks The digital blocks enable designers to create glue logic, UART, PWM, counters, and more, equipped with a programmable data path and control registers, including basic and communications blocks Meanwhile, the analog blocks facilitate the implementation of Op-Amps, PGAs, and comparators, comprising continuous time and switched capacitor blocks Both digital and analog blocks are easily configurable through user modules in PSoC Designer, enhancing design flexibility and functionality.
PSoC Designer offers an intuitive drag-and-drop design environment for programming, allowing for easy parameter setup through its user interface alongside written code This streamlined approach facilitates the quick and efficient integration of modules In our application, the microcontroller, programmed in C, is required to meet specific operational criteria.
- 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:
The analog section of the potentiostat is designed to maintain a constant voltage between the working and reference electrodes, as previously discussed Operating with small signals, the circuit measures solutions that are sensitive to ambient conditions, necessitating meticulous signal processing from component selection to signal conditioning This design enhances the original circuit philosophy by adding or improving functions to optimize measurement signals Figure 2.7 illustrates the general block diagram of this design.
To better understand the diagram, there are four functions to be explained, including Potentiostat Core, Transimpedance Amplifer, Filter and Voltage Shifter
The potentiostat core plays the role to apply the desired voltage to the working e trode lec
As we can on the Figure 2.8, the voltage generated by DAC is shifted by a voltage
41 shifter, then it was passed through a filter circuit After that, the filtered voltage is pumped to the counter electrode regarding to the reference electrode
The circuit employs a single op-amp as a voltage buffer for the reference electrode input, while another op-amp functions as a voltage shifter to reduce the voltage output from the microcontroller These two signals are subsequently combined at the control amplifier, ensuring that the voltage at the counter electrode is adjusted to the specified level.
Figure 2.9 - Trans-impedance Amp using Instrumentation Amplifier
High-side current sensing can be achieved using various configurations, including differential amplifiers, instrumentation amplifiers, and negative feedback operational amplifiers, which convert current into a proportional voltage The use of instrumentation amplifiers (In-Amps) offers significant advantages, such as high common mode rejection and high differential gain, making them effective for amplifying small signals while reducing noise from both input pins An In-Amp design typically includes two or three operational amplifiers along with carefully trimmed passive components Additionally, voltage simulation can be performed using TINA software to analyze these configurations.
Figure 2 - Simulation Result of TIA circuit 10
The INA126P is a precision instrumentation amplifier designed for accurate and low-noise differential signal acquisition, with a gain set to 100 and a bandwidth of 1.8 kHz When using a sensing resistor of 1 kΩ, the total gain reaches 100,000 The circuit's functionality was tested through simulation using TINA-TI software, where the input current through the Working Electrode is illustrated in Figure 2.10a, and the corresponding output voltage is shown in Figure 2.10b The results confirm that the output voltage is proportional to the input current, achieving the expected gain of 100,000.
In small signal conditioning circuits, noise types such as white noise, pink noise, Johnson noise, and ambient noise can significantly impact signal integrity These noises operate at various frequencies, while the desired signal is confined to a specific range, necessitating the design of filter circuits to effectively attenuate noise and preserve the signal Given that our application involves a signal at approximately DC level, implementing a low-pass filter is an effective solution for eliminating unwanted noise.
The Sallen-Key topology is a widely recognized electronic filter design employed for creating second-order active filters, appreciated for its straightforwardness This configuration features a cut-off frequency of 1 Hz, a pass band ripple of 1 dB, a gain of 1 V/V, and a stop-band frequency with -45 dB attenuation, utilizing a Butterworth response type The filter's configuration is illustrated in Figure 2.11.
Figure 2 - 11 Low-pass Filter at 1 Hz
The Filter Circuit is tested using simulation of TINA-TI at different frequencies, namely 0.1 Hz, 12.58 Hz, 25.05 Hz, 37.53 Hz and 50 Hz at 100mV The results was shown below:
Figure 2 - Filter Circuit Simulation Result 12
As we can see on Figure 2.12, only sine wave at 0.1Hz can pass the filter and retain the amplitude, the signals at higher frequencies are strongly attenuated
Figure 2 - Voltage Level Shifter Circuit 13
Figure 2.13 shows the configuration of the voltage level shifter circuit The microcontroller only operates from 0 5V while the conditioning signal varies below –
To ensure compatibility with the microcontroller's voltage range, the signal's voltage must be adjusted This adjustment begins with a reference voltage of 2.5V from the CY8C27443 The voltage is first reduced in the analog section and then increased again when it returns to the microcontroller board As illustrated in Figure 2.14, the circuit simulation shows that the applied voltage ranges from -2.5V to 0V (line (1)), while the shifted voltage varies from 0V to 2.5V (line (2)).
LabView Software in Communication with Computer
LabView, or Laboratory Virtual Instrument Engineering Workbench, is a versatile system-design platform and development environment created by National Instruments It utilizes G, a dataflow programming language that allows for intuitive graphical programming, making it easy to visualize and develop engineering systems LabView supports various communication interfaces, enabling users to write direct code or use high-level, device-specific drivers that offer native LabView function nodes for effective device control.
Figure 2 - Simulation Result of Voltage Level Shifter 14
This is also the reason why it has been the preferred solution to create, deploy and test from smart machines to recent Internet of Things applications
LabVIEW programs, known as virtual instruments (VIs), consist of two main windows: the front panel and the block diagram The front panel serves as the user interface, displaying inputs (controls) and outputs (indicators) in various formats, including numbers, Boolean values, and strings Meanwhile, the block diagram is where the algorithm is implemented, allowing users to wire terminals, incorporate subVIs, organize structures, and transfer data between different block diagram objects.
The LabView Program in our application facilitates communication between the microcontroller and the computer by establishing a connection bridge using the RS232 protocol Once the port is acquired, data is transferred from the microcontroller to the computer, where the program decrypts the information and converts it into visual representations, such as graphs This data can subsequently be stored on the computer for further analysis.
Extract DAC and ADC Value Graph Display
RESULTS AND DISCUSSION
Device Specifications
Figure 3.1 illustrates the complete components of the Data Acquisition and Processing Device (DAP) board, which comprises a power control unit, a microcontroller section, and an analog section The overall performance will be assessed through various characteristic measurements.
Figure 3.1 - Data Acquisition and Processing Device Board
The circuit can be powered via a USB port or a DC adapter, with the measured voltage at the microcontroller and Op-Amps' power pins at 4.90V This voltage is within a 5% error margin, ensuring proper circuit operation.
The applied voltage range can be from -1.3V to +1.3V Applied voltage resolution is 5 mV with accuracy ±5% Scan rate can be adjusted with the minimum equal 5mV/s c Data Acquisition:
The range of measurable voltage equals to the range of DAC voltage, from -1.3 to +1.3V The resolution is 13-bit Current range is ±170 àA d User Interface:
Figure 3.2 - LabView Computer User Interface
The software interface, developed in LabView, features a communication port and offers two selectable modes that correspond to graphs displaying real-time voltammogram measurements After completing the measurement, users can export the graph values to Excel or other file formats.
Evaluation of the Entire Data Acquisition and Processing Device (DAP)
The device is designated to implement two modes which are Linear Sweeping Mode and Cyclic Voltammetry Mode Those two functions will be tested
2.1 Evaluation of the Linear Sweep Mode
Here is a rewritten paragraph that complies with SEO rules:In a three-electrode setup, the Working Electrode was connected to one terminal of a resistor, while the Reference and Counter Electrodes were linked to the other terminal, as illustrated in Figure 3.3 This configuration enables the application of voltage across the resistor, allowing for the measurement of current flowing through it.
Figure 3.3 - Resistor Test Setup The result of the resistor test was shown on Figure 3.4:
Figure 3.4 - Result of Resistor Test
Figure 3.4 illustrates that the ADC voltage aligns with the applied DAC voltage during a full-range linear scan The DAC voltage is adjusted down by -2.5V as it passes through the analog board, and the signal from the working electrode is subsequently shifted back to fit the microcontroller's range The sensor operates within a voltage range of -1.3V to 1.3V, confirming the linearity of the applied voltage and the functionality of the data acquisition process.
2.2 Evaluation of the Cyclic Voltammetry Mode
Cyclic voltammetry (CV) is a key electrochemical measurement technique widely utilized in electro-analytical chemistry Although it is not typically used for quantitative analysis, CV is invaluable for studying redox processes, understanding reaction intermediates, and assessing the stability of reaction products This method involves varying the applied potential at the working electrode in both forward and reverse directions at a specific scan rate while monitoring the current The process often starts with an initial scan in the negative direction, followed by a reversal to the positive direction Depending on the specific analysis requirements, one full cycle, a partial cycle, or multiple cycles may be conducted.
The study utilizes an Fe 2+ / Fe 3+ solution, where the electrode is submerged in the solution A cyclic voltammetry (CV) scan is conducted, spanning from -0.2V to 0.5V at a scan rate of 25mV/s, using both the EC301 and DAP instruments.
The measurement was conducted using a three-electrode system, with integrated sensors fabricated at ITIMS, Hanoi University of Science and Technology The design of the electrode system is illustrated in Figure 3.5, while the fabrication process is detailed in Thinh’s work [31].
Figure 3.5 - Triple Electrode Sensor draw and real one The measurement setup with the DAP is shown in Figure 3.6:
Figure 3.6 - Measurement Setup with EC301
53 a CV Voltammgram in performed by EC301 b CV Voltammgram in performed by DAP Figure 3.7 - CV Voltammogram by both devices
The experimental setup was replicated using the commercial potentiostat EC301, and the results, illustrated in Figure 3.7, indicate that the voltammograms from both devices are quite similar, with only minor discrepancies in current amplitude The circuit's functionality was confirmed through real electrochemical experiments, and we will calculate and compare this value across various measurements.
For CV scan of blank electrode, between CV scan by the DAP and EC301 is
CV scan was then performed ten times for one electrode by both DAP and EC301 to verify the function of both the circuit and the sensor:
Figure 3.8 - The peak current values obtained by the DAP and the EC301
The variation between each measurement cycle is influenced by both the circuit function and the sensor used As illustrated in Figure 3.8, the average delta current peak measured with the EC301 sensor was 102.2411 µA, compared to 89.9176 µA for the PSoC Circuit-based DAP The standard deviation for the PSoC Circuit measurements was 1.68 µA (1.86%), while the EC301 exhibited a standard deviation of 1.67 µA (1.63%) The electrodes fabricated for this study demonstrated high stability and uniformity during electrochemical measurements, as indicated by standard deviations of both average values being below 2% This consistency highlights the repeatability of the DAP and the reproducibility of the sensor, given that the relative standard deviations for both measurements are closely aligned.
Recent evaluations have confirmed the capabilities of the DAP concerning applied voltage, data acquisition, and stability Moving forward, the DAP will be utilized in applications for DNA detection and heavy metal detection.
Application of the DAP in DNA sensor
This study explores the application of an electrochemical sensor for detecting DNA concentration, with increased electric signals indicating hybridization events between aptamers To enhance sensitivity and selectivity, modifications are necessary due to the small signal size Conductive polymers, known for their excellent electrical conductivity, preparation simplicity, and chemical stability, are ideal for immobilizing biomolecules and facilitating rapid electron transfer in biosensors In this research, poly-pyrrole nanowires were utilized to modify the working electrode's surface, increasing the contact area with aptamers and thereby improving the signal-to-noise ratio and overall sensor performance.
The sensor underwent initial surface modification through the electrochemical synthesis of Poly-pyrrole nanowires, followed by the immobilization of DNA probes Subsequently, the target DNA was introduced to the sensor, facilitating hybridization and resulting in a measurable change in the interface's current.
Electrochemical synthesis of Poly-pyrrole nanowire
Monomer Pyrrole solution was prepared by mixing phosphate buffer solution (pH 7.4), Gelatin Solution 0.08% weight, and Monomer Pyrrole 1M in order
Poly-pyrrole nanowires will be synthesized on a working electrode using the electrochemical deposition method The electrode is submerged in a pyrrole solution, where a constant voltage of 0.75 V, relative to an Ag/AgCl reference electrode, is applied to initiate the deposition process for a duration of 200 seconds Figure 3.9 illustrates the SEM image of the working electrode featuring the poly-pyrrole nanowires.
Figure 3.9 - SEM Image of working electrode with PPy-NWs
PPy-NWs synthesized were uniformly distributed on the surface of the electrode The size of PPy-NWs was from 60 nm to 105 nm
Both DNA Probe and DNA Target are chemicals from Integrated DNA Technologies (IDT) The aptamer order was shown in Table 3.1:
Probe Sequence Thiol-C6- -AGACCTCCAGTCTCCATGGTACGTC- 5’ 3’ Target Sequence 5’-GACGTACCATGGAGACTGGAGGTCT- 3’
The DNA probe is typically stored in a refrigerator at 4°C For the immobilization process, the probe is removed from the fridge to thaw and then placed onto the surface of a working electrode coated with poly-pyrrole nanowire The electrodes are then kept in a dry environment at room temperature for one day Following this, they are rinsed to eliminate any unbound probe.
Detection of the DNA target using DAP and DNA sensor
This thesis explores the use of cyclic voltammetry to analyze the hybridization process of DNA We will conduct a CV scan for each step, including the synthesis of pyrrole nanowire DNA probes, their immobilization, and the detection of DNA targets, utilizing both the EC301 and DAP instruments The observed changes in amplitude and the voltage at which current peaks occur will provide insights into the hybridization dynamics.
Figure 3 - CV Voltammogram after each steps at Target C = 1010 -6 mol/l by EC301
Figure 3 - CV Voltammogram after each steps at C = 1011 -6 mol/l by the DAP
A similarity between the two voltammogram summary could be seen, which is a reduction in current amplitude after each steps, as indicated on Figure 3.10 and 3.11
There are two approaches we can stand on to explain the reduction of peak current after each steps
The electrochemical approach involves the creation of a charge barrier when a chemical layer is added to the electrode surface, as discussed in the previous section on double layer capacitance Polypyrrole nanowires (PPy-NWs) are a type of semiconducting polymer that play a crucial role in this process.
The binding of DNA probes to PPy-NWs increases the capacitance and resistance at the electrode surface, leading to a decrease in peak current This phenomenon also occurs when complementary DNA targets interact with the electrode, inhibiting the charge transfer process between the bulk solution and the metal surface.
59 binds to DNA probe, the density of chemical materials increases, resulting in the inhibition of electrons transfer Thus, after each immobilization steps, the peak current decreases
From a physical perspective, PPy-NWs act as p-type semiconductors with holes as their primary charge carriers Initially, when these nanowires are deposited on the working electrode's surface, the movement of electrical charge carriers is restricted Conversely, DNA aptamers predominantly utilize electrons as their charge carriers; thus, when a DNA probe attaches to the PPy-NWs, the concentration of holes, the fundamental charge carriers in the system, diminishes.
When the DNA target attaches to the DNA probe, electrons are increasingly transferred to the interface between PPy-NWs and the bulk solution, significantly reducing hole concentration This reduction in hole concentration subsequently decreases the conductivity of the materials, resulting in a diminished peak current observed after each immobilization step.
A series of tests were conducted on various concentrations, specifically focusing on the values calculated in equation 3.1 These results will be analyzed to observe the changes after each step of the process.
Figure 3.12 shows the dependence of target concentration to the peak current for both device:
Figure 3 - 12 Relation between ∆Ip to concentrations of DNA target when measured with EC301 and PSoC circuit
The analysis reveals a consistent trend between the two regression lines, indicating that as the concentration of DNA decreases, the peak current also diminishes This decline is attributed to a reduction in the binding interactions between the DNA probe and the DNA target Consequently, fewer DNA bindings lead to decreased inhibition of the charge transfer process, resulting in a smaller difference between the probe peak current and the target peak current.
The detection limit of the DAP is lower than that of the EC301, suggesting that the noise level of the EC301 is lower, as the detection limit is typically three times the noise magnitude Additionally, the steeper slope observed in the EC301 indicates that its sensitivity is greater than that of the DAP.
Detection of heavy metal ion for food safety application
Anodic Stripping Voltammetry (ASV) is an effective electrochemical method for the quantitative determination of ions in solution, offering advantages such as simplicity and high sensitivity Utilizing the DAP Linear Sweeping Mode, this technique involves accumulating the analyte onto the electrode surface during a deposition step, followed by oxidation in the stripping step The resulting current peaks during stripping indicate the presence of analytes The scanning voltage can be applied in various forms, including linear, staircase, and square wave.
This technique includes three main steps:
- Accumulation/Deposition Step: Metal ions in the solution will be deposited on the electrode surface, reduced to the metal atoms
- Stripping Step: At this stage, metal on the electrode surface will be oxidized into ions Electrons exchanged with electrode surface will raise the current
- Cleaning Step: The voltage will keep up at a high value to make sure that all the metal atoms are oxidized back to ions and go back to solution
This thesis employs anodic stripping voltammetry to detect arsenic ions (As 3+), utilizing a linear voltage sweep A stock solution of As 3+ at 1 ppm was prepared and subsequently diluted to various concentrations of 10 ppb, 30 ppb, 50 ppb, 70 ppb, and 100 ppb, in accordance with the WHO standard of 10 ppb for arsenic in water The concentrations of these solutions were confirmed using Atomic Absorption Spectroscopy (AAS) with the Agilent 200 Series AA at Vietnam National University The measurements were conducted using a three-electrode system, and the electrode was initially cleaned with K2CrO7 to ensure accurate results.
62 contamination on the surface, then it was activated in HCl 2M in 2 minutes After that, the electrode went through three steps in the process
The measurements were conducted using both the EC301 Potentiostat device and the DAP, with optimized conditions to achieve the most stable signal and highest peak value A 3+ solution was mixed with HCl acid to adjust the pH to 2.5, and the pH level was confirmed using a Metrohm pH Meter 691.
The applied voltage form for the measurement was shown in Figure 3.13:
Figure 3 - Voltage form for ASV measurement for Arsenic Detection 13
The experiment involved a fixed voltage of -0.45V maintained for 110 seconds, followed by a stripping step where the voltage was increased to 0.6V at a scan rate of 1V/s Finally, during the cleaning step, the voltage was held steady at 0.6V for an additional 60 seconds.
Figure 3 - ASV Measurement at As3+ 14
Figure 3 - ASV Measurement at As3+ 15
The measurement with As 3+ at concentration 50 ppb was performed with two similar electrodes on two devices The results were shown on Figure 3.14 and 3.15
The voltammograms presented in Figures 3.14 and 3.15, measured by the DAP and EC301 respectively, reveal a current peak during the stripping steps as the voltage transitions from -0.45 to +0.6V The analysis of these voltammograms, plotted against time and current axes, highlights a discrepancy in the timing of peak occurrence, indicating variations in scan rates between the two devices This difference suggests potential inaccuracies in the firmware and timing algorithm of the DAP.
Figure 3 - ASV Voltammogram for 16 different As 3+ concentrations by the DAP
Figure 3 - ASV Voltammogram for 17 different As 3+ concentrations by EC301
The concentration of As 3+ was measured, with results illustrated in Figures 3.16 and 3.17 The peak current value at the working electrode surface is influenced by the number of electrons exchanged As the concentration of As 3+ rises, there is a corresponding increase in the accumulation of metal ions on the electrode surface This relationship indicates that the amount of heavy metal atoms deposited on the electrode is directly linked to the number of electrons exchanged, explaining the observed increase in peak current with higher As 3+ ion concentrations.
The regression lines of the two devices are shown below:
Figure 3 - Regression lines for both devices 18
The correlation coefficients for DAP and EC301 measurements are 0.9714 and 0.995, respectively, indicating a strong fit of the modeling line to the actual data, with EC301 demonstrating superior alignment compared to DAP While the intercepts and standard errors of both regression lines are similar, the significantly higher slope of the EC301 regression line suggests that EC301 is more sensitive than DAP.
The analysis of the regression lines in the two applications reveals two key issues: the lower sensitivity and elevated noise level of the DAP A critical factor influencing the sensitivity of a potentiostat is the I/E converter, as the cell current must pass through a sensing resistor Consequently, any noise generated by this resistor can adversely impact the measured voltage Furthermore, the sensitivity of the measurement is significantly affected by a single resistor value, while commercial potentiostats utilize a range of resistors corresponding to various current levels This contributes to the notably high noise levels observed in the DAP.
The 66 measurement may be affected by the use of a micro-controller reference voltage, as the DAC is referenced to system power, making it susceptible to noise With only an 8-bit resolution, the DAC undergoes multiple processing stages before reaching the cell Additionally, the quality of components like op-amps can introduce thermal noise, voltage drift, and DC offset voltage The ADC’s low sampling rate of 60 sps can lead to aliasing in the acquired signal Furthermore, unrecognized resistance components within the electrochemical solution contribute to a decrease in acquired current Implementing circuit measures to detect unknown impedance and utilizing feedback could help mitigate these unwanted drops, although these iR drops primarily occur at high frequencies, impacting EIS measurements.
To enhance the performance of the current device, several improvements can be made Adding resistors with varied values can boost the device's sensitivity, particularly in the I/E converter modules which are crucial for accurate current measurement A comprehensive analysis of the signal spectrum is essential for a better understanding of the impedance model and signal processing Adjusting component values in the analog circuit can reduce noise during the signal processing stage, leading to an improved signal-to-noise ratio and lower detection limits Additionally, upgrading the main processor from an 8-bit to a 32-bit micro-controller, such as the PSoC5 LP series from Cypress, will provide higher resolution in DAC and ADC functionalities Implementing Electro-Impedance Spectroscopy (EIS) is also viable, as its working principle aligns with that of a Potentiostat Furthermore, enabling wireless communication for data transmission will facilitate on-site measurements.
This chapter verified the functions of the Data Acquisition and Processing Device (DAP) by testing each module with an electrometer, including the power source, voltage generator, data acquisition system, and LabView Software The programmable voltage generator and data acquisition modules allowed for adjustable range and resolution to meet specific application needs Comprehensive testing with a resistor and redox solution confirmed that the device operates effectively as documented in the literature, making it suitable for real-world applications Additionally, this thesis explored its use in DNA sensors and heavy metal sensors for food safety analysis.
The DAP utilized cyclic voltammetry mode in its DNA sensor application to detect DNA targets at very low concentrations, achieving response times of under one minute for each measurement Simultaneous testing on the EC301 commercial potentiostat revealed that while both devices exhibited similar calibration trends, the EC301 demonstrated superior sensitivity in its regression line.
The DAP utilized linear sweeping mode for anodic stripping voltammetry in heavy metal detection for food safety analysis, with sample verification conducted via AAS spectrometer Testing on both the DAP and EC301 under identical parameters revealed that the DAP could detect heavy metal concentrations as low as 10 ppb in just two minutes While the calibration line exhibited a similar trend to that of the EC301, it demonstrated lower sensitivity, akin to findings observed in DNA sensor applications.
Some suggestions were made for the improvement of the device, which could be applied in the future
This thesis presents an overview of conventional DNA sensors and heavy metal detection methods for food safety analysis, focusing on electrochemical techniques A Data Acquisition and Processing Device was designed to facilitate analytical electrochemical measurements, operating at 5V DC and capable of generating a voltage range from -1.3V to +1.3V with a minimum resolution of 5mV The device samples data at a rate of 60 samples per second with a 13-bit resolution, achieving an obtained current range of ±170µA Communication with a computer is established via USB, allowing data to be displayed on Labview-based software and stored in various formats The system demonstrated effective electrochemical modes for DNA and heavy metal detection, with a minimum detectable target DNA concentration of 10^-12 and a heavy metal ion concentration of 10 ppb for arsenic (As 3+) Although the device's sensitivity and signal-to-noise ratio were lower than those of the commercial potentiostat EC301, the results indicate its potential for on-site measurements, offering a viable alternative to traditional laboratory electrochemical analysis.
To enhance circuit functionality, it is recommended to incorporate a series of resistors in the I/E converter to boost current sensitivity Upgrading the resolution of the DAC and ADC modules is also advisable Additionally, improving the components and the signal processing stage can significantly enhance the signal-to-noise ratio and reduce the noise level of the measured signal Furthermore, the development of the EIS function using this circuit is under consideration.
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