Introduction
The development of several electrochemical sensing technologies has been driven by the goal of enhancing and decentralising platforms to enable extremely accurate and individualized measurements. This advancement has been made possible by electrochemistry's unmatched capacity to do incredibly precise molecular measurements in intricate buffers without the use of pricey equipment. The generalizability of such platforms had been hindered for decades by the field's dependence on systems that relied on the analyte's chemical reactivity. Thus, the creation of DNA-based electrochemical sensors was prompted by the search for alternative and more universal methods (Pellitero et al. 2019).
Electrochemical DNA sensors are capable of detecting complementary DNA, RNA, or peptide sequences, creating differences in the potential of the electrochemical device. These types of sensors seem to be extremely helpful as they are more time-and cost-effective and have greater selectivity and sensitivity compared to the existing tools for detecting molecules (Rashid & Yusof et al. 2017)
The team of iGEM Thessaloniki 2019 has done the first steps to build such a device using a sensor board with a gold finish, which is able to distinguish between the active and inactive states of their molecular system. We were based on their hardware but went further by adding a temperature control system and insulation to improve the signal noise to ratio of measurements.
Components
We want to thank iGEM Thessaloniki 2019 for providing us with a lot of the components that have been used and for their instructions.
Gold Sensor Pad
We used the PCB sensor boards of the team of iGEM Thessaloniki 2019, which were with gold finish. As they have proven, each sensing board consists of 10 rows of gold microelectrodes. Microelectrodes are electrodes with tip areas of the order of micrometers. The construction of DNA sensors using gold-sputtered microelectrodes that consist of a gold reference and a counter electrode (Thiruvottriyur Shanmugam et al.2020).
Figure 1. PCB sensor board
Arduino
Arduino, as a micro-computer, is a board with multiple properties, as it can carry out any commands. We connect all the other components to this board, as it is responsible for data collection and processing. This way we manage to visualise the experimental data on a screen so that we have proper recording and evaluation. The Arduino DUE, that we used, is programmed using the Arduino Software (IDE), the Integrated Development Environment common to all our boards and running both online and offline (Due, n.d.). The board we use is one of the most powerful in computing power and is aim to increase the quality of our measurements
Figure 2. Arduino DUE
MOSFET
Transistors are semiconductor devices that behave either as signal amplifiers or as electrically controlled switches. One kind of transistor is the Metal Oxide Semiconductor Field Effect Transistor (MOSFET). On these transistors, gate voltage creates an electric field that alters the flow of charge carriers (Teja. 2022, February 21).
Temperature Control System
Temperature Sensor
For the temperature measurements, we use a sensor, a thermistor. It is a ceramic semiconductor that reacts to temperature changes by changing its resistance. Thermistors can be classified as either a Positive Temperature Coefficient (PTC) or a Negative Temperature Coefficient (NTC). When the temperature rises, the resistance of an NTC thermistor falls. On the other hand, resistance rises as the temperature falls (Wavelength Electronics. 2020, February 11).
Figure 3. Thermistor
Peltier
Peltier modules are used to maintain a certain temperature by carefully controlling the heating or cooling of a device. Two exterior ceramic plates and semiconductor pellets are used in Peltier modules. When a current is delivered through the semiconductor pellets, one of the plates absorbs heat (becomes cooler), while the other plate dissipates heat (becomes hotter) (CUI Devices. 2018, May 1).
Figure 4. Peltier
Circuit
Figure 5 shows the circuit we employed in its entirety and all its elements
Figure 5. Circuit of our hardware
Experiments
1.Experiment of immobilsation and hybridization of our structure
We immobilised DNA probes with a specific thiol acid tail, on the surface of the gold microelectrodes. When complementary DNA probes are detected, a difference in electric potential can be read. We followed the same protocols that iGEM Thessaloniki 2019 did; more detailed information is on our protocol page. Especially we used the complementary of the initiator immobilized οn the gold surface and as we add the initiator sequence, which has a reporter on it, and they bind to each other, we got a voltage difference.
Figure 6. Results from experiments of immobilisation and hybridization
Regarding the figure above, the point where it decreases abruptly we add the molecules and the reaction of hybridization happens. We wanted to do, also, the experiments with the Y-structure molecules and the microRNAs, where if the initiator was released, it will bind on the probe DNA on the gold surface, but we had the time limitation.
2. Test of the drift
As we did some experiments, we figured out the problem of drift, which is a continuous change in the relative signal of the sensors independent of target concentration.The differential MOSFET amplifier and additional coating of thiol reduced the drifting by one order of magnitude (Pellitero et al. 2019). On one row of the sensing board, there are insulated microelectrodes, where the reference electrode was insulated with PCB. In this experiment, we compare the signal results between the insulated and the non-insulated.
Figure 7. Test of the drift on the board
We did the same experiments, where on the sensor board were only the probes DNA immobilized and not the complementary sequence to bind,on the insulated microelectrodes and on the non insulated and we received the results of Figure 6. As it is shown, the insulated microelectrodes (grey line) characterized by its stability, as the non insulated (blue) increase in the period of time, although it wasn’t anything added.
3. Temperature Control
Calibration
One of the key procedures used to keep instruments accurate is instrument calibration. The calibration process involves setting up the instrument such that it can produce results for samples that are within a reasonable range. A key component of instrumentation design is eliminating or reducing conditions that lead to faulty readings. For this reason, we did the calibration method on the thermistor by determining different temperatures of double distilled water with this and concurrently with a thermometer. So, we corporated these values and made the graph below.
Figure 8.Calibration of the thermistor
We added the equation of the calibration to the code of Arduino and we used this temperature control system to make our immobilization and hybridization results more accurate. So we did the experiments in three different temperatures and as it is shown on figure 8 it is controlled.
Figure 9. Our experiments in three different controlled temperatures
References
CUI Devices. (2018, May 1) How to select a Peltier module. Retrieved October 12, 2022, from https://www.cuidevices.com/blog/how-to-select-a-peltier-module
Due. (n.d.). Arduino Documentation. Retrieved October 12, 2022, from https://docs.arduino.cc/hardware/due
Pellitero, M. A., Shaver, A., & Arroyo-Currás, N. (2019). Critical review—approaches for the electrochemical interrogation of DNA-based sensors: A critical review. Journal of The Electrochemical Society, 167(3), 037529. https://doi.org/10.1149/2.0292003jes
Rashid, J. I. A., & Yusof, N. A. (2017). The strategies of DNA immobilization and hybridization detection mechanism in the construction of electrochemical DNA sensors: A review. Sensing and bio-sensing research, 16, 19-31.
Teja, R. (2022, February 21). Introduction to MOSFET: Depletion and Enhancement Mode, applications. Electronics Hub. Retrieved October 12, 2022, from https://www.electronicshub.org/mosfet/
Thiruvottriyur Shanmugam, S., Trashin, S., & De Wael, K. (2020). Gold-sputtered microelectrodes with built-in gold reference and counter electrodes for electrochemical DNA detection. The Analyst, 145(23), 7646–7653. https://doi.org/10.1039/d0an01387k
