Hardware

The cv test introduction

 CV testing is an advanced potentiometric and voltammetric technique. During a scan, the target chemical undergoes either oxidation (loss of an electron) or reduction (gain of an electron), depending on the direction of the ramping potential^[1].
 In the cv process, key parameters must be configured, including the number of cycles, the number of points per cycle, the initial and termination voltages, the scan rate, the current threshold, and N-turn execution. The number of cycles refers to how many times the potential is cycled, while the points per cycle indicate the frequency of data recording^[2]. The initial and termination voltages define the voltage sweep range, and the scan rate specifies the rate of voltage change. The current threshold sets a safety limit to prevent exceeding the battery's safe current range. N-turn execution allows for a predetermined number of cv tests to be performed. Once these parameters are established, the test can be initiated, ultimately producing a cyclic voltammetric curve.

This is the sample curve of a cv test:

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How can CD97 and CD55 bindings be detected by the cv test system

During the interaction between CD97 and CD55, our docking results indicate that the involvement of the side chain of aspartic acid (Asp) is crucial for the binding process. Thus, broadly speaking, CD97 can be regarded as being reduced by CD55 throughout their interaction. This finding not only underscores the pivotal role of Asp in the binding mechanism but also provides novel insights into the nature of the interaction between CD97 and CD55.

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Three electrodes system of test pieces

We selected the three-electrode method, which is the most widely used approach in electrochemical analysis, for our detection process^[3]. As shown in Figure 2, the test piece features three pins corresponding to three types of electrodes: the working electrode (WE), reference electrode (RE), and counter electrode (CE). The WE acts as either an electron donor or acceptor under appropriate voltage conditions within an electrolyte environment. The CE serves as the counterpart to the WE, undergoing a reduction reaction when the WE is oxidized, and vice versa. While the CE does not participate directly in the electrochemical reaction, it provides charge balancing. In electroanalytical chemical systems, the RE must be positioned as close as possible to the WE at a fixed distance to ensure a stable reference potential.

Next: Construction of hardware

The circuit design & Device figure

Below are the circuit diagram and an image of our prototype machine.

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The usage method in testing of device

 To ensure both user-friendliness and reproducibility, we established a standard protocol for the device. For rapid testing, we coat the working electrode (WE) on the test pieces with BELO-engineered bacteria. Users can then simply apply their samples to the test pieces, which are pre-coated with BELOs, and proceed with the testing.
 Since BELOs cannot directly adhere to the carbon surface of the electrodes, we needed to find a suitable method for coating them. To address this challenge, we sought guidance from Prof. Chao-Min Cheng (for more details, see the Integrated human practice page). Through discussions with him, we learned that a slight modification to the electrode is required. We selected phosphate-buffered saline (PBS) to create a positively charged layer on the electrode. By applying a consistent voltage environment, positive ions are attracted to the electrode surface, forming the desired charge layer. Following this pre-oxidation step, BELOs can be effectively coated onto the surface of the positively charged, modified electrode.

Next: Result

Constant setting

Here’s the constant we set while testing the device:

• Positive layer: 20 mM PB
• Scanning rate: 20 mV/sec
• Scanning range: ±1 V
• Max scanning cycle: 1
• Electrode material: Carbon (WE/CE), AgCl (RE)
• Preserve environment: humidity 20% in room temperature

Testing results

A full view of the diagram does not clearly show differences between the results. Therefore, we zoomed into specific sections of the diagram to better distinguish any variations.

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Summary

 The experimental results thus far indicate that our hardware is capable of detecting differences in the system; however, the observed variations are currently subtle. We are confident that with additional experimental iterations, systematic adjustments to testing parameters, and potential modifications to the electrode materials, we can significantly enhance the precision and reliability of our hardware. These refinements will be instrumental in achieving a more robust and comprehensive cyclic voltammetry (CV) testing platform, ultimately fulfilling our objective of establishing a rapid and efficient disease detection system.

Future outlook

 From the preliminary results, it is evident that this prototype device has several areas for improvement, including enhancing the electrode materials, improving signal stability, refining the user interface on computers, and developing wireless capabilities. In the coming weeks leading up to the Jamboree, we will prioritize testing whether different materials affect the signals detected. Additionally, we will continue to improve other features to create a practical and user-friendly electrochemical analyzer for researchers.

Next: Open sources of hardware

The open sources usage

Here is our protocol and code for using the hardware. We hope it will serve as a resource for future iGEMers. Anyone interested is welcome to download it from here.

Protocol for doing testing with device

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The open source code for device usage

C code for run the hardware

#include <LiquidCrystal.h>
int pwmPin = 10;             // PWM pin for voltage control
int adcPin = A0;             // ADC pin to read current
float Potstep = 0.0078;      // Potential step, fixed due to DAC resolution
float current = 0.0;         // Variable to store measured current
float voltage = 0.0;         // Variable to store applied voltage
int vevals[] = {100, 20, 50, 100, 200, 250, 300}; // Scan rates in mV/s
int const count = 6;         // Number of different scan rates
long intervalos[count];      // Time intervals for each scan rate
int val = 0;
int n = 0;
int maxScans = 1; // Number of forward-reverse scans
int scanCount = 0; // Counter for scans
void setup() {
  TCCR1B = TCCR1B & B11111000 | B00000001; // Set dividers to change PWM frequency
  Serial.begin(9600);
  pinMode(pwmPin, OUTPUT);
  pinMode(adcPin, INPUT);
  for (int pos = 0; pos < count; pos++) {
    intervalos[pos] = (1000000L / ((vevals[pos]) * 128L));
  }
  // Output headers for CSV format
  // Serial.println("Voltage(V),Current(µA)");
}
void loop() {
  for (int pos = 0; pos <= count; pos++) {
    n = 0;
    while (n <= 1 && scanCount < maxScans) {
      // Forward scan
      for (val = 0; val <= 255; val++) {
        analogWrite(pwmPin, val);
        delay(intervalos[pos]);
        voltage = map(val, 0, 255, -1.0 * 1000, 1.0 * 1000) / 1000.0;  // Calculate the applied voltage
        current = readCurrent(adcPin);  // Function to convert ADC reading to current
        // Output the data in CSV format to Serial Monitor
        Serial.print(voltage, 6); // Voltage with 6 decimal places
        Serial.print(",");
        Serial.println(current, 6); // Current with 6 decimal places
        delay(100); // Short delay between readings
      }
      // Reverse scan
      for (val = 255; val >= 0; val--) {
        analogWrite(pwmPin, val);
        delay(intervalos[pos]);
        voltage = map(val, 0, 255, -1.0 * 1000, 1.0 * 1000) / 1000.0;  // Calculate the applied voltage
        current = readCurrent(adcPin);  // Function to convert ADC reading to current
        // Output the data in CSV format to Serial Monitor
        Serial.print(voltage, 6); // Voltage with 6 decimal places
        Serial.print(",");
        Serial.println(current, 6); // Current with 6 decimal places
        delay(100); // Short delay between readings
      }
      n++;
      scanCount++;  // Increment scan counter
    }
    
    // Exit loop if maxScans is reached
    if (scanCount >= maxScans) {
      break;
    }
  }
  // Stop execution after reaching max scans
  if (scanCount >= maxScans) {
    while (true);  // Infinite loop to halt execution
  }
}
// Function to read current based on ADC reading (modify based on actual circuit)
float readCurrent(int pin) {
  int adcValue = analogRead(pin);
  float voltage = adcValue * (5.0 / 1023.0);
  return (voltage - 2.5) / 0.12; // Assuming a transimpedance amplifier with a gain of 1/0.12 µA/V
}

Python code to load data from the arduino code

import serial
import time
import csv
import matplotlib.pyplot as plt
# Setup serial connection (adjust port and baudrate as needed)
try:
    ser = serial.Serial('COM8', 9600, timeout=1)
    time.sleep(2)  # Wait for the connection to be established
except serial.SerialException as e:
    print(f"Error opening serial port: {e}")
    exit()
# Open CSV file for writing
with open('cv_data.csv', mode='w', newline='', encoding='utf-8') as file:
    writer = csv.writer(file)
    writer.writerow(["Voltage (V)", "Current (µA)"])
    print("Reading data...")
    # Read data until stopped manually or until the Arduino stops sending
    try:
        while True:
            line = ser.readline().decode('utf-8').strip()
            if line:
                print(line)
                writer.writerow(line.split(","))
            else:
                # Timeout reached or no data available
                break
    except KeyboardInterrupt:
        print("Stopped by user")
    finally:
        # Ensure the serial connection is closed on exit
        ser.close()
        print("Serial connection closed.")
# Load the data and plot the CV curve
voltages = []
currents = []
with open('cv_data.csv', mode='r', encoding='utf-8') as file:
    reader = csv.DictReader(file)
    for row in reader:
        try:
            voltages.append(float(row["Voltage (V)"]))
            currents.append(float(row["Current (µA)"]))
        except ValueError:
            # Handle any potential conversion errors
            print("Skipping row with invalid data:", row)
# Plotting as a scatter plot
plt.figure(figsize=(10, 6))
plt.scatter(voltages, currents, label='CV Data', marker='o')
plt.xlabel('Voltage (V)')
plt.ylabel('Current (µA)')
plt.title('Cyclic Voltammetry (CV) Data')
plt.grid(True)
plt.legend()
plt.show()