Team:Rochester/Measurement
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Measurement
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Introduction
We manufactured an aptasensor to detect the presence of sepsis-related soluble biomarkers. However, detecting the binding between biomarkers and their receptors - aptamers, in this case - is a challenging task, as the concentration of biomarkers in sweat is very low. To reliably detect the binding of biomarkers to aptamers, we conducted cyclic voltammetry using a low-cost potentiostat that we made. Cyclic voltammetry is a method used to study mechanism and kinetics of reactions on electrodes.1 This method allows us to monitor current changes at working electrode while maintaining the constant voltage difference. In our project, cyclic voltammetry was essential for characterizing modified sensing devices. It is conducted using a potentiostat, which was not accessible for our team and, therefore, we decided to manufacture a low-cost potentiostat.
Testing Sensitivity
To test if our potentiostat was sensitive enough and could reliably detect different concentrations of the analyte in the electrode, we prepared different concentrations of salt (NaCl) and ran cyclic voltammetry. We prepared the following concentrations of NaCl: 1M, 0.5M, 0.1M, 0.05M, 0.001M, 5uM, 1uM, 100nM, 50nM, 5nM, and 1nM. Then, we connected the screen-printed electrode to the potentiostat circuit. The circuit was connected to JUAMI, which was a software that showed the current changes at the fixed potential difference. We set the voltage scan rate to be 0.1 V/s, from -2 V to 2 V for 10 cycles - 200 seconds. The electrode was immersed into the solution and we started cyclic voltammetry. We plotted the results (Figure 1).
Figure 1: Testing sensitivity of our potentiostat using NaCl.
Figure 1 shows that, at the concentrations of NaCl at 100nM, 5nM, and 1nM, the peak current shifted. The peak current was -2 mA in 100nM, 5mA in 5nM, and around 1mA in 1nM of NaCl. We found the maximum current and inferred the potential at which this current was observed. Using Ohm’s Law, we calculated the resistance from the potentiostat output. Then, we plotted the concentration of the NaCl against the resistance of the electrode. Table 1 shows the resistance values of the electrode at the range of concentrations of NaCl, but we noticed that the sensitivity was much better at lower concentrations - in nM scale (Figure 2). Figure 3 shows the resistance changes at lowest concentrations of NaCl.
| Concentration (M) | Resistance (Ohm) |
|---|---|
| 1 | 4.83E+01 |
| 0.5 | 1.17E+02 |
| 0.1 | 1.45E+02 |
| 0.05 | 1.16E+02 |
| 0.001 | 1.39E+02 |
| 5E-06 | 1.30E+02 |
| 1E-06 | 2.31E+02 |
| 100E-09 | 1.10E+02 |
| 50E-09 | 2.10E+02 |
| 5E-09 | 2.11E+02 |
| 1E-09 | 5.76E+02 |
Figure 2: Resistance values of electrode as the concentration of NaCl increases.
Figure 3: Resistance of electrode at the lowest concentration of NaCl.
Figures 2 and 3 show changes in resistance as the concentrations of NaCl change. Figure 2 shows that as the concentration of NaCl increases the resistance decreases, indicating that changes in concentration can be measured with our potentiostat. Additionally, Figure 3 shows the biggest change in resistance occurs between 1nM and 100nM, leading us to conclude that our potentiostat was most sensitive at lower concentrations of NaCl.
Running Cyclic Voltammetry Using a Potentiostat
To show that our aptasensor could detect the presence of a biomarker in sweat, we prepared concentrations of CPR and lactoferrin biomarkers from 1000nM and 1.9nM. First, we modified the working electrodes with either the solution of rGO and 10 nM of aptamer or with rGO, which was a control. We used cyclic voltammetry to detect electrical changes once the biomarker binds to the aptamer. We connected our potentiostat to JUAMI software, and we set the scan rate as 100 mV/s with voltage oscillating between -2 to 2 V. We ran 10 cycles, which was 200 seconds, for each experiment.
Figure 4 shows the potentiostat output at varying lactoferrin concentrations, where the change in current was monitored at desired voltage difference. We observed the shift of the peak current between the control (rGO) and experimental condition (rGO and aptamer) at all concentrations of biomarker, and, as example, we showed the results of cyclic voltammetry for lactoferrin concentrations of 1.9 nM and 31.25 nM. In Figure 4, when the concentration of the biomarker was 31.5nM, we observed that the peak current when the electrode was not modified with aptamer (control) was about 11mA. In contrast, the peak current on electrodes covered with both rGO and lactoferrin aptamer was 4 mA. Similarly, at the lactoferrin concentration of 1.9nM, the current peak was 6 mA in control and -1 mA in the aptamer-modified electrode.
Figure 4: Using cyclic voltammetry to detect binding between lactoferrin aptamer and biomarker.
Next, we found the maximum current and inferred the potential at which this current was observed. Using Ohm’s Law, we calculated the resistance of rGO sheet (Table 2). Then, we plotted the concentration of biomarker against the resistance of rGO sheet (Figure 5).
| Resistance (Ohm) | ||
|---|---|---|
| Concentration (nM) | rGO | rGO and aptamer |
| 1.9 | 2.25E+02 | 1.12E+02 |
| 7.8 | 2.24E+02 | 1.87E+02 |
| 15.63 | 2.30E+02 | 1.42E+02 |
| 31.5 | 2.16E+02 | 2.35E+02 |
| 62.5 | 2.16E+02 | 2.14E+02 |
| 125 | 1.53E+02 | 2.28E+03 |
| 250 | 1.90E+02 | 2.89E+02 |
| 500 | 2.22E+02 | 2.89E+02 |
| 1000 | 2.16E+02 | 2.16E+02 |
Table 2: Resistance values of rGO sheet at varying concentrations of lactoferrin biomarker.
Figure 5: Resistance of rGO sheet vs. the concentration of lactoferrin biomarker.
When lactoferrin aptamer was attached to rGO, we observed the increase in resistance with increasing biomarker concentration until about 400 nM (Figure 5). The data increased monotonically in a range from 50 nM until around 400 nM. Figure 5 also showed that the lowest concentrations of biomarker (1.9 nM - 50 nM) gave resistance values that fell under the curve for the negative control, indicating that the signal was not significant compared to background for the low biomarker range. At biomarker concentrations higher than 400 nM, the resistance decreases again which could be explained by aptamers being saturated with biomarkers. The control showed the flat line, as the resistance values were around the same value for all the biomarker concentrations. This showed that there was not any non-specific binding and that binding occurred only in the presence of an aptamer attached to the electrode.
Similarly, we ran the same experiments for CRP biomarker and aptamer. We prepared concentrations of CRP biomarker from 1000nM and 1.9nM. As before, we tested adding a biomarker solution to the electrode modified with either the solution of rGO and 10 nM of aptamer or with rGO, which was a control. In Figure 6, when the concentration of CRP was 62.5 nM, we observed that the peak current when the electrode was not modified with aptamer (control) was about 5 mA. In contrast, the peak current on electrodes covered with both rGO and CRP aptamer was 1.5 mA. Similarly, at the CRP concentration of 7.8 nM, the current peak was 10 mA in control and 1 mA in the aptamer-modified electrode.
Figure 6: Using cyclic voltammetry to detect binding between CRP aptamer and biomarker.
Then, we found the maximum current and inferred the potential at which this current was observed. Using Ohm’s Law, we calculated the resistance of the rGO sheet (Table 3). We plotted the concentration of the biomarker against the resistance of the rGO sheet (Figure 7).
| Resistance (Ohm) | ||
|---|---|---|
| Concentration (nM) | rGO | rGO + aptamer |
| 1.9 | 1.97E+02 | 2.67E+02 |
| 3.9 | 1.94E+02 | 1.65E+02 |
| 7.8 | 2.20E+02 | 2.54E+02 |
| 15.63 | 2.43E+02 | 2.54E+02 |
| 31.5 | 2.25E+02 | 1.91E+02 |
| 62.5 | 2.34E+02 | 2.99E+02 |
| 125 | 2.26E+02 | 2.35E+02 |
| 250 | 2.18E+02 | 2.07E+02 |
| 500 | 2.59E+02 | 2.32E+02 |
| 1000 | 2.16E+02 | 2.13E+02 |
Table 2: Resistance values of rGO sheet at varying concentrations of CRP biomarker.
Figure 4: Concentration of CRP against the resistance of rGO sheet.
Results did not show a clear difference between electrodes treated with rGO and rGO and CRP aptamer. The change in resistance at varying concentrations of biomarker was a bit smoother in the control, but the difference in resistance change was not as clear as with lactoferrin. One reason for observing these results might be due to the imprecise attachment of rGO and aptamer to the working electrode. The limitation of attachment of aptamer to the rGO could be optimized by using spacers and a covalently linking method, such as azide linking to PEI-modified rGO. We didn’t want to do this in order to keep our device as accessible as possible. Additionally, the binding affinity of CRP is 6.2 pM, and we prepared the concentrations of biomarkers in the nM range. Using such a high concentration of biomarker compared to its binding affinity might have led to the saturation of electrodes with the biomarker. Therefore, the potentiostat could not have detected any change in binding between aptamer and biomarker as all the biomarker concentrations were too high.
Control
We used two types of controls: no aptamer attached to the rGO and no biomarker in sweat. In previous experiments, we deposited either rGO and aptamer or just rGO on the top of the working electrode. Attaching just rGO to the electrode would show whether there is any non-specific binding of biomarker to the electrode. Then, we prepared the second control, where we added only sweat (without biomarkers) to the electrode that was either modified with rGO and aptamer or modified with just rGO. If different current peaks were detected on the electrode modified with aptamer, as compared to electrodes without aptamer, then our aptasensor was not actually detecting the binding of biomarkers in the previous experiment. The change in peak current detected earlier could have just been due to the aptamer modification interacting with the buffer in which the biomarker was dissolved in. We prepared sweat solutions at two different pH values, added them to the electrode and ran cyclic voltammetry, using the same parameters for voltage scan rate, voltage difference and number of cycles as before. Figure 8 shows that the current peak did not shift when sweat was added to the electrode without an aptamer, and electrode with aptamer.
Figure 5: Cyclic voltammetry showed the same current peak in both electrode modified with aptamer and rGO and electrode modified with rGO when they were treated with sweat.
Resistance values for each condition were found using the peak current and corresponding voltage (Table 4). Figure 9 showed that the current peak did not shift when sweat was added to the electrode with rGO, and electrode with rGO and aptamer. This shows that the change in peak current detected in aptamer-biomarker binding was not due to the aptamer modification interacting with the buffer in which the biomarker was dissolved in, but rather that the current shift only occurred when biomarker was present in sweat.
Resistance values for each condition were found using the peak current and corresponding voltage (Table 3).
| pH 6 | pH 4.7 | |||
|---|---|---|---|---|
| rGO | rGO +Aptamer | rGO | rGO+ aptamer | |
| Resistance (Ohm) | 2.38E+02 | 2.40E+02 | 2.26E+02 | 2.07E+02 |
Figure 6: Resistance values of rGO using sweat without biomarker.
Ease of Use
To show that our potentiostat is user-friendly, we asked students majoring in software engineering, psychology, math and data science to use our potentiostat to conduct cyclic voltammetry. We prepared the solution of 5mM potassium ferrocyanide and 0.1 M potassium chloride in 5 ml of water, according to the protocol we got from our gold partner. Then, we explained to students what potentiostat does and how we built it. Then, as we ran a demonstration of the experiment, we explained what cyclic voltammetry is and why it was important for our project. The students asked about what the potentiostat output was, how we analyzed the data and where the potentiostat was generally used.
Undergraduate students at University of Rochester test our potentiostat.
We asked students to run cyclic voltammetry of the solution we prepared. Figure 7 shows the results of three students running cyclic voltammetry. In the graph A, the student accidentally dropped the electrode and we did not observe the smooth decrease in current as we did in other two graphs.
Figure 6: Cyclic voltammetry showed the same current peak in both electrode modified with aptamer and rGO and electrode modified with rGO when they were treated with sweat.
Conclusion
We conducted cyclic voltammetry, a technique relevant for characterizing sensing devices, using an accessible potentiostat manufactured by our team. The design of the potentiostat did not require expensive materials and it was easy to manufacture. When working with developing sensing devices to monitor concentrations of any analyte, cyclic voltammetry is essential to understand whether the device is able to detect concentration changes of the target analyte. Our team developed a simple, accessible method to manufacture the potentiostat and we conducted cyclic voltammetry to characterize a sensing device.
- Metrohm. Electrochemistry – a workbook for 910 PSTAT Mini. https://www.metrohm.com/en/documents/81085020 (accessed Oct 21, 2021).