Introduction
In project SPYKE, we created a completely novel measurement technique that is quick, cheap, easy to use, and modular. The capacitive Arduino circuit that we built for under 50 euros can easily be adapted to any set of allosteric transcription factors and their DNA binding sequence. With this method, the response of allosteric transcription factors to an effector molecule can be recorded through modulation of their (un)binding to DNA. The measurement relies on capacitance changes, down to the scale of picofarad sensitivity. In our project, we used this technique to measure a capacitance change within seconds after the addition of SSA to a biosensor in which the transcription factor BlcR was bound to immobilized DNA. Figure 1 shows the advantages of our measurement approach.
Figure 1. Advantages of the capacitive Arduino circuit as a biosensing measurement technique.
The measurement and its dedicated read-out circuit were validated using models and experiments. The protocols, the assembly manual, future prospects, and prospective applications are described below.
Overview of the method
Capacitance change
We used a capacitance measurement as the final detection method for GHB. DNA-immobilized electrodes were incubated with the allosteric transcription factor BlcR which binds to the DNA and is responsive to GHB and the GHB-analog SSA. When GHB is added to the solution, BlcR unbinds from the DNA, resulting in a capacitance change measured by the dedicated read-out circuit. This change in capacitance is caused by the displacement of BlcR molecules by the water molecules. Figure 2 shows the mechanism used to detect GHB. By analyzing this change in capacitance, the allosteric transcription factor and the (un)binding to DNA can be characterized. More information about the biosensor design can be found here.
Figure 2. Simplified overview of the mechanism used to detect GHB.
Read-out circuit
To measure the capacitance changes resulting from the addition of GHB we developed a simple dedicated read-out circuit built around the Arduino Nano board. The system continuously (thus passively) measures the electrode's capacitance and outputs the data to a computer. The read-out circuit is shown in Figure 3. For more information on the design and the workings of the circuit visit the hardware page.
Figure 3. The read-out circuit used to measure the capacitance changes.
However, capacitance is not only related to the binding and unbinding of BlcR. Capacitance is also inherently sensitive to environmental influences such as temperature [1] , pH differences [2] , or even charges present in your body [3] . Minimizing these influences on the capacitance would result in a system in which the targeted signal can be analyzed more accurately. Two additional features were included in the measurement setup to minimize the environmental influences:
- Two capacitor system: An extra electrode was included in the measurement setup and we measured the signal difference between the two electrodes. The second electrode is identical to the ‘working electrode’ except that the immobilized DNA doesn’t contain the BlcR binding site. The capacitance of the electrode is therefore not responsive to GHB since there is no binding and unbinding of BlcR. Both of the electrodes respond similarly to external influences except for the addition of GHB. Measuring the difference between the two electrodes minimizes the effects of environmental perturbations, while still measuring GHB [4] . See Figures 4 & 5.
Figure 4. Schematic overview of the factors that influence the capacitance of the electrodes.
Figure 5. Schematic overview of the factors that influence the biosensor read-out in a two-capacitor configuration. The electrode on the left contains DNA with the BlcR binding sequence. The electrode on the right contains scrambled DNA. Only BlcR (un)binding influences the system significantly, which increases the signal-to-noise ratio.
- Offset reduction button: As the two electrodes are not identical, an inherent capacitance difference is measured resulting in an offset. We included a button in the read-out circuit which normalizes the offset. This can be used before the actual measurement.
Validation
To validate the effectiveness of the measurement setup in detecting GHB, multiple experiments and simulations were conducted.
Model
We used Python to create a model simulating the binding state of the oligonucleotides to BlcR. The model predicts the fraction of DNA bound to BlcR, which directly relates to the capacitance of the electrode. The model showed that upon addition of GHB the fraction of DNA bound to BlcR decreases from over 99% to less than 1%. The results are shown in Figure 6. For a full description of the model and results, please visit the model page.
Figure 6. Simulation results showing that the fraction of DNA bound to BlcR is strongly influenced by GHB concentrations.
LTspice simulations
We used the software LTspice to simulate the read-out circuit. We analyzed how capacitance changes influence the Arduino output. As can be seen in Figure 7, the read-out circuit responds strongly to capacitance changes. For a more elaborate description of the LTspice simulations and results, visit the hardware page.
Figure 7. The results of an LTspice simulation of the output voltage measured by the Arduino for different capacitor values of the electrode (purple line C = 0.5 pF, light blue line C = 0.6 pF, blue line C = 0.7 pF, black line C = 0.8 pF, and pink line C = 0.9 pF)
Experimental Validation of the read-out circuit
The measurement setup was validated using capacitors with known values. The output of the Arduino (difference between two capacitors) was then measured. As shown in Figure 8, the Arduino output shows the expected linear increase resulting from capacitance differences.
Figure 8. Validation experiments for the read-out circuit.
GHB measurements
As due to legal reasons we could only use the GHB analog SSA during the Arduino measurements at our university. We validated whether the addition of SSA would indeed result in a capacitance change. In addition, we visited another institute owing a license to work with GHB, and we repeated the same experiment this time with GHB. We used another system, a universal transducer interface (UTI), to measure a capacitance change after the addition of GHB. An increase in capacitance after GHB addition was successfully observed. See Figure 9.
Figure 9. Validation of GHB-specific capacitance changes during UTI measurements. (a&b) Electrode A with 1 µM immobilized DNA containing Blc operator. (c&d) Electrode B with 1 µM immobilized scrambled DNA. Capacitance has been measured after incubation with 4 µM BlcR: ‘BlcR’ and after the addition of GHB to a final concentration of 55 mM GHB: ‘GHB’ ( Notebook 4 , 04/10/2022)
Proof of concept
With our Arduino circuit, we were able to measure instant (within seconds) capacitance changes after the addition of SSA. The results can be found in Figure 10.
Figure 10. Capacitance measurements with Arduino circuits. Baseline electrode with immobilized 1 µM DNA; Electrode A: immobilized Blc operator sequence; Electrode B: immobilized scrambled DNA, and 4 µM BlcR. Electrode B showed no change in capacitance. Capacitance difference is measured in presence 150 µM SSA. The measurement was succesfully repeated ( Notebook 4 , 27/09/2022).
Protocols
The measurement consists of two steps:
- Immobilization: In this step, thiol-modified DNA is first immobilized to the gold surface of the electrode. Secondly, BlcR is incubated in a bath with the electrode (causing BlcR to bind to the immobilized DNA). The full protocol can be found here .
- Capacitance measurements: The capacitance is measured when BlcR is added (no SSA or GHB additon) and when SSA or GHB is added. This change in capacitance is measured with our Arduino system. The full protocol can be found here .
Recreation manual
The design of the read-out circuit can be recreated for under €50. We share the recreation manual. In addition, all the software necessary to operate the read-out device is provided. For the full building guide click here. For the software to operate the read-out circuit, visit our gitlab repository.
Future improvements and prospective applications
Quantitative measurements
Due to the inherent variability of the capacitance values between electrodes it was hard to measure differences caused by molecular events, such as binding affinities and protein sizes, during this project. However, if the quality of the electrodes would be improved, precise measurements with absolute capacitance changes could easily be done. At that point, differences in molecular characteristics could be identified.
One could for instance take the measured capacitance when GHB is added and divide that by the measured capacitance when no GHB is present. These values could give quantitative insights about how much BlcR unbinds when GHB is added. One could screen many electrodes with DNA mutants and/or BlcR mutants and measure which combination results in the largest increase in capacitance after GHB or SSA, thus which combination results in the highest responsiveness to the drug. One could also analyze the binding affinity of BlcR and/or DNA mutants by measuring the capacitance when no BlcR is added and comparing that value to the capacitance when BlcR has been incubated. The largest change in capacitance would indicate that on that electrode, the highest fraction of DNA is bound to BlcR.
Other allosteric transcription factors
The measurement method could easily be adapted to detecting the presence of other molecules than GHB. In the end, a different allosteric transcription factor and its corresponding DNA binding sequence can be engineered to respond to the presence of a specific molecule. The Arduino read-out circuit remains the same, only the immobilization and incubation steps would differ. Allosteric transcription factors could also be subjected to directed evolution to bind to new ligands, further expanding our biosensor applicabilities [5] .
Sensors
This measurement setup could be implemented in a variety of on-site sensors as described on our hardware page. As the measurement setup can be downsized significantly and the electrical output signal can be easily altered, various sensing applications are possible, see proposed implementation.