Since the E-CRISPR system involves the reaction of the nucleic acid chain on the interface between the solution and the screen-printed electrode plate, and the electrical signal conversion process generated by the reaction, it is very different from the state (appearance and characteristics) of the electrode plate itself. There are huge difficulties in theoretical modeling, so this part focuses on simple data processing.
Figure 1: Though modification of the G nucleic acid chain with methylene blue fluorescent group on the screen-printed electrode, and then using the electrochemical workstation to measure the volt-ampere signal change on the electrode plate after 8hr, 10hr, 24hr, and 36hr, it can be seen that there is no obvious peak in the current signal in 8 hours after 10 hours, indicating that the chain modification has not been fully completed at this time and the interface is still unstable. From 24 hours to 36 hours, the peak height increased significantly, indicating that the G chain is indeed constantly modifying onto the surface of the electrode plate, and the longer the time, the more and more stable the modified chain are on the electrode plate.
Cutting the G chain with the inactivated Cas9 nuclease at the HNH site will produce changes in the current signal. A batch of plates that have been modified for 24 hours was tested for voltammetric signals (Figure 2). It was found that although the electrical signals generated on different plates were different in size, the amplitude and trend of the signal change are the same. Besides, a continuous test of the voltammetric signal on the same electrode plate which has undergone a 36-hour modification of the G chain (Figure 3) found that the peak position was stable, the peak height was continuously reduced, and the signal was continuously attenuated, that is, the speed of the enzyme digestion reaction was continuously reduced.
G chain modification occurs on the surface of the electrode, and a chemical reaction occurs between the nucleic acid chain and the electrode plate, and electrons are transferred to generate electrical signals. The more chains are combined on the plate, the stronger the current signal can be seen. There are two types of plates used in the experiment, and the difference lies in the size of the electrode. Set two gradients of the duration of binding nucleic acid strands on the two plates, as shown in Figure 4. It can be seen that as the seed strand time increases, the current signal has an upward trend, which confirms that the number of strands does increase with the passage of time.
If the G chain modification is a stable accumulation process on the surface of the electrode plate, the elution in the chain modification stage will show a substantially linear elution curve with increasing time. Use buffer for the electrode plate after the chain modification lasts for 8 hours. When the elution time continues to increase, the voltammetric signal is tested (Figure 5), and the data is fitted to obtain the logistic equation instead of simple linearity. It may be that chain modification is not a simple amount accumulation, and the G chain may not all be inserted straight on the plate. Some chains may lie on the plate or entangle each other, which is difficult to elute, thus affecting the current signal.
Combining the C chain complementary to G to the G chain that has been planted on the plate, adding Cas9 nuclease, etc., a cleavage chemical reaction can occur, and the peak signal is significantly attenuated. After testing 7 sample plates, it is found that the attenuation ratio of current signal is concentrated in 30%~60%. We believe that the attenuation in the range of 30%~50% is reliable. (Figure 6)
Reaction Kinetic Model
References: Li, L., Zhang, W., Tang, X., Li, Z., Wu, Y., & Xiao, X. (2020). Fine and bidirectional regulation of toehold-mediated DNA strand displacement by a wedge-like DNA tool. Chemical Communications. doi:10.1039/d0cc03722b  C.-P. Liang, P.-Q. Ma, H. Liu, X. Guo, B.-C. Yin, B.-C. Ye, Angew. Chem. Int. Ed. 2017, 56, 9077.