2.4. Electrochemical impedance spectroscopy and PEC characterization of the PEC biosensor
Electrochemical impedance spectroscopy (EIS) is employed as a valuable technique to investigate the electrode interface, enabling the monitoring of the sequential assembly process of photoelectrochemical (PEC) biosensors. EIS tests were carried out in a 5 mM K3Fe(CN)6/K4Fe(CN)6(1:1) solution with 0.1 M KCl serving as the supporting electrolyte. The tests were conducted at a bias potential of 0.14 V, employing a frequency range of 10−2–105 Hz and an amplitude of 5 mV. Figure 6A showed that, among these electrodes, the Au NPs/rGO electrode presented the most negligible interfacial electron transfer impedance value (Ret) of about 84.95 Ω (curve a), which can be ascribed to the exceptional conductivity of vertical graphene and Au NPs. When the probe ssDNA 1, 6-mercaptohexanol (MCH), and ssDNA 1 complementary chains (ssDNA2 and ssDNA3) were fabricated in turn on the electrode surface, the Ret value gradually increased (curves b, c, and d, respectively). These results were attributed to the high steric hindrance and low conductivity of biological macromolecules (DNA, RNA) and MCH[38]. The introduction of CdTe/ZnS QDs further hindered electron transfer (curve) due to the steric steric hindrance effect of CdTe/ZnS QDs and their low conductivity. After incubating the electrode with the Cas12a–crRNA–target DNA complex, the Ret value decreased again (curve f). This decline happened because the Cas12a–crRNA duplex indiscriminately cut the DNA probe on the electrode surface, which resulted in a reduction in both the quantities of CdTe/ZnS QDs and DNA, leading to a decrease in the steric hindrance effect and an increase in conductivity on the electrode surface.
The PEC response to electrode surface modifications was continuously monitored to assess the construction process of the proposed biosensor. In Figure 6B, the Au NPs/rGO electrode demonstrated a noticeable photocurrent response (curve a) as a result of the surface plasmon resonance (SPR) effect. In the case of SPR excitation, the energetic hot electrons generated in Au NPs can be directly transferred to vertical graphene and the external circuit, causing photocurrent generation[39,40]. When the probe ssDNA1, MCH, and ssDNA1 complementary chains (ssDNA2 and ssDNA3) were fabricated in turn on the electrode surface, the photocurrent signal gradually decreased through each of these steps (curves b, c, and d, respectively), owing to the high steric hindrance and low conductivity of DNA and MCH. After CdTe/ZnS QDs were fabricated on the MCH/dsDNA/Au NPs/rGO electrode, the photocurrent signal significantly increased (curve e) due to the excellent photoelectric characteristics of CdTe/ZnS QDs. When the electrode was incubated with the Cas12a–crRNA–target DNA complex, the photocurrent signal decreased again (curve f), because the Cas12a–crRNA duplex nonspecifically cut the DNA probe on the electrode surface, resulting in a decreased quantity of CdTe/ZnS QDs and subsequently a decrease in the photocurrent originating from CdTe/ZnS QDs. In summary, the successful fabrication of the biosensor was confirmed by all results obtained from EIS and PEC characterization.