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.