2.6. Selectivity, stability, and reproducibility
For practical biosensors, it is vital to have outstanding selectivity, stability, and reproducibility. To investigate the selectivity of PEC biosensors, a range of potential interferents including Omicron BA.2, Wild-type, MERS, H1N1, H3N2, Influenza B, and HRSV were utilized.
The selectivity of the PEC biosensor was determined by holding the cDNA concentration constant at 1.0 nM and comparing the ∆I value produced by the Omicron BA.5 cDNA template with the ∆I values produced by these potential interferents. According to Figure 7A, the photocurrent change generated by the Omicron BA.5 cDNA template was significantly greater compared to other potential interferents, demonstrating the notable selectivity of the PEC biosensor.
The investigation of the PEC biosensor’s stability was also conducted. The photocurrent of the CdTe/ZnS QDs/dsDNA/Au NPs/rGO biosensor was evaluated in the detection buffer through multiple consecutive off–on–off irradiation cycles (10 cycles, 200 s; Figure 7B). The photocurrent response remained largely unchanged throughout the cycle test, with a minimal relative standard deviation (RSD) of only 0.58%, indicating the high stability and reliability of the PEC signal. To further study the biosensor’s stability, a long-term storage stability study was performed (Figure 7C). Upon storage in a dark and humid environment at 4°C for 1, 2, 3, and 4 weeks, the PEC biosensor exhibited photocurrent retention rates of 96.20%, 90.66%, 85.63%, and 73.99% of its initial response, highlighting its exceptional stability. The reproducibility of the PEC biosensor was evaluated by measuring the photocurrents of six biosensors assembled from the same batch and comparing their results. The photocurrents obtained from the six individual PEC biosensors exhibited negligible variation (RSD = 1.96%), demonstrating the remarkable reproducibility of the biosensor (Figure 7D).
2.7. Real sample analysis
To assess the sensitivity and specificity of the photoelectrochemical sensing platform for detecting the BA.5 variant in sewage, practical sample analysis was conducted. Initially, 24 sewage samples collected by automated robots were identified using RT-PCR and Sanger sequencing. The sequencing results revealed that among the collected sewage samples, only the BA.2 and BA.5 variants were detected. Specifically, there were 6 negative samples, 6 samples positive for the BA.2 variant, and 12 samples positive for the BA.5 variant. The RT-PCR analysis for the 18 positive sewage samples (Figure 8A) all showed positive results, with Ct values ranging from 15 to 31. Conversely, the results for the 6 negative sewage samples were all negative. Additionally, using the same categorized samples, the photoelectrochemical sensing platform was employed to detect the L452R mutation site (unique to the BA.5 variant and absent in the BA.2 variant). The positive threshold was defined as a ΔI value of 6.37 nA, representing the average of the blank signals plus triple its standard deviation. Positive results (ΔI > 6.37 nA) were observed exclusively in the samples containing the BA.5 variant (Figure 8B). These findings demonstrate that the photoelectrochemical sensing platform can specifically monitor the crucial L452R variant of SARS-CoV-2. In summary, the photoelectrochemical sensing platform demonstrates remarkable sensitivity and specificity in detecting the BA.5 variant in sewage.
2.8. PEC reaction mechanism
In order to elucidate the PEC reaction mechanism of the constructed PEC sensing platform, a thorough investigation into the photogenerated carrier behavior of CdTe/ZnS QDs was conducted using first-principle calculations based on density functional theory. The crystal and band structures of CdTe/ZnS QDs were depicted in the Figure 9A and 9B, respectively. The conduction band (CB) and valence band (VB) energy levels of CdTe were found to be approximately 0.80 and -0.34 eV, respectively. Besides, for ZnS, the CB and VB energy levels were approximately 2.53 and -0.19 eV. The energy gaps (Eg) of CdTe and ZnS were estimated to be 1.14 and 2.72 eV, respectively. Consequently, CdTe/ZnS QDs exhibit a type-II heterostructure, resulting in effective spatial separation of photoexcited electrons and holes. This spatial separation leads to prolonged carrier lifetimes due to reduced recombination rates. Based on these research findings, CdTe/ZnS QDs exhibit exceptional photoelectric response characteristics, which significantly enhance the sensitivity of the constructed biosensor.
The accompanying Figure 9C illustrates the specific mechanism of charge carrier transfer. Upon illumination, electrons from the VB of the semiconductors transition to the CB, resulting in an accumulation of holes in the VB. Subsequently, there is an electron transfer from ZnS to CdTe, while concurrently, holes are transferred from CdTe to ZnS due to the existing potential difference, thus realizing efficient transfer of photo generated carriers. Meanwhile, photoexcited electrons undergo a transfer process from the conduction bands of CdTe to Au NPs. Ultimately, the electrons are conveyed to vertically aligned graphene, resulting in a remarkable photocurrent response.