2.1. The underlying principle of an automated robot-driven PEC biosensing platform
Considering Omicron BA.5 as an example, the principle of an automated robot-driven photoelectrochemical biosensing platform based on a CRISPR/Cas12a system is illustrated. The L452R mutation in the spike gene serves as an important marker for detecting Omicron BA.5 infection in the population. However, there is no protospacer adjacent motif (PAM) sequence near the L452R mutation, making it challenging to use Cas12a for detection of dsDNA amplification products associated with Omicron BA.5. A previous study reported that a Cas12a–crRNA duplex can specifically recognize and bind to single-stranded DNA (ssDNA) in the absence of a PAM, initiating Cas12a’s cleavage activity[35]. Based on this report, we utilized the specificity of the Cas12a–crRNA duplex for recognizing target cDNA to detect the L452R mutation. To begin with, the automated Shennong-1 robot was responsible for performing sewage sampling, enrichment, concentration, nucleic acid extraction, and reverse transcription (Figure 1). Subsequently, the resulting reverse transcription products were subjected to PEC analysis based on CRISPR/Cas12a-mediated CdTe/ZnS QDs–dsDNA nonspecific cleavage (Figure 2). The surface of the Au NPs/rGO base electrode was modified with a CdTe/ZnS QDs–dsDNA reporter. The Cas12a–crRNA duplex was designed to precisely recognize Omicron BA.5 based on the complementarity between the target DNA and crRNA[36]. In the absence of target cDNA, the cleavage activity of Cas12a remained inactive, causing CdTe/ZnS QDs-dsDNA to remain intact on the modified electrode’s surface and leading to a noticeable photoelectrochemical signal. In the presence of target cDNA, the activity of Cas12a was initiated, resulting in trans-cleavage activation. Consequently, non-specific cutting of CdTe/ZnS QDs-dsDNA occurred on the electrode surface, leading to a decline in the photoelectrochemical signal. Therefore, the prepared CdTe/ZnS QDs-assisted CRISPR–PEC biosensor successfully converts the target sequence recognition into the large-scale cutting of dsDNA on the electrode followed by a change in photoelectric signal for ultrasensitive, highly specific photoelectrochemical nucleic acid biosensing. In summary, the automated robot-driven PEC biosensing platform constructed in this study has the potential for the rapid, ultrasensitive, and highly specific detection of Omicron BA.5 in sewage.
2.2.Morphological and compositional characterization of the CdTe/ZnS QDs–dsDNA/Au NPs/rGO electrode
The electrode morphology was assessed through scanning electron microscopy (SEM) measurements, both prior to and after the assembly process. Prior to assembly, the base electrode was composed of vertically oriented graphene with Au NPs deposited on its surface, which presented sharp edges and channels ranging in width from tens of nanometers to micrometers (Figure 3A). However, after assembly, the location of CdTe/ZnS QDs on the electrode surface was difficult to distinguish in SEM images due to their minimal particle size (Figure 3B). To address this issue, the elemental mapping was performed, confirming the homogeneous distribution of Cd, Te, Zn, and S on the CdTe/ZnS QDs–dsDNA/Au NPs/rGO electrode (Figure 3C–G). Furthermore, the surface element composition of the electrode was evaluated by X-ray photoelectron spectroscopy (XPS). The assembled electrode exhibited increased C 1s and O 1s peak areas compared to the base electrode, which can be attributed to the presence of DNA probes and carboxyl groups on the surfaces of CdTe/ZnS QDs (Figure 4A). The N 1s peak observed in the assembled electrode was likely due to the presence of DNA, while the appearance of Cd 3d, Te 3d, Zn 2p, and S 2p peaks were attributed to the CdTe/ZnS QDs (Figure 4A). Furthermore, the characteristic peaks of CdTe were observed in the Cd 3d spectra (Figure 4B) at 411.6 eV and 404.9 eV, as well as in the Te 3d spectra (Figure 4C) at 582.4 eV and 572.0 eV. Characteristic peaks of ZnS were identified in the Zn 2p spectra (Figure 4D) at 1044.9 eV and 1022.0 eV, as well as in the S 2p spectra (Figure 4E) at 162.8 eV and 161.6 eV. It should be pointed out that the peaks at 163.5 eV and 162.3 eV were derived from Au-S bonds[37]. All of the physical measurements and analysis described above confirmed the successful assembly of the CdTe/ZnS QDs–dsDNA/Au NPs/rGO electrode.