Figure Legends
Figure 1. The Shennong-1 robot for sewage sampling, enrichment, concentration, nucleic acid extraction, and reverse transcription.
Figure 2. Principle of the PEC biosensor for the detection of cDNA using CRISPR/Cas12a system.
Figure 3. Morphological and compositional characterization of the electrodes. Scanning electron microscopy images of (A) the base electrode and (B) the assembled electrode. Elemental mappings of (C) the assembled electrode, (D) Cd, (E) Te, (F) Zn, and (G) S.
Figure 4. X-ray photoelectron spectroscopy survey spectra of the electrodes. (A) X-ray photoelectron spectroscopy survey spectra of (i) the base electrode and (ii) the assembled electrode. High-resolution XPS spectra of the assembled electrode for (B) Cd 3d, (C) Te 3d, (D) Zn 2p, and (E) S 2p.
Figure 5. Optimization of crRNAs. Fluorescence analysis of crRNAs introduced with (A) a single mismatch at different sites targeting the SARS-CoV-2 wild-type cDNA template and (B) a dual mismatch at different sites targeting the BA.2 and BA. 5 cDNA templates
Figure 6. Electrochemical and PEC characterization, and detection performance of the fabricated biosensor. (A) Stepwise electrochemical impedance spectroscopy and (B) photocurrent response analysis of the fabricated photoelectrochemical biosensor (a: Au NPs/rGO; b: ssDNA/Au NPs/rGO; c: MCH/ssDNA/Au NPs/rGO; d: MCH/dsDNA/Au NPs/rGO; e: CdTe/ZnS QDs/MCH/dsDNA/Au NPs/rGO; and f: CdTe/ZnS QDs/MCH/dsDNA/Au NPs/rGO + Cas12a–crRNA­–target DNA complex in [Fe(CN)6]3−/4−. (C) Photoelectrochemical responses of the biosensor to various concentrations of target DNA (a: 0; b: 1.0 aM; c: 10.0 aM; d: 100.0 aM; e: 1.0 fM; f: 10.0 fM; g: 100.0 fM; h: 1.0 pM; i: 10.0 pM; j: 100.0 pM; and k: 1.0 nM). (D) Relationship between the change in signal (ΔI) and the logarithm of the concentration of target DNA. Error bars represent the standard deviation of triplicate measurements. Cas12a: CRISPR-associated protein 12a; crRNA: CRISPR RNA; dsDNA: double-stranded DNA; MCH: 6-mercaptohexanol; NPs: nanoparticles; QDs: quantum dots; rGO: reduced graphite oxide; ssDNA: single-stranded DNA.
Figure 7. Selectivity, stability, and reproducibility of the biosensor. (A) Selectivity of the photoelectrochemical biosensor incubated with various sequences: Omicron BA.5, Omicron BA.2, Wild-type, MERS, H1N1, H3N2, Influenza B, and HRSV. (B) Photocurrent stability of the biosensor under consecutive off−on−off illumination for 10 cycles. (C) Responses of the photoelectrochemical biosensor’s photocurrent after different storage periods. (D) Reproducibility of the fabricated photoelectrochemical biosensor. Columns with different superscript letters show significant differences (p < 0.05).
Figure 8. Evaluation of the photoelectrochemical biosensing platform by real sample analysis. Detection of the L452R mutation in the spike gene of Omicron BA.5 in 24 sewage samples via (A) RT-PCR and (B) the proposed biosensor.
Figure 9. PEC reaction mechanism elucidated via density functional theory. (A) Top view and front view of the CdTe/ZnS QDs crystal structure model. (B) Calculated band structures of the CdTe/ZnS QDs. (C) The specific transfer mechanism of charge carriers.