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.