*2.9. Recovery Experiments*

A 25 g chicken sample was placed in a sterile homogenization bag containing 225 mL of LB broth without additives and homogenized continuously on a flapping homogenizer for 2 min, without incubation of the chicken homogenate after contamination. A quantity of 1 mL of 1.3 × 10<sup>1</sup> CFU/mL, 1.3 × 10<sup>4</sup> CFU/mL, or 1.3 × 10<sup>7</sup> CFU/mL *Listeria monocytogenes* liquid was added to 9 mL of sample homogenate, and the test tube was shaken to mix it evenly. The sample homogenate containing different concentrations of pathogenic bacteria was centrifuged for 5 min under low-speed centrifugation at 2000 r/min. The supernatant was absorbed and added into the sterilization centrifuge tube and centrifuged at a speed of 12,000 r/min for 3 min. After that, the supernatant was discarded, and 1 mL of normal saline was added for full mixing. Then the *Listeria monocytogenes* pollution solution of the adult model was configured, and the prepared sensor was used for analysis and detection.

## **3. Results and Discussion**

#### *3.1. Photoelectrochemical Reaction Mechanism of the Sensor*

The charge transfer of the working electrode is displayed in Figure 2. WO3 has a high electron-hole recombination rate and a low photoelectric conversion efficiency. Therefore, sensitizing the CdTe quantum dots with a small band gap can broaden the spectral response range, improve the photoelectric conversion efficiency, and increase the photocurrent intensity. Au NPs mainly connect aptamers through Au-S bonds and can also increase the absorption of light by quantum dots. AA is used as the electron donor in the electrolyte solution.

Under illumination, the electrons generated by the CdTe quantum dots' transition from the valence band to the conduction band flow through the Au NPs and are injected into the conduction band of WO3. Then, together with the photogenerated electrons generated inside WO3, they are finally transferred to the FTO electrode to generate the current signal. The WO3 and CdTe valence bands leave a large number of photogenerated holes. At this time, the AA in the solution provides electrons and is oxidized by the holes.

When there were no *Listeria monocytogenes*, the CdTe QDs were close to the electrode surface and produced sensitization, and the photocurrent increased. In the presence of *Listeria monocytogenes*, Ap specifically combined with *Listeria monocytogenes*, causing the CdTe QDs to leave the electrode surface and reduce the photocurrent intensity. Meanwhile, Exo I could recognize and cut Ap. *Listeria monocytogenes* bound to Ap continued to bind to Ap on the electrode, and the QDs were far away from the electrode. In this cycle, the QDs

on the electrode gradually decreased, and the photocurrent decreased significantly. The sensitive detection of *Listeria monocytogenes* was achieved.

**Figure 2.** Photogenerated electron transfer mechanism.

## *3.2. Characterization of Synthesized Materials*

Figure 3a shows the XRD patterns of FTO, WO3/FTO, and Au/WO3/FTO. The WO3/FTO diffraction peaks (002), (200), (020), (-112), (202), (222), (140), (240), and (420) are obvious in the figure, which proves the formation of monoclinic crystal WO3 (JCPDS no. 43-1035). When the Au NPs were modified, no new peaks appeared, but the intensity of the WO3 peaks was significantly reduced. This may be because of the Au NP layer covering the surface of WO3, but the content of Au NPs was too small. Figure 3b shows the XRD pattern of CdTe QDs. The 2θ value corresponds to the three crystal planes (111), (220) and (311) of the cubic crystal CdTe on the standard card no. 65-1046. The above results indicate that the sensor electrode material was successfully prepared.

Figure 4 shows the SEM images of WO3 and Au/WO3 modified on FTO conductive glass. It can be seen that WO3 nanosheets were arranged vertically on the surface of the FTO glass (Figure 4a) with regular shapes and a thickness of 70.8–74.7 nm. Figure 4b shows a cross-sectional SEM image of WO3/FTO. The WO3 layer deposited on the surface of FTO was relatively uniform, with a thickness of about 422 nm. The vertically arranged structure increased the surface area of the WO3 layer and could improve the utilization rate of light. After modification with Au NPs, as shown in Figure 4c, the basic morphology of the WO3 layer did not change, and the surface of the smooth nanosheet became rough, indicating that the Au NPs were successfully modified. There was no change in the cross-sectional thickness after the modification of Au NPs.

The TEM image (Figure 5a) of Au NPs shows that the prepared Au NPs were spherical and uniform in size, and the particle size was between 10 and 15 nm. Obvious lattice fringes can be seen in Figure 4b, and the lattice spacing of the Au (111) lattice plane was about 0.235 nm after software analysis.

To further confirm the surface element composition, we performed an XPS test on WO3/Au. Figure 6a shows the full spectrum of WO3/Au. W, Au, O, and C were observed. Figure 6b shows the XPS spectrum of W 4f. It shows that the characteristic peaks of the binding energy of W 4f7/2 and W 4f5/2 were 35.7 eV and 37.7 eV, consistent with W6+. The XPS spectrum (Figure 6c) of O 1s showed two characteristic peaks of the binding energies of 530.3 eV and 531.9 eV. The peak at 530.3 eV aligns with the O2− ions characteristic of the WO3 phase, corresponding to lattice oxygen. The peak at 531.9 eV corresponds with the chemically adsorbed oxygen species at the oxygen vacancy in WO3 [41,47,48]. The XPS

spectrum of Au 4f consisted of two parts, as shown in Figure 6d. The characteristic peaks at 83.8 eV and 87.5 eV can be ascribed to Au 4f7/2 and Au 4f5/2, respectively.

**Figure 3.** XRD pattern of FTO, WO3/FTO, and Au/WO3/FTO (**a**) and XRD pattern of CdTe QDs (**b**).

**Figure 4.** SEM images of the WO3/FTO front (**a**) and cross-section (**b**) and Au/WO3/FTO (**c**).

**Figure 5.** TEM image (**a**) and HRTEM image (**b**) of Au NPs.

**Figure 6.** XPS spectra of WO3/Au: survey spectrum (**a**), high-resolution XPS of W 4f (**b**), O 1s (**c**), Au 4f (**d**).

Figure 7 shows the UV–vis (a) and fluorescence spectra (b) of CdTe QDs. The curves a, b and c in the figure represent quantum dots prepared through reactions for 30 min, 1 h and 3 h, respectively. With the extension of the reaction time, the peaks of the ultraviolet– visible absorption spectrum and the fluorescence spectrum gradually shifted to the longwavelength direction, which enhanced the absorption range of visible light. This may be due to the quantum confinement effect, through which the emission wavelength was red-shifted. The stronger the peak in the fluorescence spectrum, the higher the electron-hole recombination rate. After optimization experiments, CdTe QDs with a reflow time of 3 h were selected to construct the sensor. The exciting absorption peak was 538 nm, and its maximum fluorescence emission wavelength was 582 nm.

#### *3.3. Photocurrent Characterization of the Aptamer Sensor*

In order to prove the successful preparation of the working electrode of the aptamer sensor, a photocurrent response test was performed in a 10 × PBS buffer containing 0.12 mol/L of AA with a pH of 7.4. As shown in Figure 8, the light source was turned on and off at 20 s and 40 s, and photocurrent changes within 60 s were observed. The blank FTO had no change in photocurrent (curve a), and when modified with WO3, the photocurrent significantly increased (curve b). This is because WO3 had good photoelectric activity and generated current under light conditions. When Au NPs were modified on the electrode surface, the photocurrent decreased (curve c). Since the Fermi level of gold nanoparticles is lower than that of WO3, the work function of gold nanoparticles is greater than that of WO3. To balance the two Fermis levels, part of the photogenerated electrons on the conduction band of WO3 is transferred to the gold nanoparticles, which causes a decrease in the photocurrent. When cDNA and MCH were modified, the photocurrent had a slight increase (curve d), which may have been due to the modification of cDNA and MCH, weakening the balance trend of the Fermi level [49]. Therefore, the tendency of photogenerated electrons on the conduction band of WO3 to transfer to gold nanoparticles was weakened. After the QD–Ap conjugate was modified on the electrode, the photocurrent was significantly enhanced because of the sensitization of the quantum dots (curve e). When 30 μL of 1.3 × 10<sup>6</sup> CFU/mL *Listeria monocytogenes* (LM) solution containing 20 U of Exo I was added dropwise and incubated for 60 min, the photocurrent decreased significantly (curve f). This is because the specific binding of *Listeria monocytogenes* and Ap caused the QD–Ap conjugate to detach from the electrode surface, and the quantum dot sensitization was weakened. The shearing effect of Exo I also released the *Listeria monocytogenes* that had been bound to Ap, which re-attached to the electrode surface. The binding of Ap further weakened the sensitization effect. To verify the shear cycle amplification effect of Exo I, a control experiment was carried out. As shown in Figure 9, the photocurrent of the aptamer electrode (curve b) with 30 μL of 1.3 × 10<sup>6</sup> CFU/mL *Listeria monocytogenes* containing Exo I was 26% lower than that of the electrode without Exo I (curve a). This shows that Exo I has a significant signal-amplifying effect on the photocurrent detection process. In summary, this shows that the aptamer sensor was successfully constructed and can be used for *Listeria monocytogenes* detection.

**Figure 7.** UV-vis (**a**) and fluorescence spectra (**b**) of CdTe QDs, a: 30 min, b: 1 h, c: 3 h. Inset in Figure (**a**) shows the image of CdTe QDs under UV lamps with different reflow times.

**Figure 8.** Photocurrent graphs of working electrodes modified with different materials; a: FTO, b: WO3/FTO, c: Au/WO3/FTO, d: MCH/cDNA/Au/WO3/FTO, e: QD–Ap/MCH/cDNA/Au/WO3/FTO, f: LM-Exo I/QD–Ap/MCH/cDNA/Au/WO3/FTO.

**Figure 9.** Photocurrent graph of aptamer electrode with or without Exo I modification, a: LM/QD– Ap/MCH/cDNA/Au/WO3/FTO, b: LM-Exo I/QD–Ap/MCH/cDNA/Au/WO3/FTO.

#### *3.4. EIS and CV Characterization of Aptamer Sensors*

Electrochemical impedance spectroscopy (EIS) was used to further prove the successful construction of the aptamer sensor. EIS was used to a different frequency AC signal to the system; analyze the change in impedance with frequency; analyze the electrode dynamics, diffusion, and electric double layer; and study the mechanism of the solid electrolyte and corrosion protection electrode materials. Electrochemical impedance analysis was performed on working electrodes with different modification processes in solutions. A typical EIS spectrum is a curve with a semicircle and a "tail", which correspond to the high-frequency region and the low-frequency region, respectively. The high-frequency area is dominated by charge transfer, and the low-frequency area is dominated by mass transfer. Among them, the diameter of the high-frequency region circle is equal to the charge transfer resistance (Rct). The larger the diameter, the greater the obstruction of the oxidation–reduction probe on the electrode surface. In Figure 10, FTO has the smallest diameter (curve a), indicating that FTO without any modification can transfer electrons more effectively. After the deposition of WO3, the impedance increased (curve b). Because of the excellent conductivity of gold nanoparticles, the impedance decreased after modification with Au NPs (curve c). The impedance value increased after cDNA and MCH were modified (curve d). This is because the oligonucleotide was negatively charged, and the oxidation–reduction probe Fe (CN)6 3−/4− was also negatively charged; the repulsive force between the two caused an increase in the resistance of electron transport, and MCH is non-conductive, which led to an increase in impedance. When the QD–Ap conjugate was modified on the electrode, the impedance was further increased because of the weak conductivity of QDs and the increase in oligonucleotides. The impedance decreased after incubating Exo I and *Listeria monocytogenes*. This is because many oligonucleotides and QDs left the electrode surface after the specific binding of *Listeria monocytogenes* and Ap and Exo I shearing. Therefore, the change of impedance value proves that the aptamer sensor was constructed successfully.

**Figure 10.** Electrochemical impedance spectroscopy, a: FTO, b: WO3/FTO, c: Au/WO3/FTO, d: MCH/cDNA/Au/WO3/FTO, e: QDs-Ap/MCH/cDNA/Au/WO3/FTO, f: LM-Exo I/QDs-Ap/MCH/cDNA/Au/WO3/FTO. Inset is the equivalent circuit model.

Cyclic voltammetry is also a commonly used electrochemical analysis method, as shown in Figure 11. On the bare FTO electrode (curve a), a pair of obvious redox anode and cathode peaks can be observed at −0.15 V and 0.5 V, and the two peaks are symmetrical, which proves that the reaction is reversible. After being modified with WO3, the redox peak current decreased (curve b). When Au NPs were modified, the current value increased (curve c), indicating that Au NPs can accelerate electron transfer. When cDNA, MCH (curve d), and QD–Ap conjugate (curve e) were modified sequentially, the redox peak current gradually decreased, and the current value increased after incubating Exo I and *Listeria monocytogenes* (curve f). The above test results were consistent with the EIS results, providing further evidence for the successful construction of the sensor.

#### *3.5. Optimization of Experimental Parameters*

The test conditions of the sensor were optimized, and the concentration of AA, the pH value of the electrolyte, the reflow time of the quantum dots, and the incubation time of the electrode with *Listeria monocytogenes* and Exo I were investigated.

AA acts as an electron donor and plays a significant role in increasing photocurrent. As shown in Figure 12a, when there was no AA in the electrolyte, the photocurrent of WO3/FTO was the smallest. With the gradual increase in AA concentration, the photocurrent reached its maximum value at 0.10 mol/L. When the AA concentration exceeded 0.10 mol/L, the current value gradually decreased. It may be that the excessive AA concentration increases the absorbance of the electrolyte and reduces the light intensity on the electrode surface. Therefore, 0.10 mol/L was chosen as the optimum concentration of AA.

In addition, aptamers can only remain active in relatively neutral solutions. Figure 12b shows that the WO3/FTO electrode photocurrent reached the maximum when the electrolyte pH was 7.4, so 7.4 was selected as the optimal pH.

**Figure 11.** Cyclic voltammogram, a: FTO, b: WO3/FTO, c: Au/WO3/FTO, d: MCH/cDNA/Au/WO3/FTO, e: QD–Ap/MCH/cDNA/Au/WO3/FTO, f: LM-Exo I/QD–Ap/MCH/cDNA/Au/WO3/FTO.

**Figure 12.** Photocurrent graphs of different AA concentrations (**a**); pH (**b**); quantum dot reflux times (**c**), a: 30 min, b: 1 h, c: 3 h; and incubation time (**d**).

Three CdTe quantum dots with different reflow times were prepared. It can be seen from the above that the positions of the strongest peaks of the ultraviolet–visible absorption spectra and fluorescence spectra of CdTe quantum dots with different reflow times are different. As time increased, the positions of the strongest peaks moved toward the longwavelength direction. To further prove the size of the photocurrent of quantum dots at different reflow times, three different QDs were used to construct aptamer electrodes and perform photocurrent detection. Figure 12c shows that the photocurrent was the largest when the reflux time was 3 h. Therefore, CdTe QDs with a reflow time of 3 h were chosen to construct an aptamer sensor.

The incubation time of Exo I and *Listeria monocytogenes* also affects the change in photocurrent. Proper incubation time not only reduces the time used in the preparation process but also enables the aptamer sensor to achieve optimal performance. As can be seen from Figure 12d, the photocurrent gradually decreased with an increase in time before the incubation time of 60 min. After 60 min, the photocurrent decreased, but it gradually stabilized. Therefore, the optimal incubation time of Exo I and *Listeria monocytogenes* was 60 min.

#### *3.6. Analytical Performance of Aptamer Sensor*

The prepared aptamer sensor relies on the changes in the photocurrent of the electrodes before and after the addition of pathogenic bacteria to quantitatively and qualitatively detect the pathogenic bacteria. The change in sensor photocurrent is directly related to the concentration of pathogenic bacteria. Figure 13a shows the photocurrent change curve of different concentrations of *Listeria monocytogenes*. It shows that as the *Listeria monocytogenes* concentration increased, the photocurrent gradually decreased. This is because of the combination of a large amount of *Listeria monocytogenes* and Ap on the electrode surface, which weakened the sensitization effect of QDs and reduced the photocurrent. Figure 13b shows that from 1.3 × 10 CFU/mL to 1.3 × 10<sup>7</sup> CFU/mL, the change in photocurrent had a very good linear relationship with the concentration of pathogenic bacteria. The linear equation obtained was ΔI = 9.76logCLM − 4.44 (R<sup>2</sup> = 0.9980), and the detection limit was 45 CFU/mL. This may be due to the sensitization of quantum dots and the auxiliary amplification effect of Exo I, allowing the aptamer sensor to have a wider detection range and a smaller detection limit.

**Figure 13.** Photocurrent curve (**a**) and a calibration curve (**b**) of the aptamer sensor with different concentrations of *Listeria monocytogenes* (a→g: 1.3 × 101→1.3 × 107CFU/mL).

#### *3.7. Selectivity, Stability, and Reproducibility of the PEC Sensing Platform*

Specificity is a significant indicator of the sensor. The specificity of the sensor was characterized by the change of photocurrent before and after incubation with different interferents. A quantity of 1.3 × 10<sup>5</sup> CFU/mL *Listeria monocytogenes* (A) was selected for detection; *Staphylococcus aureus* (B), *Escherichia coli O157:H7* (C), and *Salmonella typhimurium* (D) at the same concentration were selected as interferents, and physiological saline (E) was used as a blank control. As can be seen from Figure 14a, the photocurrent changed significantly before and after the incubation of *Listeria monocytogenes*. After calculation, the change value RSD was 2.94%. The photocurrent changes in the interferents were similar to those in the blank group. This result indicates that the prepared aptamer sensor has satisfactory specificity for *Listeria monocytogenes*.

To investigate the stability of the sensor, the electrode was incubated with 1.3 × 10<sup>5</sup> CFU/mL *Listeria monocytogenes*, and Exo I was repeatedly switched on and off within 400 s to observe the change in photocurrent to judge the stability of the sensor. The light source switch interval was 20 s, as shown in Figure 14b. It can be seen from the figure that there was almost no change in the photocurrent within 400 s. After the second electrode was placed in a refrigerator at 4 ◦C for a week, the photocurrent detection was performed again under the same conditions, and the current value dropped by about 5%, which proved that the sensor had good stability.

Five electrodes were constructed under the same conditions, 1.3 × 10<sup>5</sup> CFU/mL *Listeria monocytogenes* and Exo I were incubated with photoelectric detection, and the reproducibility of the sensor was analyzed. The experimental calculation showed that the RSD was 2.1%, proving that the prepared aptamer sensor has good accuracy and reproducibility.

## *3.8. Analysis of Real Samples*

To verify the feasibility of the sensor to detect *Listeria monocytogenes* in actual samples, we added 1 × 10<sup>2</sup> CFU/mL, 1 × 10<sup>5</sup> CFU/mL, and 1 × 10<sup>7</sup> CFU/mL of *Listeria monocytogenes* to chicken samples that did not contain *Listeria monocytogenes* for photocurrent detection. The *Listeria monocytogenes* concentration was obtained and compared with the actual addition to calculate the recovery rate. As shown in Table 1, for three different dilutions of *Listeria monocytogenes*, the prepared aptamer sensor was used for detection, and the recovery rate met the requirements. This shows that the sensor has grea<sup>t</sup> potential in practical applications.

**Figure 14.** *Cont*.

**Figure 14.** (**a**) Photocurrent response of the sensor to 1.3 × 10<sup>5</sup> CFU/mL of *Listeria monocytogenes* (A), *Staphylococcus aureus* (B), *Escherichia coli O157:H7* (C), *Salmonella typhimurium* (D), saline (E); (**b**) photocurrent response of the aptamer electrode when the light was turned on and off within 400 s continuously.


**Table 1.** Detection of *Listeria monocytogenes* in chicken samples with sensors.
