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Transmission electron microscopy (FEI Tecnai G2 F30, USA) was used to study the morphological characteristics of microgels (Figure 1A). The sample was dropped on the copper mesh and imaged by using a transmission electron microscope after natural drying. The observed microgel diameter was approximately 0.5 μm, and nanoscale AuNPs were uniformly encapsulated inside the microgel. The images clearly showed that the microgel composite had a large specific surface area and porous network structure. The voids with an average size of 0.5–1 μm among these porous network microgels can provide a suitable microenvironment for S protein to bind and conduct biological activity and induce protection against interference from other proteins. X-ray photoelectron spectroscopy (Thermo ESCALAB 250XI, USA) was used to evaluate the successful synthesis of the microgels. Figure 1B shows the characteristic signals of the NIPAM-co-AAc/AuNPs microgels, in which signals at 284.8, 399.4, 531.8, and 88.0 eV represent C(1 s), N(1 s), O(1 s), and Au(4 f), respectively[29].
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In confirming the successful construction of the sensing platform of microgels, CV and EIS were used to describe the modification of the electrode surface. Figure 2 shows the obtained CV redox curves. When the microgel, antibody, and S protein were gradually bound to the electrode surface, the peak continuously decreased because the electron exchange between the gold electrode and the electrolyte was blocked. The peak current was highest on the bare electrode, and it decreased when the microgel was bound to the upper electrode. When the S protein antibody was bound to the microgel-modified electrode surface, the current signal intensity decreased significantly, indicating that a number of negatively charged phosphate groups hindered electron exchange at the electrode surface. When the S protein was precisely captured by the antibody, the peak current signal intensity decreased. This trend was also demonstrated by EIS. Therefore, the S protein was loaded onto the modified electrode by specifically binding to the antibody.
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In obtaining optimal conditions for the assay, the key factors (volume of the microgel, concentration of the antibody, and incubation time of the antibody with the S protein) were tested and optimized in a PBS solution with 5 mmol/L [Fe(CN)6]3−/[Fe(CN)6]4− to achieve a satisfactory signal. (current difference: ΔI = I0 − Ip; Ip is the peak current when the S protein is detected, and I0 is the peak current when no S protein is present.)
Figure 3A shows the effect of microgel dosage on the experiment. The results indicated that the current response ΔI increased rapidly to 28.01 μA as the volume of microgel on the gold electrode surface increased from 2 to 6 μL. Afterward, the microgel continuously increased, and ΔI decreased gradually. This result was due to the fact that antibody binding decreased when the microgel material was insufficient, which led to a less sensitive detection of the S protein. Moreover, when the electrode surface contained excessive materials, the increment of the thickness of the microgel film would hinder electron transfer, which would negatively affect detection. Therefore, the appropriate volume of the microgel material was 6 μL to form the most suitable film on the electrode surface for detection.
Figure 3. Assessment of the effects of different factors on the ability of the sensor to detect S proteins: (A) microgel volumes, (B) antibody concentration, and (C) incubation time.
In determining the most suitable antibody concentration, the effect of different antibody concentrations on S protein detection was investigated (Figure 3B). When the antibody concentration increased from 0.5 to 10 μg/mL, the difference in the electrochemical signal widened. On the contrary, the change in electrochemical signal stabilized when the antibody concentration continuously increased. These results indicated that the sensing device modified with 10 μg/mL of antibody on the microgel material was the suitable concentration for capturing S protein.
When the incubation time of SARS-Cov-2 S protein was 50 min, the DPV response of the sensor reached the maximum (Figure 3C). The electrochemical signal gradually increased with the increase of the incubation time of the nanoprobe and started to decrease after 50 min, indicating that binding saturation between the S protein and the nanoprobe was achieved. Hence, subsequent experiments were conducted under these optimal conditions.
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[Fe(CN)6]3−/4− (5 mmol/L) was adopted as the electrolyte, and the DPV determination of the S protein standard sample was carried out to determine its detection linear range and detection limit. As shown in Figure 4A, when the S protein concentration varied from 10−13 to 10−9 mg/mL (10−13, 10−12, 10−11, 10−10, and 10−9 mg/mL), good DPV was observed. The peak redox current decreased with the increase of S protein concentration. Antibodies could recognize and capture S protein and form antibody S protein complexes on the surface of the electrode. Thus, when the complex was formed, the electron transport channels were blocked, preventing electron transfer and resulting in a weakened electrical signal. The changing electrical signal showed a linear relation to the negative logarithm (−Log) of the S protein concentration over a linear range of 10−13 to 10−9 mg/mL, which was confirmed by plotting the negative logarithm of S protein concentration versus current ΔI, which was obtained from multiple measurements (n ≥ 3, Figure 4B). The linear regression equation was presented as follows: ΔI = −9.135lgC + 125.604. The correlation coefficient (R2) was 0.998. Based on the International Union of Theoretical and Applied Chemistry calculation, the limit of detection (LOD) is three times the standard deviation of the blank/slope, and the LOD of the sensor is 9.55 fg/mL.
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In examining the long-term stability of the biosensors, the sensors were stored at 4 °C, and the DPV current responses of the sensors to the same concentration of S protein were evaluated every 3 days. The preservation rate of the sensor was 98.72% after 3 days of storage, 95.80% for 9 days, and 89.47% for 15 days. As shown in Figure 5A, the sensor has competent stability.
Figure 5. Assessment of microgel sensors’ performance: (A) reproducibility, (B) stability at 4 °C, and (C) specificity.
In addition, under the same operating conditions, the four electrodes were detected with the same concentration of S protein, and the signal response values were recorded to verify the repeatability of the sensor. The measurement results are shown in Figure 5B. The response values of the four electrodes were relatively close, and the relative standard deviation (RSD) was less than 5%, indicating that the sensor has evident reproducibility.
As shown in Figure 5C, four substances, including glucose, human serum albumin, α-synuclein, and Tau-441 protein, and a mixture of the four substances with S protein were selected as interference substances for the specificity experiment. Their concentration was controlled at a higher level than the detected S protein (10 pmol/L S protein and 100 pmol/L interferents). The results exhibited little difference between the peak current of S protein alone (28.61 ± 0.26 µA) and that of S protein with interfering substances (30.47 ± 0.35 µA). The binding of the S protein to the antibody remained intact, although the sensor was exposed to a mixture with a 10-fold concentration of the interferent and S protein. Simultaneously, the current exhibited by the detection of interfering substances alone was lower than that exhibited by the detection of S protein, and the difference in DPV peak currents obtained by interferents significantly narrowed. These results indicate the high selectivity of the sensing platform for the S protein.
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In evaluating the potential application of the microgel sensor, this sensor was used for the detection of a series of standard S protein solutions, which were prepared in artificial saliva. Artificial saliva samples had more complex properties than PBS, but the obtained results still showed no deviation from the standard curve (Figure 6). The linear range was 10−9–10−13 mg/mL, and the linear regression equation was presented as follows: ΔI = −12.314lgC + 170.486. The correlation coefficient (R2) was 0.984. Hence, the microgel sensor fabricated in this study is suitable for the detection of S protein in various real samples.
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In demonstrating the reliability of the presented microgel sensor for actual specimen analyses, the sensing platform was used to measure three known different concentrations of S protein (500, 25, and 5 pg/mL). Each concentration was measured at least three times. The results are listed in Table 1. The recoveries ranged from 96.33% to 103.01%, and the RSD was less than 5%. This result indicates that the electrochemical biosensor is satisfactorily accurate and precise.
Sample Added concentration
(pg/mL)Found
(pg/mL)Recoveries
(%)RSD
(%)1 500 515.67 103.01 2.67 2 25 24.08 96.33 1.64 3 5 4.89 97.75 2.17 Note. RSD, relative standard deviation. Table 1. Measurement of recoveries (%) and RSD (%)
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In this study, a microgel material-based sensor was used for the rapid and sensitive electrochemical detection of the SARS-COV-2 spike protein. The process parameters were scientifically and effectively optimized to obtain good sensing performance. The optimal conditions for the volume of microgel modification, the concentration of antibody, and the incubation time of antibody and S protein were 6, 10, and 50 min, respectively. This work achieved satisfactory results under optimal experimental conditions. The linear range was 10−13–10−9 mg/mL, and the detection limit was 9.55 fg/mL. The microgel was stabilized at room temperature and in an adjustable system that closely resembles the microenvironment of aqueous biological tissues. The microgel-based 3D electrode-modified material provides a natural microenvironment for S protein capture with better selectivity for S proteins than conventional 2D materials. Meanwhile, the activity of the sensing platform performed well within 15 days, and the results obtained from the modification of different electrodes showed favorable reproducibility. Therefore, the sensing platform can handle a large number of samples over the long term. The detection properties of the sensors in the experiments are compared with those of previous literature, and the previously reported sensors for the detection of SARS-CoV-2 are summarized in Table 2. The results show that the electrochemical sensor based on the microgel mode is an efficient and reliable detection platform for the detection of S protein.
Targets Material Method Linear range LOD Ref N protein Screen-printed electrodes DPV — 8 ng/mL [30] N protein Metalorganic frameworks
MIL-53(Al)DPV 0.025–50 ng/mL 8.33 pg/mL [31] S protein Pd-Au nanosheets DPV 0.01–1,000 ng/mL 0.72 × 10−2 ng/mL [32] S protein Molecularly imprinted polymer SWV 50–400 fmol/L 64 fmol/L [33] S protein Screen-printed carbon electrodes EIS 0.01–100 nmol/L 66 pg/mL [34] S protein Screen-printed graphene EIS 0.25–1,000 fg/mL 0.25 fg/mL [35] S protein AuNP antibodies DLS — 5.29 × 103 TCID50/mL [36] S protein — SERS — 9.3 pmol/L [37] S protein — Fiber-optic biolayer
interferometry— 36 pmol/L [38] S protein Microgel DPV 10−13–10−9mg/mL 9.55 fg/mL This work Note. DPV, differential pulse voltammetry; SWV, square wave voltammetry; EIS, electrochemical impedance spectroscopy; DLS, dynamic light scattering; SERS, surface-enhanced raman spectroscopy. Table 2. Comparison of different biosensor techniques for the detection of SARS-CoV-2 proteins
A new microgel-based electrochemical sensing system for the detection of the SARS-CoV-2 S protein has been developed. This S protein detection system displays competent sensitivity and accuracy, indicating its application potential in model biofluids. Although the practical application of this method to real-world samples needs further exploration and amelioration, our results demonstrate that this sensing platform can be integrated into portable devices to monitor the outbreak of emerging viruses. Considering that microgel synthesis can be tailored to capture any protein, this new electrochemical sensing system can be widely used to detect SARS-CoV-2 S variants of concern (VOCs) proteins and other emerging viruses. Furthermore, this method may play a role in the detection of SARS-CoV-2 VOCs and potential new outbreaks caused by emerging viruses.
The S protein detection system in this experiment showed excellent sensitivity, stability, and accuracy, with detection limits as low as 9.55 fg/mL. This sensing platform yielded more accurate reports than conventional assays at a lower cost and with a simpler and faster detection process. Despite our satisfactory results, the present sensing platform still has some limitations. Intact viruses could not be detected under the conditions used in this experiment because of the strong infectiousness of SARS-CoV-2. Therefore, further experiments must be performed to demonstrate the effectiveness of the detection of actual viruses.
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Characterization of Nanomaterials
Electrochemical Characterization of the Sensing Platform
Optimization of Experimental Conditions
Analysis of Standard Samples in PBS
Stability, Accuracy, and Reproducibility
Measurements with Artificial Saliva Sample
Accuracy and Precision
22354Supplementary Materials.pdf |