3.1 Characterization of AuNPs, Au@Pt nanozymes and Au@Pt-mAb
In accordance with Fig. 1A, Au@Pt nanozymes and Au@Pt-mAb were prepared. The AuNPs, Au@Pt nanozymes and Au@Pt-mAb were characterized with Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), UV‒vis absorption spectrophotometer, energy dispersive X-ray spectroscopy (EDS) and dynamic light scattering (DLS). Figure 2B showed the SEM image of smooth AuNPs. The SEM images proved the 3D structure of Au@Pt nanozymes (Fig. 2C), similar to litchi, which enhances the loading capacity of nanozymes and can accommodate more antibodies. TEM revealed that the AuNPs were smooth and had a diameter of 22 nm (Fig. 1D), and the Au@Pt nanozymes were spherical with average diameters of 28 nm (Fig. 1E). It was demonstrated that the surface of AuNPs was coated with Pt, resulting in an increase in size. The result verified that the Au@Pt nanozymes were synthesized. Figure 1F and 1G depict the obtained EDS spectrum and its interpretation regarding the composition of the AuNPs and Au@Pt nanozymes. These indicate the presence of an Au core (red) encircled by a Pt (green). Meanwhile, the sizes of the Au@Pt nanozymes ranged from 13.5 ~ 58.8 nm, with a mean size of 28 nm detected by DLS, which was 22.87 nm larger than that of the AuNPs, as shown in Fig. 1H. Moreover, to study the composition of the Au@Pt nanozymes, EDS was used for elemental analysis. SEM, TEM and EDS mapping verified a core-shell structure with Au core and Pt shell. To demonstrate the successful preparation of Au@Pt and Au@Pt-mAb, UV–vis absorption spectrophotometer was utilized to explore the formed nanostructures (Fig. 1I). At 520 nm, the AuNPs showed a prominent absorption peak. As previously reported, since the Pt was present, Au@Pt nanozymes exhibited no obvious peak or a weaker absorption region. The conjugation resulted in a shift in the peak of Au@Pt nanozymes, signifying successful antibody coupling [16]. To provide additional proof of the effective synthesis of Au@Pt-mAb, fourier transform infrared (FT-IR) spectra of Au@Pt nanozymes before and after antibody coupling were studied (Fig. 1J). Compared with Au@Pt nanozymes, there is a strong IR absorption bands at Au@Pt-mAb around 1550 ~ 1650cm− 1, indicating that the antibody has been successfully coupled to Au@Pt nanozymes. These investigations have shown conclusive proof that Pt was positioned on the surface of the AuNPs and that the antibody was successfully linked to the Au@Pt. The chemical states of AuNPs and Au@Pt nanozymes were further explored using X-ray photoelectron spectroscopy (XPS). As shown in Fig. 1K, compared with Au, Pt showed more obvious characteristic peaks, and the peaks shifted to the right. The XPS of Au 4f peak shows that it is made up of two peaks: Au 4f7/2 and Au 4f5/2. The binding energy were 83.4 eV and 87 eV, respectively, meaning the Au0 and Au+, respectively [17] (Fig. 1L). Similarly, the binding energies of Pt4f7/2 and Pt4f5/2 were 70.9 eV and 74.1 eV, respectively (Fig. 1M). Pt exhibited three chemical states: Pt0, Pt2+and Pt4+. Pt0 is the most significant because it has more unsaturated chemical bonds on its surface, which produce more active sites and increase catalytic efficiency [18].
3.2 Kinetic analysis of Au@Pt nanozyme
By altering the substrate concentrations, the steady-state kinetic study was performed, and the corresponding steady-state kinetic parameters (Km and Vmax) were calculated (Fig. 2A and 2B). In this work, the reaction rate showed an increasing trend with increasing concentrations of TMB and H2O2. The Au@Pt nanozymes shown good affinity for TMB and H2O2, their Km values were 0.86 mM and 0.31 mM, respectively. The Vmax values for TMB and H2O2 were 6.46×10− 7 M s− 1 and 3.51 ×10− 7 M s− 1, respectively, demonstrating that the Au@Pt nanozymes demonstrated a high catalytic rate as well as a quick reaction rate.
3.3 Comparison of catalytic stability of Au@Pt nanozymes and HRP
To further investigate the superiority of the proposed Au@Pt nanozymes, it and HPR were pretreated at various pH values (1 ~ 13) and temperatures (10℃~90℃) for 10 min, after which their peroxide-like activity was investigated. According to Fig. 2C, the Au@Pt nanozymes exhibited significant catalytic activity when they were treated at different temperatures (10°C ~ 90°C). HRP became fully inactive when the temperature exceeded 60°C. The reason might be that HRP is a kind of protein and is easily denatured at high temperatures. Furthermore, as shown in Fig. 2D, under different pH conditions, comparing Au@Pt to HRP, the former had higher catalytic activity. When the pH is less than 3, HRP loses its activity. When the pH was greater than 12, the HRP activity decreased sharply. Compared with that of HRP, the activity of Au@Pt decreased but did not decrease. According to the results above, Au@Pt nanozymes outperform HRP in terms of stability and catalytic efficiency.
3.4 Optimization Parameters of the indirect competitive immunoassay
3.4.1 Au@Pt nanozymes preparation
The uniformity and catalysis of Au@Pt nanozymes are vital factors in the sensitivity of the immunoassay. Therefore, the Au@Pt nanozymes was optimized. The particle size and uniformity of the Au@Pt nanozymes were changed by adding different amounts of 1% trisodium citrate (3 mL, 5 mL, 7.5 mL, and 10 mL). Subsequently, the AuNPs and Au@Pt nanozymes were characterized by TEM (Fig. 3A) and DLS (Fig. 3B) (Fig. S2, Supporting Information). When the volume of trisodium citrate is 3 mL, the uniformity of the Au@Pt nanozymes is terrible; When the volume of trisodium citrate is 7.5 mL, the uniformity of the AuNPs is poor; when the volume of trisodium citrate is 10 mL, the intensity is low than that at 5 mL. Therefore, 5 mL of trisodium citrate (1%) was selected to prepare the Au@Pt nanozymes.
3.4.2 Blocking agent
Protein can be attached to Au@Pt nanozymes through electronic attraction and hydrophobic interactions. However, binding of non-target proteins can reduces sensitivity and specificity. Therefore, it is necessary to block the unbound antibody sites on Au@Pt nanozymes. As the most commonly used blocking agent, BSA has a good blocking effect. In this study, various concentrations of BSA (0 ~ 2000 mg/L) were optimized. Figure 3C illustrates how N/P value increased continuously as the BSA concentration increased. According to the results, Au@Pt nanozymes could not be entirely blocked by the low concentration of BSA. When the concentration of BSA is 1700 mg/L, N/P value is the highest, OD value is about 1. Therefore, 1700mg/L was selected as the blocking agent.
3.4.3 Antigen and antibody of working concentrations
The concentrations of antigen and antibody play essential role in the sensitivity of the immunoassay to Au@Pt nanozymes, as more mAb modified on Au@Pt nanozymes can greatly improve the affinity to react with antigens. Thus, a chessboard assay was used to screen for combinations of antigens and antibodies. The OD of 1 is chosen to save antigen and antibody utilized while maintaining the sensitivity requirements. As shown in Table 1 in Supporting Information, with increasing antigen and antibody concentrations, the OD also increased gradually. With a fixed antibody concentration, the OD value grew progressively with increasing antigen concentration (0.5 ~ 5 mg/L). As the antibody concentration range from 0.5 ~ 5 mg/L, the OD value gradually increased, while the antigen concentration remained constant. The antigen concentration was 1 mg/L, and the OD values were all approximately 1 at different antibody concentrations. The OD value reached its maximum when the antibody concentration was 5.26 mg/L, and the OD value was 1 when the antibody concentration was 0.55 mg/L. Therefore, the optimal antibody concentration on the Au@Pt nanozymes was determined to improve the sensitivity of the method. The optimization approach used the OD value of negative well (N), positive well (P), and N/P as the evaluation criteria to identify the appropriate antibody concentrations (Fig. 3D). With increasing antibody concentrations, the OD values of the positive wells and the N/P value increased. The positive well OD value reached a maximum when the antibody concentration was 5.26 mg/L. The N/P value reached its maximum when the antibody concentration was 7.69 mg/L. A concentration of 5.26 mg/L was selected based on cost-effectiveness. Thus, the optimal antigen-antibody concentrations were 1 mg/L and 5.26 mg/L, respectively.
Table 1
For the immunoassay, the ideal antigen and antibody concentrations.
Antibody Concentration(mg/L) | OVA-Hapten Concentration (mg/L) |
5 | 2.5 | 1 | 0.5 |
10.00 | 1.88 | 1.57 | 1.08 | 0.81 |
7.69 | 1.95 | 1.53 | 1.11 | 0.79 |
5.26 | 1.96 | 1.64 | 1.21 | 0.86 |
2.79 | 1.70 | 1.40 | 1.05 | 1.00 |
1.37 | 1.36 | 1.26 | 1.11 | 0.93 |
0.55 | 1.17 | 1.10 | 1.06 | 0.94 |
3.5 Calibration curves
Under the optimized conditions, calibration curves were established for the Au@Pt-assisted indirect competitive immunoassay. The study demonstrated that a decreased in OD values and a rise in pesticides inhibition with higher concentrations of pesticides, because the pesticide competed with the antigen to bind the antibody on the Au@Pt nanozymes. The calibration curves and regression equations were built, with inhibition (%) as Y-axis the ordinate axis and the logarithm (LOD) concentration of pesticides as the X-axis. Figure 4A shows the immunoassay standard curve for acetamiprid. A good linear relationship was acquired (R2 = 0.9911) over a wide linear range of 1.85 ~ 327.19 µg/L, exhibiting a low LOD of 0.78 µg/L and good sensitivity (24.58 µg/L). The studies show that the immunoassay has higher sensitivity, good sensitivity and a low LOD for the detection of acetmiprid residue.
3.6 Accuracy and precision
The accuracy, precision, and LOD were assessed to determine the reliability and applicability of the assay. The pesticide standard solutions at concentrations (2.5, 25, and 100 µg/L) were added to blank samples (Chinese cabbage, cucumber, and zucchini) to assess the recovery rate, indicating the accuracy of the immunoassay. In addition, the precision was evaluated under optimal conditions. As demonstrated in Table 2, the average recoveries of the developed the immunoassay based on the Au@Pt nanozymes ranged from 72.5–115.18%, with RSDs between 0.46% and 10.35%. For the Chinese cabbage samples, the average recoveries ranged from 72.5 to 108.45% with RSDs (0.46 ~ 7.49%), and a low LOD (1. 60 µg/L) and IC50 (20.10 µg/L) were acquired in the competitive assay. Similarly to the Chinese cabbage samples, the average recoveries, RSDs, LODs, and IC50 of cucumber samples were 87.64 ~ 115.18%, 5.1 ~ 10.35%, 1.64 µg/L and 17.75 µg/L, respectively. The average recoveries, RSDs, LODs, and IC50 of Zucchini were 81.99 ~ 112.77%, 0.75 ~ 9.99%, 0.65 µg/L, and 11.63 µg/L, respectively. The results show that the Au@Pt-amplified the immunoassay meets the criteria for pesticide residue analysis approaches.
Moreover, for the blank matrix supernatant, regression equations were established. As shown in Fig. 4B, the regression equations were y = 0.3636x + 0.0262, y = 0.3859x + 0.0179, and y = 0.3194x + 0.1589 for Chinese cabbage, cucumber, and zucchini, respectively. Hence, good correlations (R2 = 0.9944 for Chinese cabbage, R2 = 0.9901 for cucumber and R2 = 0.9779 for zucchini) were obtained using the competitive assay. Therefore, the competitive assay of the immunoassay based on Au@Pt nanozymes show promise as a sensitive and reliable assay for detecting acetamiprid.
Table 2
The immunoassay was utilized to recover spiked acetamiprid from vegetable samples (Chinese cabbage, cucumber, and zucchini).
Samples | Spiked (µg/L) | Nanozyme-immunoassay (µg/L) | Recovery (%) | RSD (%) |
Chinese cabbage | 2.5 | 1.81 | 72.50 | 7.49 |
25 | 27.11 | 108.45 | 0.46 |
100 | 81.40 | 81.40 | 6.04 |
cucumber | 2.5 | 2.05 | 87.64 | 5.10 |
25 | 28.19 | 115.18 | 5.66 |
100 | 95.04 | 98.21 | 10.35 |
zucchini | 2.5 | 1.60 | 81.99 | 9.99 |
25 | 28.79 | 112.77 | 0.75 |
100 | 98.21 | 95.04 | 2.30 |
3.7 Specificity
Specificity is crucial for assessing performance as it involves identifying the target molecule [19]. Several commonly used pesticide (100 ng/mL) solutions of imidacloprid, thiamethoxam, fenthion, chlorpyrifos, malathion, and acetamiprid mixed with these pesticides were conducted on these pesticides to assess the specificity of the proposed competitive assay for detecting acetamiprid. As indicated by Fig. 4C, the interfering substrates alone showed a low detectable signal relative to the target acetamiprid. Interestingly, the coexistence of interfering substances with the acetamiprid did not result in a notable alteration in the absorbance of the target analyte, demonstrating the high specificity of the immunoassay, which is based on Au@Pt nanozymes.
3.8 Comparison the HRP-assisted immunoassay with Au@Pt-assisted immunoassay
To verify the reliability of the Au@Pt-assisted indirect competitive immunoassay, we established calibration curves of HRP-assisted immunoassay, as shown in Fig. 4D. The R2 was 0.9643, and the LOD, sensitivity, and linear range of the immunoassay were 1.11 µg/L, 10.08µg/L, and 1.93 µg/L ~ 57.72 µg/L, respectively. Compared with those of the HRP-assisted immunoassay, the LODs of the Au@Pt-assisted immunoassay were lower, and the linear range was wider. Therefore, Au@Pt-assisted immunoassay is a reliable method for detecting trace pesticide residues. Replacing HRP with Au@Pt nanozymes offers several advantages, including lower preparation costs, simpler preparation processes, and improved stability. This substitution not only eliminates the need for IgG but also simplify experimental procedures.