*3.3. Quantitative Detection of Cr6+ in Tea Samples*

The detection and quantitative analysis of Cr6+ in tea samples were obtained based on the redox reaction between carbimazole and Cr6+. The hydrolytic product (methimazole) of carbimazole was adsorbed on the surface of Au@Ag via Ag-S and Ag-N bonds, and its Raman signal was greatly enhanced. At the same time, the aggregation of these functionalized substrates caused by NaCl also resulted in significant SERS enhancement [16]. In order to identify the characteristic peaks of Cr6+, the spectra of high concentration of Cr6+ solution (100 μg/L) were compared with the blank tea sample (Cr6+ = 0 μg/L) (Figure 4a). Carbimazole was hydrolyzed to methimazole under acidic media that causes carbimazole to have a similar Raman spectrum to methimazole. The strongest characteristic peak was shown at 595 cm−<sup>1</sup> due to the enhancement of Au@AgNPs on methimazole. The presence of Cr6+ causes a redox reaction between Cr6+ and methimazole, forming a disulfide compound and resulting in a decrease in Raman intensity at 595 cm<sup>−</sup>1. Therefore, the quantitative analysis of Cr6+ can be realized by using the change of Raman intensity at 595 cm−1. The main peaks of the methimazole contribution are listed in Table 1. As can be seen from Figure 4a, the characteristic peak at 595 cm−<sup>1</sup> was attributed to the vibration of the C–N–S bend.

**Figure 4.** (**a**) SERS spectra of Cr6+ (0 μg/L) and Cr6+ (100 μg/L); (**b**) SERS spectra of Cr6+ in tea at different concentrations ranging from 0 to 100 μg/L; (**c**) the SERS intensity at 595 cm−<sup>1</sup> at various concentration of Cr6+; (**d**) calibration curve between the SERS intensity at 595 cm−<sup>1</sup> and logarithm of the concentration of Cr6+.



The SERS spectra of different concentrations of Cr6+ ranging from 0 to 100 μg/L were obtained and are shown in Figure 4b. The Raman intensity at 595 cm−<sup>1</sup> significantly decreased with increasing Cr6+ concentration in the tea samples (Figure 4c) as the methimazole was reduced to disulfide. The calibration curve of Cr6+ was obtained using the relationship between the Raman intensity at 595 cm−<sup>1</sup> and the logarithm of the concentration of Cr6+ (Figure 4d). The calibration curve showed a good linear relationship in the range of 5~100 μg/L (R2 = 0.99863). The linear quantitative relationship was described by the equation y = −32,207.76X + 67,161.47. Furthermore, the limit of detection (LOD) was

defined as the concentration of Cr6+, resulting in a 3% decrease in Raman intensity related to the blank tea sample (0 μg/L) [28]. According to the established equation, the LOD was calculated to be 3.78 μg/L, indicating that the immunosensor had a good sensitivity. Due to the dilution factor of 250 times in the pretreatment of tea samples, the detection range of Cr6+ in tea sample was 1.25~25 mg/kg, with a LOD of 0.945 mg/kg, which was much lower than the recommended tolerable level (5 mg/kg) of Cr6+ in tea.
