All of these reagents were of analytical grade, and all aqueous solutions were prepared with Milli-Q water (>18.2 MΩ⋅cm). 3,3′,5,5′-Tetramethylbenzidine (TMB) and hydrogen peroxide (H₂O₂) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Cu(NO₃)₂·3H₂O was obtained from Tianjin Bodi Chemical Industry Co. Ltd. (Tianjin, China). Na₂HPO₄·12H₂O, NaH₂PO₄·2H₂O, NaCl, and glucose were all purchased from Tianjin Best Chemical Co. Ltd. (Tianjin, China). L-alanine (L-Ala), L-phenylalanine (L-Phe), L-proline (L-Pro), L-histidine (L-His), and urea were all purchased from Beijing Solaibao Technology Co. Ltd. (Beijing, China). The pH of the solution was measured with a PB-10 pH meter (Sartorius, 91 Germany). UV-Vis absorption spectroscopic measurements were carried out on a TU-1900 spectrophotometer (Beijing Pu Analysis General Instrument Co., Ltd.) with an optical path length of 10 mm.

The coordination-driven chemistry of L-His and Cu²⁺ ions was studied for colorimetric reaction. First, the TMB–H₂O₂ system was constructed. Two hundred microliters of 420 μM Cu²⁺ions, 40 μl of 160 mM TMB, and 40 μl of 300 mM H₂O₂ were added to 2 ml of phosphate buffer saline (PBS) solution (C = 0.1 M, pH = 5.7) in the absence and presence of 200 μl of 80 μM L-His. The mixture was incubated at 45°C for 20 min. Finally, the adsorption spectrum of the mixture was measured on UV-Vis spectrophotometer equipped with 1-cm path length quartz cuvettes.

The coordination-driven chemistry of L-His and Cu²⁺ ions was dramatically influenced by pH. We evaluated the effect of pH on the colorimetric reaction of the TMB–H₂O₂ system. Different pH of phosphate buffer saline (PBS) solution (C = 0.1 M) were prepared, including 4.0, 5.0, 5.3, 5.7, 6.0, 7.0, 8.0, and 9.0. Then, 40 μl of 160 mM TMB and 40 μl of 300 mM H₂O₂ were added to 2 ml of phosphate buffer saline (PBS) solution with various pH.

Then various concentrations of L-His and equal concentration of Cu²⁺ ions were incubated in PBS buffer solution (pH 5.7) at room temperature for 10 min, and the mixture was added into the TMB–H₂O₂ system. The final concentration of Cu²⁺ was 30 μM with various concentration of L-His. The mixture was incubated at 45°C for 20 min and then measured by UV-vis.

Meanwhile, the effect of interferents on the detection system was investigated with the above conditions with the substitution of L-His into the interferents. As urine detection probe, L-Ala, L-Phe, L-Pro, glucose, NaCl, and urea, as main constituents of urine, were evaluated as interferents (Sadighi et al., 2006; Dutta et al., 2014; Zheng et al., 2015). Besides, the main molecules were all added into the solution of PBS (c = 0.1 M, pH 7.4) to prepare the simulated urine for recovery testing.

Our biosensing system consisted of two critical reactions: the coordination-driven chemistry of L-His and Cu²⁺ ions and the colorimetric TMB–H₂O₂ system (Figure 1). TMB, which served as a colorimetric probe, was oxidized by H₂O₂ to oxTMB in the presence of Cu²⁺ ions. During this process, Cu²⁺ ions presented intrinsic catalytic activity for the oxidation of TMB (colorless) to oxTMB (blue) in the presence of H₂O₂ (Figure 1A). Upon adding L-His to the biosensing system, the amino groups and hydroxyl groups of L-His chelated with Cu²⁺ ions and formed stable L-His–Cu²⁺ complexes (Zhang et al., 2013). Hence, L-His inhibited the catalytic activity of Cu²⁺ for the oxidation of TMB in the presence of H₂O₂, and the color of the TMB–H₂O₂ system did not change significantly (Figure 1B). Therefore, the change in color and decrease in absorbance were directly related to the L-His concentration. During the detecting process for the detecting system, the change in color and the decrease in absorbance were both taken as the detection signal of L-His. Hence, we constructed a colorimetric L-His detection sensor based on this mechanism.

To validate the feasibility of the colorimetric sensor for L-His detection, we compared the color and UV-vis spectra of a pristine state of the probe with those of the probe after exposing the TMB–H₂O₂ system to L-His. The color of the TMB-H₂O₂ was blue, and the highest peak in the UV-vis spectrum of the probe in the presence of Cu²⁺ ions was observed at 652 nm (Figure 1C). The color of the TMB–H₂O₂ system changed to light blue in the presence of L-His, indicating that the coordination-driven chemistry of L-His and Cu²⁺ ions affected the oxidation of the TMB–H₂O₂ system. Infrared spectrometry (IR) analysis was utilized to confirm the coordination-driven chemistry of L-His and Cu²⁺ ions, and the results indicated that the proposed method was suitable for L-His detection.

Because the colorimetric reaction and the coordination of Cu²⁺ ions with L-His were significantly affected by pH, we first evaluated the effects of reaction pH on L-His detection (Figure 2). The colorimetric reaction of the TMB–H₂O₂ system was significantly affected by pH (Figure 2A). The color of the TMB–H₂O₂ system was blue at pH 4, 5, and 6 and changed to yellow and colorless with the increasing pH to 7, 8, and 9. This indicated that the optimum pH for colorimetric reaction ranged between 4 and 6. Therefore, we subsequently evaluated the effect of pH in the range of 4–6 on the detection assay (Figures 2B–G). The color-changing rate on the TMB–H₂O₂ system increased rapidly with increasing pH, reached a plateau at pH >5.7, and then it decreased. The UV-vis absorbances of the highest peak (λ ₆₅₅) in the absence and presence of L-His were defined as A₀ and A, respectively. The UV-vis spectra indicated that the absorbance-changing rate of the TMB–H₂O₂ system [(A₀-A)/A₀] was the highest at pH is 5.7 (Figure 2H). Therefore, pH 5.7 was used as the optimum pH value of the assay buffer of the sensing system.

Owing to the catalytic effect of Cu²⁺ ions on the TMB–H₂O₂ system and the chelating interaction of Cu²⁺ ions and L-His, an assay for rapid and simple L-His detection was fabricated. The change in absorbance at 652 nm of the TMB–H₂O₂ system with increasing L-His concentration was analyzed under the optimized experimental conditions (Figure 3A). The absorbance of sensing system decreased with the addition of L-His. Moreover, the photographs of the corresponding solution color were inserted into Figure 3A. Upon increasing L-His concentration, the color of the TMB–H₂O₂ system changed from deep blue to colorless. Furthermore, the containment level of 10 μM L-His can be clearly distinguished with the naked eye. In addition (A₀-A)/A₀ depended on the concentration of L-His and increased linearly upon increasing L-His concentration in the range of 60 nM to 1 μM and 1 μM to 1 mM, respectively (inset in Figure 3B). The limit of detection of the sensing system was 50 nM (S/N = 3), and the linear equations describing the dependence of (A₀-A)/A₀ on the L-His concentration in the aforementioned L-His concentration ranges were y = 22.5 × −52.6 and y = 10.4 × −4.3, respectively (regression coefficient of 0.976 and 0.969, respectively).

To effectively exhibit the proposed method, the method was listed to compare with other methods (colorimetric and fluorescent platform based on nanomaterials) in Table 1. Compared with other colorimetric or fluorescent assay, the colorimetric strategy in this study has lower detection and wider analytical range.

The selectivity of the proposed colorimetric sensing system should be evaluated for real or simulated samples because real samples are complex. In particular, amino acids present in real samples can interfere with the quantitative analysis of L-His because their properties and structure are similar to those of L-His. Therefore, we selected L-alanine, L-proline, and L-phenylalanine as interferents to be evaluated because they are the primary components of urine. Meanwhile, the effect of glucose, urea, and NaCl were also studied because they are also the main component in urine (Figure 4). The change of absorbance and (A₀-A)/A₀ were presented in Figures 4A,B, respectively. Because the tested amino acids and biomolecules did not present significant signals, we concluded that our platform presented good selectivity for L-His, which is the most distinct difference in the structure between L-His and other amino acids. Other amino acids cannot chelate interaction with Cu²⁺ ions and have no effect on the colorimetric reaction of the TMB–H₂O₂ system.

To illustrate the practical application of colorimetric sensor for L-His detection, the simulated urine was prepared according to the previous literature (Sadighi et al., 2006; Dutta et al., 2014; Zheng et al., 2015). Briefly, L-Ala, L-Phe, L-Pro, glucose, NaCl, and urea were all added into the solution of PBS (c = 0.1 M, pH 7.4). Besides, different L-His concentrations were also added into the simulated urine. We used the standard addition method to obtain the L-His concentrations of simulated samples (Table 2). The recovery values were 102.0%, 95.3%, and 98.5%, respectively. Meanwhile, the relative standard deviations (RSDs) were 3.8, 4.2, and 2.1. The recovery values were between 95.3% and 102.0%, and the RSDs were no more than 4.2%. These results demonstrated that the proposed colorimetric sensor had a promising application for glyphosate detection in real samples.