All reagents were purchased commercially and further purified when used. All the reactions involved were monitored by thin-layer chromatography (TLC) and analyzed with ultraviolet lamps at 254 and 365 nm. Products were separated and purified by column chromatography (200–300 mesh silica gel). NMR were recorded by Bruker avance II instrument in deuterium chloroform (400 MHz for ¹H and 100 MHz for ¹³C). Chemical shifts are reported in ppm, versus internal tetramethylsilane (TMS) as a standard. The mass spectrum was obtained on a Thermo LXQ by liquid chromatgraphy-ion trap mass spectrometry. Absorption spectra were recorded with a UV-2550 spectrophotometer. The fluorescence emission spectra were measured by Shimazu RF-5301PCS fluorescence spectrophotometer with excitation wavelength of 500 nm. The molecular weight by MS-ESI (Jeol LTD JMS-HX 110/110A) was performed.

Ultraviolet-visible (UV-vis) spectra were recorded using a 1 cm quartz tube on a UV-2550 spectrophotometer, and fluorescence spectra were performed at room temperature on a Shimadzu RF-5301PCS fluorescence spectrophotometer. The making phosphate buffers with pH values of 3.0, and 4.0 each buffer was mixed with different equivalents of amino acids. Appropriate amount of RBTPA to be measured was weighed and dissolved in EtOH to prepare a solution with a concentration of 20 μg/mL for use. Different concentrations of amino acids in solvents were detected by ultraviolet and fluorescence methods. The wavelength range of UV-Vis is 450–700 nm. The fluorescence of the compound was obtained at the optical path of 10 mm and the excitation wavelength of 560 nm, and the emission wavelength was recorded in the range of 400–700 nm.

The fluorescence stability data was determined using a Japanese Shimadzu RF-5301PC fluorescence spectrophotometer, DMSO was used as the solvent, and the sample concentration was continuously diluted with DMSO until the fluorescence intensity of the RBTPA remained stable. Fluorescence intensity stability over 5 min and 10 min was measured, respectively.

The mass spectrums were obtained at the Thermo LXQ by liquid chromatgraphy-ion trap mass spectrometry. To verify this mechanism, a random selection of RBTPA solutions containing 1.0 mg/mL of different amino acid solutions was performed by mass spectrometry to confirm our proposed mechanism.

The design concept of the amino acid fluorescence probe in this paper is to consider the double response effect. Triphenylamino aldehyde, the most widely used luminescent electron donor in the semiconductor field, is selected as the terminal binding point, and the fluorophore rhodamine B is introduced. This design not only greatly reduces the fluorescence quenching problem of the probe, but also has the rapid response function in the acid environment in the overall structure, the best of both worlds (Scheme 1).

The rhodamine B structure’s iminopentane nitrogen heterocyclic ring is easily opened, and the pentane nitrogen heterocyclic ring is extremely unstable, forming an open ring rhodamine B structure and releasing strong fluorescence when the external electron is combined with imine through the Michael addition reaction. Furthermore, the conjugation of the big link is enhanced and given a high fluorescence effect and stability when triphenylamino aldehyde is combined with amino acids. Due to the change of the overall structure, different amino acid compounds produce different color changes under the irradiation of 365 nm wavelength, and the qualitative analysis of their mass spectrometry was determined by LC-MS, so as to detect the identification of different amino acids (Figure 1).

Continuous testing from 0 to 40 equivalents were conducted in order to examine the variations of RBTPA with different equivalents and different amino acids at pH = 3 and 4 in ethanol solvents. As can be seen in Figure 2, RBTPA’s blank absorption wavelength is 562 nm at pH = 3. The absorption strength of RBTPA is marginally decreased when mixed with other amino acids, yet it exhibits a clear wavelength redshift of 665 nm, with a gap of approximately 100 nm between the blank and control groups. The blank group and the control group showed no change in absorption wavelength at pH = 4, but there was a considerable drop in absorption intensity to around 0.8, with the total difference remaining at the same level. The outcomes demonstrated that the fluorescence impact and amino acid double response binding capacity improved with increasing acidity of RBTPA. Conversely, at high pH values, the reaction is less and fluorescence quenching occurs (Figures 2, 3) (Table 1).

Figures 4, 5 show that the total fluorescence spectrum’s wavelength range remains largely constant and that the outcomes are comparable. The intensity of the fluorescence emission is clearly visible at pH = 3, and it is essentially between 1,150 and 1,750, with histamine having the lowest intensity. The intensity of the fluorescence emission is essentially between 400 and 500 at pH = 4. The best effect occurs at pH = 3, and the emission intensity is roughly three times different from the former, which is compatible with the aforementioned UV spectrum data (Figures 4, 5).

Compared with the above four figures, there are obvious curve changes in Figures 2, 3. Each of the corresponding amino acids showed strong reactivity with RBTPA, indicating a response effect between them. Although the fluctuation decline rate of the overall curve is somewhat obvious, there are differences in the absorption intensity. In particular, it shows strong responsiveness at pH = 3 (Table 2).

By comparing the color response recognition of five different amino acids and RBTPA, it can be observed that at pH = 3, the color of RBTPA is opaque except for tryptophan at daylight and 365 nm, and the others are transparent pink or dark red. The color of the blank sample is bright yellow. The results showed that the double response with imino and aldehyde group of RBTPA resulted in color change under the strong acid environment of pH = 3. On the other hand, at pH = 4, the color response recognition of five different amino acids and RBTPA is somewhat dim compared to the former, and there is almost no bright luster at daylight and 365 nm. The results show that one part of RBTPA reacts with imino and aldehyde group in double response, while the other part directly generates acid salt, which affects the fluorescence intensity. These phenomena are in complete agreement with the ultraviolet absorption and fluorescence emission intensity data explained above (Figure 6).

To observe the stability of RBTPA, a test was performed for 5–10 min at pH = 3 and 4 strong acids. It can be seen from Figure 7 that the two lines maintain a relatively flat state, indicating that the compound RBTPA does not change under the condition of strong acid, and is a stable fluorescent probe (Figure 7).

In order to further determine the binding of RBTPA to different amino acids in acidic environments for qualitative analysis, each response reaction solution was tested in detail by mass spectrometry. It can be seen from Figure 8 that the mass spectra of tyrosine and phenylalanine are 943.41, 905.43, 905.43, and 889.44, respectively. It can be analyzed as (M + Na⁺), (M + H⁺), (M + H⁺), and (M + H⁺) respectively. By analogy method, cysteine and can be resolved as respectively histidine (M + Na⁺), (M + Na⁺), (M + H⁺) and (M + Na⁺); tryptophan can be resolved as (M + H⁺) and (M + H⁺).