A fluorescence colorimetric sensor based on L-Trp@AuNCs, its fabrication method and application

Abstract

This invention discloses a fluorescence colorimetric sensor based on L-Trp@AuNCs, its preparation method, and its application, belonging to the field of detection technology. The L-Trp@AuNCs-based fluorescence colorimetric sensor is prepared by mixing HAc-NaAc buffer, L-Trp@AuNCs, H2O2, and TMB. Furthermore, this invention proposes a preparation method for the aforementioned L-Trp@AuNCs-based fluorescence colorimetric sensor, comprising: mixing HAc-NaAc buffer, L-Trp@AuNCs, H2O2, and TMB to obtain the fluorescence colorimetric sensor. In addition, this invention proposes the application of the aforementioned ratiometric fluorescence sensor or the ratiometric fluorescence sensor prepared by the aforementioned method in the detection of tetracycline antibiotics. The fluorescence colorimetric sensor proposed in this invention exhibits a wider linear range and a lower detection limit in both its colorimetric and fluorescence modes.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically to a fluorescence colorimetric sensor based on L-Trp@AuNCs, its preparation method, and its application. Background Technology

[0002] L-Trp@AuNCs, or tryptophan-modified gold nanoclusters, have attracted considerable attention in the field of materials science. During their preparation, specific functional groups of L-Trp interact with gold ions, forming stable structures through reduction and other methods. They possess unique optical properties, with fluorescence emission peaks located within a specific wavelength range, and exhibit superior stability compared to ordinary gold nanoparticles. In biosensing, L-Trp@AuNCs can serve as sensitive probes for the detection of specific biomolecules and metal ions, achieving highly selective recognition based on changes in fluorescence signals. In bioimaging, their excellent biocompatibility enables clear imaging of cells and tissues, aiding in early disease diagnosis and pathological research. Future breakthroughs in more fields are expected.

[0003] How to detect TCs using L-Trp@AuNCs is a technical problem that needs to be solved by existing technologies. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide a fluorescence colorimetric sensor based on L-Trp@AuNCs, its preparation method and application, thereby solving the technical problem of how to detect TCs using L-Trp@AuNCs in the prior art.

[0005] To achieve the above-mentioned technical objectives, the present invention provides a fluorescence colorimetric sensor based on L-Trp@AuNCs, which is obtained by mixing HAc-NaAc buffer, L-Trp@AuNCs, H2O2 and TMB.

[0006] In any embodiment, the concentration of H2O2 is 0.8-1.0 M, and the concentration of TMB is 1.0-2.5 mM.

[0007] Furthermore, the present invention also proposes a method for preparing the above-mentioned fluorescence colorimetric sensor based on L-Trp@AuNCs, comprising: mixing HAc-NaAc buffer, L-Trp@AuNCs, H2O2 and TMB to obtain the fluorescence colorimetric sensor.

[0008] In any embodiment, the L-Trp@AuNCs are prepared by the following steps: stirring L-Trp solution and HAuCl4 at 95-100°C, then centrifuging to collect the supernatant, and then filtering.

[0009] Furthermore, the present invention also proposes the application of the above-described ratio fluorescence sensor or the ratio fluorescence sensor prepared by the above-described method in the detection of tetracycline antibiotics.

[0010] In any embodiment, the above application includes: adding H2O2, TMB and the test solution to HAc-NaAc buffer and reacting, then adding concentrated H2SO4 to terminate the reaction, and then measuring the absorbance of the solution at 450 nm and the fluorescence intensity at 400 nm at an excitation wavelength of 290 nm using an ELISA reader.

[0011] In any embodiment, the reaction time for adding TMB to the HAc-NaAc buffer is 6-8 min; and / or the reaction time for adding the test solution is 5-10 min.

[0012] In any embodiment, the tetracycline antibiotic is one or more of TC, OTC, and CTC.

[0013] In any implementation, the linear regression equations for the colorimetric and fluorescence patterns of TC are Y = 0.061X + 1.899 (R²). 2 = 0.991) and Y = 0.251X + 1.389(R) 2 = 0.997); the linear regression equations for the colorimetric and fluorescence patterns of OTC drugs are Y = 0.401X + 1.001 (R² = 0.997). 2 = 0.991) and Y = 0.097X + 2.090(R 2 = 0.991); the linear regression equations for the colorimetric and fluorescence patterns of CTC are Y = 0.055X + 1.968 (R = 0.991). 2 = 0.996) and Y = 0.206X + 1.461 (R 2 = 0.992); In colorimetric mode, X represents the concentration of TC, OTC or CTC, and Y represents the ratio A / A0 of the absorbance of the solution at 450 nm to that of the blank solution at 450 nm. In fluorescence mode, X represents the concentration of TC, OTC or CTC, and Y represents the ratio F0 / F of the fluorescence intensity of the blank solution at 400 nm under an excitation wavelength of 290 nm to that of the solution at 400 nm under an excitation wavelength of 290 nm. Substitute the absorbance or fluorescence intensity of the test solution into the corresponding formula to obtain the concentration value of the corresponding TCs.

[0014] Compared with existing technologies, the advantages of this invention include: The fluorescence colorimetric sensor based on L-Trp@AuNCs proposed in this invention is prepared by mixing HAc-NaAc buffer, L-Trp@AuNCs, H2O2, and TMB. This fluorescence colorimetric sensor can be used for the detection of TCs in various samples. The colorimetric and fluorescence modes of the fluorescence colorimetric sensor proposed in this invention have a wider linear range and a lower detection limit. The fluorescence colorimetric sensor proposed in this invention achieves both dual signal output and highly sensitive and rapid detection of TCs, and the detection process can be completed in just over ten minutes. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the principle of L-Trp@AuNCs dual-mode detection of TCs in this invention.

[0016] Figure 2 These are TEM images of L-Trp@AuNCs in Embodiment 1 of the present invention; wherein, (A) is a TEM image of L-Trp@AuNCs; and (B) is a TEM image of L-Trp@AuNCs + TCs.

[0017] Figure 3 The fluorescence spectra of L-Trp@AuNCs prepared in Example 1 of this invention are shown below; (A) fluorescence spectrum of L-Trp@AuNCs; (B) fluorescence spectrum of L-Trp@AuNCs under different treatments.

[0018] Figure 4 These are the ultraviolet absorption and fluorescence spectra of L-Trp@AuNCs prepared in Example 1 of the present invention; wherein, (A) are the ultraviolet absorption spectra of L-Trp@AuNCs under different treatments; and (B) are the fluorescence spectra of L-Trp@AuNCs under different treatments.

[0019] Figure 5 This is a diagram showing the feasibility analysis results of Example 1 of the present invention; wherein, (A) ultraviolet absorption spectra under different treatments, and inset: color changes of the solution under different treatments; (B) fluorescence spectra under different treatments.

[0020] Figure 6 This is a diagram showing the volume optimization results of L-Trp@AuNCs in Embodiment 1 of the present invention.

[0021] Figure 7 This is a graph showing the optimized H2O2 concentration results of Example 1 of the present invention.

[0022] Figure 8 This is a graph showing the optimization results of the TMB concentration of the fluorescence colorimetric sensor in Embodiment 1 of the present invention.

[0023] Figure 9This is a graph showing the optimized incubation time of TCs for the fluorescence colorimetric sensor of Embodiment 1 of the present invention.

[0024] Figure 10 This is a graph showing the optimized TMB reaction time of the fluorescence colorimetric sensor according to Embodiment 1 of the present invention.

[0025] Figure 11 This is a graph showing the changes in A / A0 and F0 / F under different TC concentrations in Example 1 of the present invention; wherein, (A) A / A0 under different TC concentrations, inset: linear graph in the range of 1.5-24 μM; (B) Changes in F0 / F under different TC concentrations, inset: linear graph in the range of 0.375-37.5 μM.

[0026] Figure 12 This is a graph showing the changes in A / A0 and F0 / F under different OTC concentrations in Example 1 of the present invention; wherein, (A) A / A0 under different OTC concentrations, inset: linear graph in the range of 0-7.5 μM; (B) Changes in F0 / F under different OTC concentrations, inset: linear graph in the range of 1.5-60 μM.

[0027] Figure 13 The graphs show the changes in A / A0 and F0 / F under different CTC concentrations in Example 1 of the present invention; (A) A / A0 under different CTC concentrations, inset: linear graph in the range of 1.5-30 μM; (B) Changes in F0 / F under different CTC concentrations, inset: linear graph in the range of 0.375-37.5 μM.

[0028] Figure 14 This is a specific analysis result diagram of the fluorescence colorimetric sensor of Embodiment 1 of the present invention, wherein (A) is the colorimetric mode and (B) is the fluorescence mode.

[0029] Figure 15 This is a graph showing the stability analysis results of L-Trp@AuNCs in Example 1 of this invention. (A) Fluorescence stability; (B) Absorption stability.

[0030] Figure 16 The diagram shows the results of TC stability analysis in Example 1 of this invention; where (A) is the colorimetric stability analysis result of TC in tap water, (B) is the fluorescence stability analysis result of TC in tap water, (C) is the colorimetric stability analysis result of TC in honey, and (D) is the fluorescence stability analysis result of TC in honey.

[0031] Figure 17The figure shows the results of OTC stability analysis in Example 1 of the present invention; wherein, (A) colorimetric stability analysis results of OTC in tap water, (B) fluorescence stability analysis results of OTC in tap water, (C) colorimetric stability analysis results of OTC in honey, and (D) fluorescence stability analysis results of OTC in honey.

[0032] Figure 18 The figure shows the results of CTC stability analysis in Example 1 of the present invention; wherein, (A) is the colorimetric stability analysis result of CTC in tap water, (B) is the fluorescence stability analysis result of CTC in tap water, (C) is the colorimetric stability analysis result of CTC in honey, and (D) is the fluorescence stability analysis result of CTC in honey. Detailed Implementation

[0033] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60~120 and 80~110 are listed for a specific parameter, it is also expected that ranges of 60~110 and 80~120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1~3, 1~4, 1~5, 2~3, 2~4, and 2~5. In this application, unless otherwise stated, the numerical range "a~b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0034] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0035] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0036] This specific embodiment provides a fluorescence colorimetric sensor based on L-Trp@AuNCs, which is obtained by mixing HAc-NaAc buffer, L-Trp@AuNCs, H2O2 and TMB, wherein the concentration of H2O2 is 0.8-1.0 M and the concentration of TMB is 1.0-2.5 mM.

[0037] This specific embodiment also proposes a method for preparing the above-mentioned fluorescence colorimetric sensor based on L-Trp@AuNCs, including: mixing HAc-NaAc buffer, L-Trp@AuNCs, H2O2 and TMB to obtain the fluorescence colorimetric sensor; the L-Trp@AuNCs are prepared by the following steps: stirring L-Trp solution and HAuCl4 at 95-100℃, then centrifuging to collect the supernatant, and then filtering.

[0038] This specific embodiment also proposes the application of the above-mentioned ratio fluorescence sensor or the ratio fluorescence sensor prepared by the above-mentioned method in the detection of tetracycline antibiotics, wherein the tetracycline antibiotics are one or more of TC, OTC and CTC.

[0039] In some embodiments, the above application includes: adding H2O2, TMB and the test solution to HAc-NaAc buffer and reacting, then adding concentrated H2SO4 to terminate the reaction, and then measuring the absorbance of the solution at 450 nm and the fluorescence intensity at 400 nm at an excitation wavelength of 290 nm using an ELISA reader.

[0040] In some embodiments, the reaction time for adding TMB to the HAc-NaAc buffer is 6-8 min; the reaction time for adding the test solution is 5-10 min.

[0041] In some embodiments, the linear regression equations for the colorimetric and fluorescence patterns of TC are Y = 0.061X + 1.899(R² + π²) / π². 2 = 0.991) and Y = 0.251X + 1.389(R) 2 = 0.997); the linear regression equations for the colorimetric and fluorescence patterns of OTC drugs are Y = 0.401X + 1.001 (R² = 0.997). 2 = 0.991) and Y = 0.097X + 2.090(R 2 = 0.991); the linear regression equations for the colorimetric and fluorescence patterns of CTC are Y = 0.055X + 1.968 (R = 0.991). 2 = 0.996) and Y = 0.206X + 1.461 (R = 0.996) and Y = 0.206X + 1.461 (R 2=0.992); In colorimetric mode, X represents the concentration of TC, OTC or CTC, and Y represents the ratio of the absorbance of the solution to the absorbance of the blank solution, A / A0. In fluorescence mode, X represents the concentration of TC, OTC or CTC, and Y represents the ratio of the fluorescence intensity of the solution to the fluorescence intensity of the blank solution, F0 / F. Substitute the absorbance or fluorescence intensity of the test solution into the corresponding formula to obtain the concentration value of the corresponding TCs.

[0042] The mechanism of action of L-Trp@AuNCs involves the proximity of H2O2 molecules to the L-Trp@AuNCs surface. The active sites of the gold nanoclusters interact with the oxygen atoms of H2O2, weakening the OO bonds in H2O2. Subsequently, the functional groups of L-Trp assist in substrate localization and promote electron transfer through electrostatic interactions or hydrogen bonding. The gold nanoclusters act as electron transport mediators, transferring electrons from the substrate to H2O2, causing it to decompose into reactive oxygen species, such as hydroxyl radicals. These reactive oxygen species possess strong oxidizing properties and can further oxidize the substrate, for example, oxidizing colorless TMB to blue oxTMB, thus exhibiting peroxidase-like activity. TCs possess a unique conjugated polycyclic structure, which endows them with special electronic properties, enabling them to synergistically interact with L-Trp@AuNCs and significantly enhance their peroxidase-like activity.

[0043] Combination Figure 1 Figure A shows the synthesis of L-Trp@AuNCs and how TCs can induce the aggregation of L-Trp@AuNCs; Figure B shows how TMB can enhance the fluorescence of L-Trp@AuNCs; and Figure C illustrates the experimental principle: without the target compound TCs, the oxidative activity of L-Trp@AuNCs is relatively weak and cannot oxidize TMB to produce color. The addition of TMB enhances the fluorescence of L-Trp@AuNCs, resulting in a lower color (absorbance) and a stronger fluorescence signal. When the target compound TCs are added, TCs can cause L-Trp@AuNCs to aggregate. The aggregated L-Trp@AuNCs have significantly enhanced oxidative activity, allowing them to oxidize TMB to oxTMB. oxTMB then rapidly transforms into a yellow diimine salt compound under acidic conditions. Because the absorption spectrum of the diimine salt overlaps significantly with the fluorescence emission spectrum of L-Trp@AuNCs, the fluorescence of L-Trp@AuNCs is quenched. As a result, the signal output shows enhanced color (absorbance) and weakened fluorescence signal.

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0045] In this invention, the terms "some embodiments," "this embodiment," and examples are used to describe a subset of all possible embodiments. However, it is understood that "some embodiments" can be the same subset or different subsets of all possible embodiments and can be combined with each other without conflict.

[0046] If the application documents contain similar descriptions such as "first / second", the following explanation shall be added: In the following description, the terms "first / second / third" are used only to distinguish similar objects and do not represent a specific ordering of objects. It is understood that "first / second / third" may be interchanged in a specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0047] In this embodiment, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, object A and / or object B can represent three situations: object A exists alone, object A and object B exist simultaneously, and object B exists alone.

[0048] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0049] The instruments used in the following embodiments are shown in Table 1.

[0050] Table 1 The main reagents used in the following examples are shown in Table 2.

[0051] Table 2 Example 1

[0052] Solution preparation (1) TMB solution (15 mM): Weigh 0.7210 g TMB and dilute to 200 mL according to the ratio of DMSO to distilled water of 4:6.

[0053] (2) NaAc-HAc buffer (50 mM, pH 4.0): Mix 0.2 M 180 mL NaAc solution and 0.2 M 820 mL HAc solution and adjust the pH to 4.0.

[0054] (3) Concentrated sulfuric acid (2 M): Take 55 mL of concentrated sulfuric acid and dilute it to 500 mL with distilled water.

[0055] Preparation of L-Trp@AuNCs: First, 2 mL of 80 mM L-Trp solution and 8 mL of 6.0 mM HAuCl4 were stirred at 100 °C for 3 hours. Then, the supernatant was collected after centrifugation at 13751 g for 15 minutes and centrifuged three times. Finally, the solution was simply filtered through a 0.22 μm filter and stored at 4 °C.

[0056] Experimental feasibility verification In this embodiment, the colorimetric signal output is triggered by the oxidation of TMB by L-Trp@AuNCs under H2O2 catalysis. The conjugated polycyclic structure of TCs enhances the peroxidase-like activity of L-Trp@AuNCs, accelerating the TMB oxidation process. To increase the colorimetric sensitivity, concentrated sulfuric acid is added to the solution, converting oxTMB into a diimine salt, which exhibits a strong UV absorption peak at 450 nm. To verify the feasibility of the colorimetric signal output in this experiment, two sets of experiments were set up. After incubation at 37℃ for 15 min in both sets of experiments, the UV absorption spectra of the two sets of samples in the range of 330-550 nm were measured using a microplate reader. In this method, the fluorescence signal output is achieved by the conversion of oxTMB into a diimine salt under acidic conditions, which quenches the fluorescence emission spectrum of L-Trp@AuNCs at 400 nm. To verify the feasibility of the fluorescence signal output experiment, the following two sets of experiments were set up. After incubation at 37℃ for 15 min in both sets of experiments, the fluorescence emission spectra of the two sets of samples in the wavelength range of 330-550 nm under an excitation wavelength of 290 nm were measured using a microplate reader: (1) 10 μL L-Trp@AuNCs + 20 μL H2O2 (10 M) + 20 μL TMB (20 mM) + 10 μL H2SO4 (2M).

[0057] (2) 10 μL L-Trp@AuNCs + 20 μL H2O2 (10 M) + 20 μL TMB (20 mM) + 60 μLTCs (100 μM) + 10 μL H2SO4 (2M).

[0058] Optimization of experimental conditions L-Trp@AuNCs size optimization In the microplate, six volume gradients of L-Trp@AuNCs were set up with volumes of 2, 6, 10, 14, 18, and 22 μL of L-Trp@AuNCs. Then, L-Trp@AuNCs, 10 μL of 10 M H₂O₂, 20 μL of 20 mM TMB, and 60 μL of 100 μM TCs were added to 50 mM pH 4.0 HAc-NaAc buffer. Specifically, the TCs and L-Trp@AuNCs were reacted together first, then H₂O₂ and TMB were added for oxidation and color development. Finally, 10 μL of 2 M H₂SO₄ was added to terminate the reaction, resulting in a final volume of 200 μL. In the blank solution, the same volume of ultrapure water was used instead of TCs, and four parallel controls were set up for each data set. The solution was incubated at 37°C for 15 minutes, and the absorbance at 450 nm and the fluorescence intensity at 400 nm (excitation wavelength of 290 nm) were measured using a microplate reader. In this experiment, the absorbance at 450 nm of the solution containing TCs was denoted as A, and the fluorescence intensity at 400 nm was denoted as F; the absorbance at 450 nm of the blank solution was denoted as A0, and the fluorescence intensity at 400 nm was denoted as F0. The ratio of absorbance (A / A0) and fluorescence intensity (F0 / F) were used to represent the amount of diimine salt generated and the quenching effect of diimine salt on the fluorescence of L-Trp@AuNCs, respectively. The optimal volume of the L-Trp@AuNCs solution was selected when the A / A0 and F0 / F values ​​were maximized or when the A / A0 and F0 / F values ​​reached a plateau.

[0059] H2O2 concentration optimization In the microplate, six H2O2 concentration gradients were set up with final H2O2 concentrations of 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 M. Then, 10 μL of L-Trp@AuNCs, H2O2, 20 μL of 20 mM TMB, and 60 μL of 100 μM TCs were added to 50 mM pH 4.0 HAc-NaAc buffer. Specifically, TCs and L-Trp@AuNCs were reacted together first, then H2O2 and TMB were added for oxidation and color development. Finally, 10 μL of 2 M H2SO4 was added to terminate the reaction, resulting in a final system volume of 200 μL. In the blank solution, the same volume of ultrapure water was used instead of TCs, and four parallel controls were set up for each data set. The solution was incubated at 37°C for 15 minutes, and the absorbance at 450 nm and the fluorescence intensity at 400 nm (excitation wavelength of 290 nm) were measured using a microplate reader. In this experiment, the absorbance at 450 nm of the solution containing TCs was denoted as A, and the fluorescence intensity at 400 nm was denoted as F; the absorbance at 450 nm of the blank solution was denoted as A0, and the fluorescence intensity at 400 nm was denoted as F0. The ratio of absorbance (A / A0) and fluorescence intensity (F0 / F) were used to represent the amount of diimine salt generated and the quenching effect of diimine salt on the fluorescence of L-Trp@AuNCs, respectively. The optimal concentration of H2O2 was selected when the A / A0 and F0 / F values ​​were maximized or when the A / A0 and F0 / F values ​​reached a plateau.

[0060] TMB concentration optimization Six TMB concentration gradients were set up in the ELISA plate, with final TMB concentrations of 0.25, 0.5, 1.0, 1.5, 2.0, and 2.5 mM, respectively. Then, 10 μL of L-Trp@AuNCs, 10 μL of 9 M H₂O₂, TMB, and 60 μL of 100 μM TCs were added to 50 mM pH 4.0 HAc-NaAc buffer. Specifically, TCs and L-Trp@AuNCs were reacted together first, then H₂O₂ and TMB were added for oxidation and color development. Finally, 10 μL of 2 M H₂SO₄ was added to terminate the reaction, resulting in a final system volume of 200 μL. In the blank solution, the same volume of ultrapure water was used instead of TCs, and four parallel controls were set up for each data set. The plates were incubated at 37°C for 15 minutes, and the absorbance at 450 nm and the fluorescence intensity at 400 nm (excitation wavelength of 290 nm) were measured using an ELISA reader. In this experiment, the absorbance of the solution containing TCs at 450 nm is represented by A, and the fluorescence intensity of the solution at 400 nm is represented by F. The absorbance of the blank solution at 450 nm is represented by A0, and the fluorescence intensity of the blank solution at 400 nm is represented by F0. The ratio of absorbance A / A0 and the ratio of fluorescence intensity F0 / F represent the amount of diimine salt generated and the effect of diimine salt quenching on the fluorescence of L-Trp@AuNCs, respectively. The optimal concentration of TMB is selected when the A / A0 and F0 / F values ​​are maximized or when the A / A0 and F0 / F values ​​reach a plateau.

[0061] Time optimization (1) Optimization of TC incubation time In the microplate, six TC incubation time gradients were set up with incubation times of 0, 5, 10, 15, 20, and 25 min for TCs (i.e., the reaction time between TCs and L-Trp@AuNCs). Then, 10 μL of L-Trp@AuNCs, 10 μL of 9 M H2O2, 20 μL of 20 mM TMB, and 60 μL of 100 μM TCs were added to 50 mM pH 4.0 HAc-NaAc buffer. Specifically, TCs and L-Trp@AuNCs were reacted together first, then H2O2 and TMB were added for oxidation and color development, and finally 10 μL of 2 MH2SO4 was added to terminate the reaction, resulting in a final system volume of 200 μL. In the blank solution, the same volume of ultrapure water was used instead of TCs, and four parallel controls were set up for each data set. The solution was incubated at 37°C for 15 minutes, and the absorbance at 450 nm and the fluorescence intensity at 400 nm (excitation wavelength of 290 nm) were measured using a microplate reader. In this experiment, the absorbance at 450 nm of the solution containing TCs was denoted as A, and the fluorescence intensity at 400 nm was denoted as F; the absorbance at 450 nm of the blank solution was denoted as A0, and the fluorescence intensity at 400 nm was denoted as F0. The ratio of absorbance (A / A0) and fluorescence intensity (F0 / F) were used to represent the amount of diimine salt generated and the effect of diimine salt quenching on the fluorescence of L-Trp@AuNCs, respectively. The optimal incubation time for TCs was determined by the time when A / A0 and F0 / F values ​​reached their maximum or the time when A / A0 and F0 / F values ​​reached a plateau.

[0062] (2) Optimization of TMB reaction time In the microplate, six TMB reaction time gradients were set up with reaction times (times for L-Trp@AuNCs to oxidize TMB to generate oxTMB for color development) of 2, 4, 6, 8, 10, and 12 min, respectively. Then, 10 μL of L-Trp@AuNCs, 10 μL of 9 M H2O2, 20 μL of 20 mM TMB, and 60 μL of 100 μM TCs were added to 50 mM pH 4.0 HAc-NaAc buffer. Specifically, TCs and L-Trp@AuNCs were reacted together first, then H2O2 and TMB were added for oxidation and color development, and finally 10 μL of 2 M H2SO4 was added to terminate the reaction, resulting in a final volume of 200 μL. In the blank solution, the same volume of ultrapure water was used instead of TCs, and four parallel controls were set up for each data set. The solution was incubated at 37°C for 15 minutes, and the absorbance at 450 nm and the fluorescence intensity at 400 nm (excitation wavelength of 290 nm) were measured using a microplate reader. In this experiment, the absorbance at 450 nm of the solution containing TCs was denoted as A, and the fluorescence intensity at 400 nm was denoted as F; the absorbance at 450 nm of the blank solution was denoted as A0, and the fluorescence intensity at 400 nm was denoted as F0. The ratio of absorbance (A / A0) and fluorescence intensity (F0 / F) were used to represent the amount of diimine salt generated and the effect of diimine salt quenching on the fluorescence of L-Trp@AuNCs, respectively. The optimal time for the TMB reaction was determined by either the time when A / A0 and F0 / F values ​​reached their maximum or the time when A / A0 and F0 / F values ​​reached a plateau.

[0063] Establishment of standard curve A colorimetric fluorescence detection procedure for TCs was established based on the optimized conditions described above. First, 10 μL of L-Trp@AuNCs, 10 μL of 9 M H2O2, and 20 μL of 20 mM TMB were added to a 96-well plate and mixed thoroughly. Then, different final concentration gradients of TC (0, 0.375, 0.75, 1.5, 3, 6, 12, 18, 24, 30, 37.5, 45, 52.5, 60, 75, 90 μM), OTC (0, 0.375, 0.75, 1.5, 3, 6, 12, 18, 24, 30, 37.5, 45, 52.5, 60, 75, 90 μM), and CTC (0, 0.375, 0.75, 1.5, 3, 6, 12, 18, 24, 30, 37.5, 45, 52.5, 60, 75, 90 μM) were added to the plate and mixed thoroughly. The solution was thoroughly mixed with a 50 mM pH 4.0 HAc-NaAc buffer solution, and finally, 50 mM HAc-NaAc buffer was added to the microplate to bring the final reaction volume to 200 μL. The mixture was incubated at 37°C for 15 minutes. The absorbance at 450 nm and the fluorescence intensity at 400 nm (excitation wavelength of 290 nm) were measured using a microplate reader. The absorbance at 450 nm and the fluorescence intensity at 400 nm were used for quantitative analysis of TC, OTC, and CTC using a microplate reader. Colorimetric fluorescence standard curves for TC, OTC, and CTC were established based on the changes in the absorbance ratio A / A0 and the fluorescence intensity ratio F0 / F.

[0064] Specificity analysis Based on the optimized detection procedure described above, TC, OTC, and CTC (30 μM) were detected using this method along with other common antibiotic compounds (600 μM), including KAN, CAP, STS, VAH, and AMN. In this analysis, the concentrations of TC, OTC, and CTC were taken as the median of the method's detection range, while the concentrations of other antibiotic compounds were 20 times the TC concentration. Four parallel control groups were set up for each data set, along with a blank control group containing no antibiotic compounds. The specificity of this method for TC detection was evaluated by plotting the absorbance ratio (A / A0) and fluorescence intensity ratio (F0 / F).

[0065] Testing of real samples The practicality and accuracy of this method were evaluated through testing and analysis of tap water and honey samples. For the tap water sample, 50 mL of tap water was simply filtered using a 0.22 μm filter. For the honey sample, 5 mL of deionized water was added to 1.0 g of honey and stirred for 1 minute to ensure uniform mixing. Different concentrations of TC, OTC, and CTC were added to the treated tap water and honey samples. Then, under optimized experimental conditions, the absorbance ratio A / A0 and fluorescence intensity ratio F0 / F of TC, OTC, and CTC were determined. The spiked samples were then subjected to recovery analysis, and the recoveries and coefficients of variation were calculated for both the colorimetric and fluorescence modes. The formulas for calculating the recoveries and coefficients of variation are as follows: Recovery rate = (2-1) Coefficient of variation = (2-2).

[0066] Stability analysis To ensure the long-term effectiveness of this detection method, a stability analysis was conducted. The colorimetric detection mode of this method is achieved by enhancing the peroxidase-like activity of L-Trp@AuNCs with TCs to accelerate the oxidation of TMB, while the fluorescence detection mode is achieved by converting oxTMB to diimine salt under acidic conditions, quenching the fluorescence of L-Trp@AuNCs at 400 nm. Therefore, the stability of L-Trp@AuNCs plays a decisive role in the stability of the sensing system. The stability was studied by measuring the changes in fluorescence intensity and absorbance of L-Trp@AuNCs over 30 days. Simultaneously, the stability of the detection method was evaluated by detecting tap water and honey samples using this method over 20 days, with a blank control included. The absorbance ratio (A / A0) and fluorescence intensity (F0 / F) were used to assess the stability of the method. First, tap water and honey samples containing different concentrations of TC, OTC, and CTC were prepared. Then, different samples were measured every 4 days, with four parallel controls for each sample group. Finally, the changes in fluorescence signals of samples with the same concentration at different times were compared.

[0067] Experimental results Characterization of L-Trp@AuNCs Transmission electron microscopy characterization The synthesis of L-Trp@AuNCs involved reducing Au(III) of HAuCl4 to Au(I) in a high-temperature reaction. The Au(I) then coordinated with the amino, carboxyl, and indole ring groups of L-Trp to form stable L-Trp@AuNCs. The L-Trp@AuNCs samples were dropped onto a copper grid, dried overnight at 37°C, and analyzed using transmission electron microscopy. The results are as follows: Figure 2As shown in Figure A, TEM images reveal that L-Trp@AuNCs exhibit irregular spherical shapes, with the outer contour clearly showing an enclosure of L-Trp. Figure 2 As shown in B, L-Trp@AuNCs will aggregate when TCs are present.

[0068] Fluorescence spectroscopy characterization The absorption and emission spectra of L-Trp@AuNCs were observed by scanning their fluorescence spectra, and the results are as follows: Figure 3 As shown in Figure A, the excitation wavelength of L-Trp@AuNCs is 275 nm, and the emission wavelength is 365 nm. By subjecting L-Trp@AuNCs to different treatments, the following effects were observed: Figure 3 As can be seen from B, only when a high concentration of TMB is added to L-Trp@AuNCs can an antenna effect be triggered, causing a red shift in the fluorescence of L-Trp@AuNCs and the fluorescence intensity to become about 5 times the original.

[0069] UV absorption spectral characterization of L-Trp@AuNCs and TMB By scanning the UV absorption spectra of L-Trp@AuNCs, TMB, and their mixture, Figure 4 As can be seen from Figure A, the UV spectrum of L-Trp@AuNCs changed somewhat after being mixed with a high concentration of TMB. Figure 4 As can be seen from B, the fluorescence spectrum of L-Trp@AuNCs mixed with high concentration of TMB has good overlap with the absorption spectrum of TMB oxidized to diimine salt, and the two can undergo IFE.

[0070] Experimental feasibility verification Based on the proposed experimental scheme, the feasibility of this method was studied under different treatments. The feasibility of the colorimetric and fluorescence methods was verified by studying the UV absorption and fluorescence spectra of the solutions under different treatments. Figure 5 As shown in Figure A, when TCs are present, the solution exhibits a strong UV absorption peak at 450 nm, and the solution appears yellow; when TCs are absent, the solution shows almost no UV absorption at 450 nm, and the solution appears pale yellow. Figure 5 As can be seen from B, when TCs are absent, the solution exhibits strong fluorescence at 400 nm; when TCs are present, the fluorescence of the solution at 400 nm is significantly quenched.

[0071] Conditional optimization L-Trp@AuNCs size optimization The volume of L-Trp@AuNCs in the reaction process was optimized, and the results are as follows: Figure 6As shown, with the increase of L-Trp@AuNCs volume, the values ​​of A / A0 and F0 / F exhibit a trend of first increasing and then decreasing. When the L-Trp@AuNCs volume increases to 10 μL, the values ​​of A / A0 and F0 / F reach their maximum values, and at this point, the values ​​of A / A0 and F0 / F reach a plateau. When the L-Trp@AuNCs volume is greater than 10 μL, the values ​​of A / A0 and F0 / F gradually decrease with increasing L-Trp@AuNCs volume. To adhere to the principle of reagent conservation, 10 μL was selected as the optimal volume for L-Trp@AuNCs.

[0072] H2O2 concentration optimization The H2O2 concentration in the reaction process was optimized, and the results are as follows: Figure 7 As shown, when the H2O2 concentration is less than 0.9 M, the values ​​of A / A0 and F0 / F increase with increasing H2O2 concentration. When the H2O2 concentration increases to 0.9 M, the values ​​of A / A0 and F0 / F reach their maximum values. When the H2O2 concentration is greater than 0.9 M, the values ​​of A / A0 and F0 / F gradually decrease with increasing H2O2 concentration. Therefore, 0.9 M is chosen as the optimal concentration of H2O2.

[0073] TMB concentration optimization The TMB concentration in the reaction process was optimized, and the results are as follows: Figure 8 As shown, when the TMB concentration is less than 1.5 mM, the values ​​of A / A0 and F0 / F increase with increasing TMB concentration. When the TMB concentration reaches 1.5 mM, the values ​​of A / A0 and F0 / F reach their maximum values. When the TMB concentration is greater than 1.5 mM, the values ​​of A / A0 and F0 / F gradually decrease with increasing TMB concentration. Therefore, 1.5 mM is chosen as the optimal TMB concentration.

[0074] Time optimization (1) Optimization of TC incubation time The TCs incubation time for the reaction process was optimized, and the results are as follows: Figure 9 As shown, for fluorescence mode, changes in TC incubation time have almost no effect on the F0 / F value. For colorimetric mode, as TC incubation time increases, the A / A0 value first increases and then decreases, reaching its maximum at an incubation time of 5 minutes. In summary, both A / A0 and F0 / F values ​​reach their maximum values ​​at a TC incubation time of 5 minutes. Therefore, 5 minutes is selected as the optimal incubation time for TCs.

[0075] (2) Optimization of TMB reaction time The TMB reaction time of the reaction process was optimized, and the results are as follows: Figure 10As shown, with the increase of TMB reaction time, the values ​​of A / A0 and F0 / F exhibit a trend of first increasing and then decreasing. When the TMB reaction time is less than 6 minutes, the values ​​of A / A0 and F0 / F gradually increase with increasing reaction time. When the TMB reaction time increases to 6 minutes, the values ​​of A / A0 and F0 / F reach their maximum values. When the TMB reaction time is greater than 6 minutes, the values ​​of A / A0 and F0 / F gradually decrease with increasing reaction time. Therefore, 6 minutes is selected as the optimal reaction time for TMB.

[0076] Establishment of standard curve Based on the optimized experimental conditions described above, a dual-mode fluorescence colorimetric method for detecting TCs based on L-Trp@AuNCs was established. Different concentrations of TC, OTC, and CTC standards were detected.

[0077] For TC, in colorimetric mode, the concentration of TC is used as the independent variable X, and the ratio of the absorbance of the TC-containing solution to the absorbance of the blank solution (A / A0) is used as the dependent variable Y; in fluorescence mode, the concentration of TC is used as the independent variable X, and the ratio of the fluorescence intensity of the TC-containing solution to the fluorescence intensity of the blank solution (F0 / F) is used as the dependent variable Y. The results are as follows: Figure 11 As shown, the linear regression equations for colorimetric and fluorescence patterns in this method are Y = 0.061X + 1.899 (R²). 2 = 0.991) and Y = 0.251X + 1.389 ( The linear detection ranges are 1.5-24 μM and 0.375-37.5 μM, respectively, and the LODs are 0.083 μM and 0.008 μM, respectively (LOD = 3 σ / S, where σ is the standard deviation of the blank group and S is the slope of the standard curve).

[0078] For OTC, in colorimetric mode, the concentration of OTC is used as the independent variable X, and the ratio of the absorbance of the OTC-containing solution to that of the blank solution (A / A0) is used as the dependent variable Y. In fluorescence mode, the concentration of OTC is used as the independent variable X, and the ratio of the fluorescence intensity of the OTC-containing solution to that of the blank solution (F0 / F) is used as the dependent variable Y. The results are as follows: Figure 12 As shown, the linear regression equations for colorimetric and fluorescence patterns in this method are Y = 0.401X + 1.001(R² + π / 2). 2 = 0.991) and Y = 0.097X + 2.090 ( The linear detection ranges are 0-7.5 μM and 1.5-60 μM, with LODs of 0.015 μM and 0.031 μM, respectively (LOD = 3 σ / S, where σ is the standard deviation of the blank group and S is the slope of the standard curve).

[0079] For CTC, in colorimetric mode, the concentration of CTC is used as the independent variable X, and the ratio A / A0 of the absorbance of the CTC-containing solution to that of the blank solution is used as the dependent variable Y; in fluorescence mode, the concentration of CTC is used as the independent variable X, and the ratio F0 / F of the fluorescence intensity of the OTC-containing solution to that of the blank solution is used as the dependent variable Y. The results are as follows: Figure 13 As shown, the linear regression equations for colorimetric and fluorescence patterns in this method are Y = 0.055X + 1.968 (R² + π / 2)². 2 = 0.996) and Y = 0.206X + 1.461 (R 2 = 0.992), with linear detection ranges of 1.5-30 μM and , respectively. The LOD values ​​were 0.087 μM and 0.012 μM, respectively (LOD = 3σ / S, where σ is the standard deviation of the blank group and S is the slope of the standard curve).

[0080] Specificity analysis Based on the optimized experimental conditions and established standard curves described above, TC, OTC, and CTC (30 μM) were detected using this method along with other common antibiotic compounds (600 μM), including KAN, CAP, STS, VAH, and AMN. The results for this experimental method are as follows: Figure 14 As shown in A and B, the values ​​of A / A0 and F0 / F of the solution are high only when TCs are present, while the values ​​of other antibiotic compounds are not significantly different from the blank level.

[0081] Testing of real samples Based on this experimental method, its applicability was determined by detecting TCs in tap water and honey samples. The results for tap water samples are shown in Table 3. In colorimetric mode, the recoveries of TC in tap water samples were 94.03%-100.06%, OTC 95.23%-101.33%, and CTC 95.68%-96.52%. In fluorescence mode, the recoveries of TC in tap water samples were 94.32%-101.71%, OTC 98.12%-101.82%, and CTC 97.62%-101.87%.

[0082] Table 3 Analysis of spiked recovery rates in tap water samples (n = 4) N: Not detected The results of the tests on the honey samples are shown in Table 4. For colorimetric analysis, the recoveries of TC in tap water samples were 102.94%-109.87%, OTC 96.33%-102.94%, and CTC 96.73%-99.28%. For fluorescence analysis, the recoveries of TC in tap water samples were 95.55%-104.27%, OTC 98.28%-101.94%, and CTC 98.23%-100.27%.

[0083] Table 4. Analysis of spiked recoveries in honey samples (n = 4) Therefore, the colorimetric fluorescence dual-mode method established in this study exhibits excellent analytical performance in both modes, with spiked recoveries consistently ranging from 80% to 120%, and the coefficient of variation for recoveries of all samples controlled within 10%. This result not only fully meets the technical requirements for analytical method validation according to AOAC international standards, but also fully demonstrates that this method possesses both high accuracy and good reproducibility in quantitative analysis. In conclusion, the L-Trp@AuNCs-based colorimetric dual-mode method established in this experiment can be used for the detection of TCs in various samples.

[0084] Stability analysis The stability of L-Trp@AuNCs was analyzed by measuring changes in fluorescence and absorbance over 30 days. Figure 15 As shown, the fluorescence intensity of L-Trp@AuNCs remained essentially unchanged over 30 days, indicating that L-Trp@AuNCs possesses strong stability, which provides a basis for this experimental method to test TCs over a long period of time.

[0085] Meanwhile, the stability of this detection method was evaluated by measuring the ratios A / A0 and F0 / F of tap water and honey samples tested using this method compared to a blank sample over 20 days. Figure 16 As shown, when TC in tap water and honey was detected using this method over 20 days, the values ​​of A / A0 and F0 / F in the samples hardly changed, indicating that the fluorescence colorimetric dual-mode sensor has good reproducibility and long-term stability over 20 days.

[0086] like Figure 17 As shown, when OTC in tap water and honey was detected using this method over 20 days, the values ​​of A / A0 and F0 / F in the samples hardly changed, indicating that the fluorescence colorimetric dual-mode sensor has good reproducibility and long-term stability over 20 days.

[0087] like Figure 18As shown, when CTCs in tap water and honey were detected using this method over 20 days, the values ​​of A / A0 and F0 / F in the samples hardly changed, indicating that the fluorescence colorimetric dual-mode sensor has good reproducibility and long-term stability over 20 days.

[0088] Comparison with other methods The constructed dual-mode fluorescence colorimetric method for detecting TCs based on L-Trp@AuNCs was compared with previously reported methods for TC detection. Table 5 shows the results. Compared with other single-mode or dual-mode colorimetric or fluorescence methods, this method exhibits a wider linear range and a lower detection limit in both colorimetric and fluorescence modes. This method achieves both dual-signal output and highly sensitive, rapid detection of TCs, with the detection process completed in just 11 minutes.

[0089] Table 5. Comparison of our dual-mode method with previously reported methods for detecting TCs based on nanomaterials. References: [1] Song Y, Qiao J, Liu W, et al. Enhancement of gold nanoclusters-based peroxidase nanozymes for detection of tetracycline [J]. MicrochemicalJournal, 2020, 157. [2] Wang W, Yin Y, Gunasekaran S. Nanozymatic degradation and simultaneous colorimetric detection of tetracycline [J]. Food Chemistry, 2023, 426. [3] Meng L, Lan C, Liu Z, et al. A novel ratiometric fluorescence probe for highly sensitive and specific detection of chlorotetracycline among tetracycline antibiotics [J]. Analytica Chimica Acta, 2019, 1089: 144-51. [4] Uriarte D, Domini C, Garrido M. New carbon dots based on glyceroland urea and its application in the determination of tetracycline in urinesamples [J]. Talanta, 2019, 201: 143-8. [5] Chen Z, Li Z, He H, et al. Ratiometric fluorescence sensor based on deep learning for rapid and user-friendly detection of tetracyclineantibiotics [J]. Food Chemistry, 2024, 450. Other beneficial effects of the present invention: (1) A fluorescence colorimetric dual-mode method based on L-Trp@AuNCs was established for the detection of TCs.

[0090] (2) For TC, the linear regression equations for colorimetric and fluorescence patterns in this method are Y = 0.061X + 1.899 (R² + π²) and π² = π² + ... 2 = 0.991) and Y = 0.251X + 1.389(R) 2 = 0.997), the linear detection ranges are 1.5-24 μM and 0.375-37.5 μM, and the LODs are 0.083 μM and 0.008 μM, respectively. For OTC, the linear regression equations for colorimetric and fluorescence modes of this method are Y = 0.401X + 1.001 (R² = 0.997), respectively. 2 = 0.991) and Y = 0.097X + 2.090(R 2 =0.991), the linear detection range is 0-7.5 μM and 1.5-60 μM, and the LODs are 0.015 μM and 0.031 μM, respectively. For CTC, the linear regression equations for colorimetric and fluorescence modes in this method are Y = 0.055X + 1.968 (R² = 0.991), respectively. 2 = 0.996) and Y = 0.206X + 1.461 (R = 0.996) and Y = 0.206X + 1.461 (R 2 = 0.992), the linear detection ranges are 1.5-30 μM and 0.375-37.5 μM, and the LODs are 0.087 μM and 0.012 μM, respectively.

[0091] (3) For tap water samples, the recovery rates of TC, OTC, and CTC were 94.03%-100.06% in colorimetric mode, 95.23%-101.33% in colorimetric mode, and 95.68%-96.52% in fluorescence mode. For honey samples, the recovery rates were 94.32%-101.71%, 98.12%-101.82%, and 97.62%-101.87% in fluorescence mode. The results for honey samples are shown in Table 4. For honey samples, the recovery rates of TC, OTC, and CTC were 102.94%-109.87% in colorimetric mode, 96.33%-102.94% in colorimetric mode, and 96.73%-99.28% in fluorescence mode. For fluorescence mode, the recovery rates of TC in tap water samples were 95.55%-104.27%, OTC 98.28%-101.94%, and CTC 98.23%-100.27%.

[0092] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A fluorescence colorimetric sensor based on L-Trp@AuNCs, characterized in that, It was prepared by mixing HAc-NaAc buffer, L-Trp@AuNCs, H2O2 and TMB.

2. The fluorescence colorimetric sensor based on L-Trp@AuNCs according to claim 1, wherein the concentration of H2O2 is 0.8-1.0 M and the concentration of TMB is 1.0-2.5 mM.

3. A method for fabricating a fluorescence colorimetric sensor based on L-Trp@AuNCs as described in any one of claims 1-2, characterized in that, include: The fluorescence colorimetric sensor was obtained by mixing HAc-NaAc buffer, L-Trp@AuNCs, H2O2, and TMB.

4. The preparation method according to claim 3, characterized in that, The L-Trp@AuNCs were prepared by the following steps: stirring L-Trp solution and HAuCl4 at 95-100℃, then centrifuging to collect the supernatant, and then filtering.

5. The application of the ratio fluorescence sensor according to any one of claims 1-2 or the ratio fluorescence sensor prepared by the preparation method according to any one of claims 3-4 in the detection of tetracycline antibiotics.

6. The application according to claim 5, characterized in that, include: H2O2, TMB, and the test solution were added to the HAc-NaAc buffer and reacted. Then, concentrated H2SO4 was added to terminate the reaction. The absorbance of the solution at 450 nm and the fluorescence intensity at 400 nm under an excitation wavelength of 290 nm were then measured using an ELISA reader.

7. The application according to claim 6, characterized in that, The reaction time for adding TMB to HAc-NaAc buffer is 6-8 min.

8. The application according to claim 6, characterized in that, The reaction time after adding the test solution is 5-10 minutes.

9. The application according to claim 6, characterized in that, The tetracycline antibiotics are one or more of TC, OTC, and CTC.

10. The application according to claim 6, characterized in that, The linear regression equations for the colorimetric and fluorescence patterns of TC are Y = 0.061X + 1.899 (R²). 2 = 0.991) and Y = 0.251X + 1.389(R) 2 = 0.997); the linear regression equations for the colorimetric and fluorescence patterns of OTC drugs are Y = 0.401X + 1.001 (R² = 0.997). 2 = 0.991) and Y = 0.097X + 2.090(R 2 =0.991); the linear regression equations for the colorimetric and fluorescence patterns of CTC are Y = 0.055X + 1.968 (R² = 0.991). 2 = 0.996) and Y=0.206X + 1.461(R 2 = 0.992); In colorimetric mode, X represents the concentration of TC, OTC or CTC, and Y represents the ratio A / A0 of the absorbance of the solution at 450 nm to the absorbance of the blank solution at 450 nm. In fluorescence mode, X represents the concentration of TC, OTC or CTC, and Y represents the ratio F0 / F of the fluorescence intensity of the blank solution at 400 nm under an excitation wavelength of 290 nm to the fluorescence intensity of the solution at 400 nm under an excitation wavelength of 290 nm. Substitute the absorbance or fluorescence intensity of the test solution into the corresponding formula to obtain the concentration value of the corresponding TCs.

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