A coumarin fluorescent probe for detecting kynurenine, a visual fluorescent test paper and a preparation method and application thereof

Abstract

The application provides a coumarin fluorescent probe for kynurenine detection, a visual fluorescent test paper, and a preparation method and application thereof, and belongs to the technical field of kynurenine detection. The fluorescent probe can be specifically combined with kynurenine, has good linear relationship, and has low detection limit. The prepared visual fluorescent test paper presents obvious color gradient change with the change of kynurenine concentration under ultraviolet light irradiation. The application has the advantages of sensitivity, convenience, low cost, good selectivity and the like for kynurenine detection, and is suitable for rapid screening of kynurenine in body fluid samples and auxiliary diagnosis of related diseases.

Description

Technical Field

[0001] This invention belongs to the field of kynurenine detection technology, and particularly relates to a coumarin-based fluorescent probe, a visual fluorescent test strip, and their preparation method and application for kynurenine detection. Background Technology

[0002] Kynurine (Kyn) is a key metabolite in the kynurine pathway, a major metabolic pathway for tryptophan (Trp). Produced by the rate-limiting enzyme's breakdown of Trp, Kyn is the initial metabolite of the kynurine pathway and plays a crucial role in multiple physiological processes. Kyn can serve as an indicator of various immune system-related diseases, such as depression, Alzheimer's disease, stroke, schizophrenia, sepsis, glioblastoma, and breast cancer. It inhibits immune responses through multiple pathways, acting as an important immunomodulatory factor, including inhibiting dendritic cell maturation, suppressing NK cell killing ability, reducing T cell proliferation and activation and inducing T cell apoptosis, inhibiting B cell receptor-mediated B cell proliferation and antibody synthesis, and promoting the differentiation of T cells and M2 macrophages. Therefore, kynurine is a highly promising immunosuppressive biomarker. Thus, developing a sensitive, convenient, and highly specific method for detecting humoral Kyn concentrations is of significant value for the early diagnosis of related diseases and the study of related immune mechanisms.

[0003] Currently, the main detection methods for Kyn include chromatography and immunoassay. The most widely used chromatographic method is to use high-performance liquid chromatography coupled with ultraviolet, fluorescence, or mass spectrometry. Although these methods have their advantages, they have limitations such as complex sample pretreatment, difficulty in obtaining equipment, and high cost. Immunoassay methods, such as enzyme-linked immunosorbent assay (ELISA), rely on antibodies, conjugates, and enzymes, which are relatively expensive and require long culture periods and special conditions. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a coumarin-based fluorescent probe for kynurenine detection, a visual fluorescent test strip, its preparation method, and its application. The resulting fluorescent test strip is loaded with a coumarin fluorescent probe that can specifically detect Kyn. After binding with Kyn, it causes a change in the fluorescence color of the test strip under ultraviolet light, enabling sensitive and convenient qualitative and semi-quantitative detection of Kyn.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a coumarin-based fluorescent probe for the detection of kynurenine, the structural formula of which is shown in Formula I: .

[0006] The coumarin compounds of this invention with specific chemical structures possess molecular recognition sites that specifically bind to kynurenine, and after binding, a significant change in fluorescence signal (fluorescence enhancement) occurs.

[0007] Furthermore, after the fluorescent probe binds to kynurenine, the maximum emission wavelength is 600 nm under 570 nm excitation light; within the kynurenine concentration range of 0-1000 µM, the fluorescence intensity increases with increasing kynurenine concentration; within the 0-10 µM range, the fluorescence intensity shows a good linear relationship with the kynurenine concentration, and the detection limit is 71 nm.

[0008] The present invention also provides a method for preparing the above-mentioned coumarin-based fluorescent probe for kynurenine detection, comprising the following steps: (1) Bis-(2,4,6-trichlorophenyl)-malonate was reacted with 8-hydroxyjuloridin in toluene under reflux. After cooling, the mixture was filtered, washed, and dried to obtain intermediate compound IV. The structural formula of intermediate compound IV is as follows: ; (2) POCl3 was added dropwise to dimethylformamide (DMF) and stirred. Intermediate compound IV was added to react with the mixture. After quenching, the pH was adjusted to neutral. The mixture was then filtered, washed, and dried to obtain intermediate compound V. The structural formula of intermediate compound V is as follows: ; (3) Dissolve the intermediate compound V in dichloromethane, add ethanethiol and triethylamine, stir at room temperature, concentrate and purify by column chromatography to obtain the coumarin fluorescent probe for kynurenine detection.

[0009] Further, in step (1), the molar ratio of bis-(2,4,6-trichlorophenyl)-malonate to 8-hydroxyjuloridin is 1:1; the temperature of the heating reflux reaction is 110°C and the time is 5h.

[0010] Further, in step (2), the ratio of POCl3 to intermediate compound IV is 1 mL: 3.25 mmol, the reaction temperature is 60 °C, and the reaction is carried out overnight.

[0011] Further, in step (3), the molar ratio of the intermediate compound V to the ethanethiol and triethylamine is 1:2:2.

[0012] In the preparation process of this invention, the first step involves heating bis-(2,4,6-trichlorophenyl)-malonate and 8-hydroxyjuloridin in toluene under reflux to construct a hydroxyl-containing coumarin intermediate IV via esterification / condensation reaction, providing reaction sites for subsequent functional group modification; the second step involves formylation modification of intermediate IV using a Vilsmeier-Haack reaction system composed of POCl3 and DMF to introduce an aldehyde functional group, thereby obtaining intermediate V and enriching the molecular recognition and fluorescence regulation sites; the third step involves nucleophilic substitution reaction of ethanethiol and intermediate V to introduce a thioether functional group under the basic catalysis of triethylamine, followed by column chromatography purification to obtain the probe of formula I, ultimately forming a complete molecular structure that can specifically bind to kynurenine.

[0013] The present invention also provides a visual fluorescent test strip comprising the coumarin fluorescent probe described above for the detection of kynurenine.

[0014] The present invention also provides a method for preparing the above-mentioned visual fluorescent test strip, wherein the paper substrate is immersed in the coumarin fluorescent probe solution for kynurenine detection and dried to obtain the visual fluorescent test strip.

[0015] This invention utilizes the porous adsorption properties of paper substrates to load probe molecules with kynurenine recognition and fluorescence response capabilities onto the paper substrate, achieving probe solidification. This transforms liquid-phase fluorescence detection into solid-phase visual test strip detection. The paper substrate provides a solid-phase carrier for the probe, and the probe molecules are fixed in the fibrous pores of the paper substrate through physical adsorption / intermolecular interactions. This preserves the fluorescence response characteristics of the probe's specific binding with kynurenine while also making the detection tool portable, breaking through the dependence of liquid-phase detection on instruments.

[0016] Furthermore, the coumarin fluorescent probe solution for kynurenine detection is prepared by dissolving the coumarin fluorescent probe for kynurenine detection in a buffer solution with pH=1; the soaking time is 30-60 min; and the drying temperature is 30-40℃.

[0017] The present invention also provides an application of the above-mentioned visual fluorescent test strip in the detection of kynurenine, wherein the detection concentration of kynurenine is 10nM-10mM.

[0018] Furthermore, under 254nm ultraviolet light irradiation, as the concentration of kynurenine increased from 0 to 10mM, the color of the fluorescent test strip changed from dark purple to light green, light orange, and rose red in sequence.

[0019] In this invention, 254nm ultraviolet light is the effective excitation light for the probe-kynurenine conjugate, which can excite the conjugate to produce characteristic fluorescence. When the kynurenine concentration is 0, the free probe molecules on the paper substrate emit dark purple fluorescence under 254nm ultraviolet light. As the concentration increases, the amount of probe binding to kynurenine increases, the electronic transition energy of the molecular conjugated system changes, the fluorescence emission wavelength red-shifts / blue-shifts, and the macroscopic color changes successively to light green, light orange, and magenta. The color change is positively correlated with the concentration, realizing visualized qualitative detection.

[0020] Furthermore, the method also includes the step of preparing a standard colorimetric card: adding a series of concentrations of kynurenine solution to the visualization fluorescent test paper, and obtaining a colorimetric card with color gradient changes after drying, which is used for semi-quantitative detection of kynurenine.

[0021] In existing technologies, the detection of Kyn mainly relies on high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assay (ELISA), but these methods have limitations such as expensive equipment, complex operation, long processing time, and reliance on antibodies. This invention proposes for the first time a visual detection method based on coumarin-based fluorescent probes, which has advantages such as high sensitivity, simple operation, low cost, and no need for large instruments, making it particularly suitable for rapid screening in resource-limited environments. Compared with existing technologies, this invention has the following advantages and technical effects: The coumarin fluorescent probe provided by this invention can specifically bind to Kyn, generating a significant fluorescence enhancement signal at 600 nm under 570 nm excitation light, with a detection limit of 71 nM. The method provided by this invention for preparing a visual fluorescent test strip by loading the probe onto a paper substrate exhibits a clear color gradient change with Kyn concentration under 254 nm ultraviolet light irradiation, enabling qualitative and semi-quantitative detection of Kyn. This method is characterized by its simple operation, low cost, high sensitivity, and good selectivity. Attached Figure Description

[0022] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 Compound I prepared in Example 1 1 H NMR spectrum; Figure 2 Compound I prepared in Example 1 13 C NMR spectrum; Figure 3 The fluorescence emission spectra of compound I prepared in Example 1 reacting with different concentrations of Kyn; Figure 4 The graph shows the linear relationship between the fluorescence intensity of compound I prepared in Example 1 and the concentration of Kyn. Figure 5 The bar chart shows the fluorescence response of compound I prepared in Example 1 to different amino acids. Figure 6 The visualization of the fluorescent test strip prepared in Example 2 shows the color change under natural light and ultraviolet light; Figure 7 The fluorescence emission spectra of the coumarin analogue without ethanethiol in Comparative Example 1 reacted with different concentrations of Kyn. Figure 8 The fluorescence emission spectra of the reaction between the Kyn fluorescent probe and different concentrations of Kyn in Comparative Example 2 are shown. Figure 9 The graph shows the linear relationship between the fluorescence intensity of the Kyn fluorescent probe and the Kyn concentration in Example 1, Comparative Example 1, and Comparative Example 2. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] This invention provides a coumarin-based fluorescent probe for the detection of kynurenine (Kyn), the structural formula of which is shown in Formula I: .

[0029] In a preferred embodiment of the present invention, after the fluorescent probe binds to kynurenine, the maximum emission wavelength is 600 nm under 570 nm excitation light; the fluorescence intensity increases with increasing kynurenine concentration in the range of 0-1000 µM; and the fluorescence intensity shows a good linear relationship with kynurenine concentration in the range of -0-10 µM, with a detection limit of 71 nm.

[0030] This invention also provides a method for preparing the above-mentioned coumarin-based fluorescent probe for kynurenine detection, specifically including the following steps: (1) Bis-(2,4,6-trichlorophenyl)-malonate (compound II, 5.28 mmol) and 8-hydroxyjuloridin (compound III, 5.28 mmol) were dissolved in 25 mL of toluene and heated under reflux at 110 °C for 5 h. After the reaction was completed, the reaction solution was cooled to room temperature and filtered. The precipitate was collected and washed with n-hexane and dried under vacuum to obtain a gray solid, which was intermediate compound IV. The structural formula of bis-(2,4,6-trichlorophenyl)-malonate is as follows: ; The structural formula of 8-hydroxyjuloridin is: ; The structural formula of intermediate compound IV is: ; (2) POCl3 (1 mL) was added dropwise to anhydrous DMF (N,N-dimethylformamide, 1 mL) and stirred at 50 °C for 30 min to obtain an orange-red solution. Intermediate compound IV (3.25 mmol) was dissolved in 5 mL of DMF and added dropwise to the above solution to obtain a scarlet suspension. The mixture was reacted overnight at 60 °C. After the reaction was completed, the reaction mixture was poured into 50 mL of ice water to quench the reaction. The pH of the solution was adjusted to neutral with NaOH solution (volume concentration of 20%) to obtain a large amount of precipitate. The crude product (i.e., the precipitate) was filtered, washed with water, and vacuum dried to obtain a brick-red solid, which is intermediate compound V. The structural formula of intermediate compound V is: ; (3) Dissolve intermediate compound V (0.99 mmol) in 18 mL of dichloromethane, add ethanethiol (1.98 mmol) and stir, then add triethylamine (1.98 mmol), stir overnight at room temperature. After the reaction is complete, concentrate the mixture to obtain crude product, and further purify it by rapid column chromatography to obtain a red solid, which is the coumarin fluorescent probe for the detection of kynurenine.

[0031] This invention also provides a visualization fluorescent test strip containing the coumarin-based fluorescent probes described above for the detection of kynurenine.

[0032] This invention also provides a method for preparing the above-mentioned visual fluorescent test strip, wherein the paper substrate is immersed in a coumarin-based fluorescent probe solution for kynurenine detection and then dried to obtain the test strip.

[0033] In a preferred embodiment of the present invention, the coumarin fluorescent probe solution for kynurenine detection is prepared by dissolving the coumarin fluorescent probe for kynurenine detection in a buffer solution (0.5M HCl-KCl solution) with pH=1; the soaking time is 30-60 min; and the drying temperature is 30-40℃.

[0034] The present invention also provides an application of the above-mentioned visual fluorescent test strip in the detection of kynurenine, wherein the detection concentration of kynurenine is 10 nM-10 mM.

[0035] In a preferred embodiment of the present invention, under 254nm ultraviolet light irradiation, as the concentration of kynurenine increases from 0 to 10mM, the color of the fluorescent test strip changes from dark purple to light green, light orange, and rose red in sequence.

[0036] In a preferred embodiment of the present invention, the method further includes the step of preparing a standard colorimetric card: adding a series of concentrations of kynurenine solution onto a visual fluorescent test paper, and drying it to obtain a colorimetric card with color gradient changes, which is used for semi-quantitative detection of kynurenine.

[0037] All raw materials used in the embodiments of this invention were purchased commercially.

[0038] In this embodiment of the invention, room temperature refers to "25±2℃".

[0039] The technical solution of the present invention will be further illustrated by the following embodiments.

[0040] Example 1 A method for preparing the above-mentioned coumarin-based fluorescent probe for kynurenine detection specifically includes the following steps: (1) Bis-(2,4,6-trichlorophenyl)-malonate (compound II, 5.28 mmol) and 8-hydroxyjuloridine (compound III, 5.28 mmol) were dissolved in 25 mL of toluene and heated to reflux at 110 °C for 5 h. After the reaction was completed, the reaction solution was cooled to room temperature and filtered. The precipitate was collected, washed with n-hexane, and dried under vacuum to obtain a gray solid, which was intermediate compound IV. The reaction process of this step is as follows: ; (2) POCl3 (1 mL) was added dropwise to anhydrous DMF (N,N-dimethylformamide, 1 mL) and stirred at 50 °C for 30 min to obtain an orange-red solution; intermediate compound IV (3.25 mmol) was dissolved in 5 mL of DMF and added dropwise to the above solution to obtain a scarlet suspension. The suspension was reacted at 60 °C overnight. After the reaction was completed, the reaction mixture was poured into 50 mL of ice water to quench the reaction. The pH of the solution was adjusted to neutral with NaOH solution (volume concentration of 20%) to obtain a large amount of precipitate. The crude product was filtered, washed with water, and vacuum dried to obtain a brick-red solid, which is intermediate compound V; the reaction process of this step is as follows: ; (3) Dissolve intermediate compound V (0.99 mmol) in 18 mL of dichloromethane, add ethanethiol (1.98 mmol) and stir, then add triethylamine (1.98 mmol), stir overnight at room temperature. After the reaction is complete, concentrate the mixture to obtain the crude product, and further purify it by rapid column chromatography to obtain a red solid, which is the coumarin fluorescent probe (compound I) for the detection of kynurenine. The reaction process of this step is as follows: .

[0041] Compound I prepared in Example 1 1 The H NMR spectrum can be seen Figure 1 , 13 The C NMR spectrum is shown in Figure 2 .

[0042] Compound I obtained in Example 1 was used to prepare a 10 mM stock solution of compound I probe using dimethyl sulfoxide (DMSO). A 20 mM Kyn solution was prepared using ultrapure water. All spectroscopic experiments were performed at room temperature. Before testing, the stock solution of compound I was diluted to a 10 μM working solution with 0.5 M HCl-KCl buffer at pH=1.

[0043] 1. The fluorescence response of compound I to Kyn was determined by fluorescence titration, with the molar ratio of Kyn to compound I ranging from 0 to 100. The fluorescence emission spectra of each solution were measured sequentially using a fluorescence spectrophotometer (λex = 570 nm, slit width: 10 nm / 10 nm).

[0044] Figure 3 The fluorescence emission spectra of the reaction of compound I with different concentrations of Kyn are shown. It can be seen that after the probe molecule of compound I binds to Kyn, the maximum emission wavelength is at 600 nm, and the fluorescence intensity at 600 nm increases with the increase of Kyn concentration (0-1000µM).

[0045] 2. Plot a standard curve of fluorescence intensity at 600 nm for low Kyn concentrations and calculate the detection limit. The detection limit is calculated using the following formula: ; in σ The standard deviation of the fluorescence intensity of 20 blank probe solutions; K To determine the slope of the linear calibration curve for the detector, the concentration of the probe molecule was 10 µM.

[0046] Figure 4 The graph shows the linear relationship between the fluorescence intensity of compound I and the concentration of Kyn. It can be seen that at low concentrations of Kyn (0–10 µM), the maximum fluorescence emission of compound I at 600 nm exhibits a certain linear relationship, with a correlation coefficient of 0.9986. This demonstrates a good linear relationship, with a detection limit of 71 nM, indicating that compound I has good sensitivity to Kyn.

[0047] 3. Prepare 1mM valine (Val), asparagine (Asn), tryptophan (Trp), arginine (Arg), phenylalanine (Phe), tyrosine (Tyr), and cysteine ​​(Cys) respectively. Add compound I obtained in Example 1 to other amino acids and detect the fluorescence intensity of compound I and different amino acids at 600nm.

[0048] Figure 5 The bar chart shows the fluorescence response of compound I to different amino acids. It can be seen that after adding other amino acids (1mM), the fluorescence intensity of probe compound I at 600nm only changes slightly or not at all. However, after adding Kyn, a significant fluorescence signal can be observed, indicating that the probe is specific to Kyn.

[0049] Example 2 Compound I obtained in Example 1 was used to prepare a 10 mM stock solution of compound I probe using dimethyl sulfoxide (DMSO). The stock solution was then diluted to a 10 μM working solution using 0.5 M HCl-KCl buffer (pH=1). Multiple rectangular strips of filter paper were cut and dried in a 90°C oven. The filter paper was then soaked in water for 15 min and dried using a forced-air dryer. The dried filter paper was then soaked in the prepared probe solution for 30 min and subsequently dried in a 35°C oven to obtain a visual fluorescent test strip.

[0050] The fluorescent test strips prepared in Example 2 were reacted with different concentrations of Kyn (0-10 mM), and the color changes of the fluorescent test strips detecting different concentrations of Kyn were observed under irradiation with 254 nm ultraviolet light (UV light) or natural light (Day light). The color changes of the visualized fluorescent test strips prepared in Example 2 under natural light and ultraviolet light are shown in [the figure]. Figure 6 As can be seen, under 254nm ultraviolet light irradiation, when the Kyn concentration is 0, the fluorescent test paper appears dark purple. With increasing Kyn concentration (0-10mM), the color of the fluorescent test paper gradually changes from dark purple to light green, light orange, and then to magenta, showing a significant fluorescence change and sensitive reaction. Therefore, adding a series of concentrations of kynurenine solution to the visual fluorescent test paper and obtaining a colorimetric card showing a color gradient after drying can be used for the semi-quantitative detection of kynurenine and has good application value.

[0051] Comparative Example 1 The coumarin analogue without ethanethiol (i.e., compound V in Example 1) has the following structural formula: .

[0052] Fluorescence emission spectra of coumarin analogs without ethanethiol reacting with different concentrations of Kyn are shown below. Figure 7 As can be seen, the fluorescence change is not obvious at low concentrations of Kyn (<10µM). At Kyn >10µM, the probe binds to Kyn and has maximum absorption at 588nm under 560nm excitation.

[0053] Comparative Example 2 The existing Kyn fluorescent probe (prepared according to reference Org. Lett. 2013, 15, 2, 235–237) has the following structural formula: .

[0054] The fluorescence emission spectra of the reaction between the Kyn fluorescent probe and different concentrations of Kyn are shown in the figure. Figure 8 The linear relationship between the fluorescence intensity of the Kyn fluorescent probe and the Kyn concentration in Example 1, Comparative Example 1, and Comparative Example 2 is shown in the figure. Figure 9As can be seen, in Comparative Example 2, the Kyn fluorescent probe binds to Kyn and has maximum absorption at 585 nm under 555 nm excitation, with a detection limit of 99 nM, which is lower than that of Example 1.

[0055] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A coumarin-based fluorescent probe for the detection of kynurenine, characterized in that, The structural formula of the coumarin-based fluorescent probe is shown in Formula I: 。 2. A method for preparing the coumarin-based fluorescent probe for kynurenine detection as described in claim 1, characterized in that, Includes the following steps: (1) Bis-(2,4,6-trichlorophenyl)-malonate was reacted with 8-hydroxyjuloridin in toluene under reflux. After cooling, the mixture was filtered, washed, and dried to obtain intermediate compound IV. The structural formula of intermediate compound IV is as follows: ； (2) POCl3 was added dropwise to dimethylformamide and stirred. Intermediate compound IV was then added to react with the mixture. After quenching, the pH was adjusted to neutral. The mixture was then filtered, washed, and dried to obtain intermediate compound V. The structural formula of intermediate compound V is as follows: ； (3) Dissolve the intermediate compound V in dichloromethane, add ethanethiol and triethylamine, stir at room temperature, concentrate and purify by column chromatography to obtain the coumarin fluorescent probe for kynurenine detection.

3. The method for preparing the coumarin-based fluorescent probe for kynurenine detection according to claim 2, characterized in that, In step (1), the molar ratio of bis-(2,4,6-trichlorophenyl)-malonate to 8-hydroxyjuloridin is 1:1; the temperature of the heating reflux reaction is 110°C and the time is 5h.

4. The method for preparing the coumarin-based fluorescent probe for kynurenine detection according to claim 2, characterized in that, In step (2), the ratio of POCl3 to intermediate compound IV is 1 mL: 3.25 mmol; In step (3), the molar ratio of the intermediate compound V to the ethanethiol and triethylamine is 1:2:

2.

5. A visual fluorescent test strip, characterized in that, It includes the coumarin-based fluorescent probe for kynurenine detection as described in claim 1.

6. A method for preparing the visual fluorescent test strip according to claim 5, characterized in that, The paper substrate is immersed in the coumarin-based fluorescent probe solution for kynurenine detection and then dried to obtain the visual fluorescent test paper.

7. The method for preparing the visual fluorescent test strip according to claim 6, characterized in that, The coumarin fluorescent probe solution for kynurenine detection is prepared by dissolving the coumarin fluorescent probe for kynurenine detection in a buffer solution with pH=1; the soaking time is 30-60 min; and the drying temperature is 30-40℃.

8. The application of the visual fluorescent test strip of claim 5 in the detection of kynurenine for non-diagnostic purposes, characterized in that, The detection concentration of kynurenine was 10 nM-10 mM.

9. The application of the visual fluorescent test strip according to claim 8 in the detection of kynurenine for non-diagnostic purposes, characterized in that, Under 254nm ultraviolet light irradiation, as the concentration of kynurenine increases from 0 to 10mM, the color of the fluorescent test strip changes from dark purple to light green, light orange, and rose red in sequence.

10. The application of the visual fluorescent test strip according to claim 8 in the detection of kynurenine for non-diagnostic purposes, characterized in that, It also includes the step of preparing a standard colorimetric card: adding a series of concentrations of kynurenine solution to the visualization fluorescent test paper, and obtaining a colorimetric card with color gradient changes after drying, which is used for semi-quantitative detection of kynurenine.

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