Method for detecting dopamine of different provenances based on efficient in-situ fluorescence reaction system initiated by imidazole substances

Abstract

The invention relates to a method for detecting dopamine of different provenances based on an efficient in-situ fluorescence reaction system initiated by imidazole substances, and belongs to the technical field of biosensing. 1-(3-aminopropyl) imidazole is used as a catalyst, and can initiate rapid in-situ fluorescence reaction between dopamine and 1, 5-dihydroxynaphthalene and generate a strong yellow fluorescent compound (AFC). The system realizes high-sensitivity dopamine detection in a range of 0-200 [mu] M, the limit of detection (LOD) is 33.3 pM, then a buffer solution is replaced by Tris-HCl + 1% TW 80, and the limit of detection can be as low as 66.7 fM. In addition, obvious selectivity can also be shown on dopamine at the concentration as low as 10 nM. The detection on different provenances of dopamine is realized. The system can also be used for detecting dopamine in clinical sample urine, and has good consistency with a traditional ELISA kit.

Description

Technical Field

[0001] The present invention relates to a preparation method of a detection kit for different source dopamine by using an efficient in-situ fluorescence reaction initiated by an imidazole-based substance, and belongs to the field of biosensing technology. Background Art

[0002] Dopamine (DA) is an important catecholamine neurotransmitter, mainly present in the central nervous system of humans. It plays an important role in many physiological activities such as sensation, memory, cognition, attention, information transduction, addiction, and motor control. Abnormal dopamine levels lead to a series of physiological and psychological diseases. Research shows that DA has been used in the diagnosis and treatment of various neurological diseases, such as schizophrenia, depression, Parkinson's disease, restless legs syndrome, Alzheimer's disease, etc. Therefore, rapid and accurate detection of DA is of great significance for the clinical diagnosis, treatment, and prevention of these diseases. In recent years, a variety of methods for detecting DA have been reported, including chemiluminescence (CL), high performance liquid chromatography (HPLC), surface enhanced Raman scattering (SERS), electrochemistry, fluorescence, and colorimetry. Among them, fluorescence method has attracted much attention due to a series of advantages such as simplicity, rapidity, sensitivity, and accuracy. The Sun group provided a genetically encoded fluorescence sensor (GRABDA) that can detect endogenous DA in Drosophila, fish, and mice in real time. The Li group reported a genetically encoded fluorescence sensor that can rapidly and specifically detect extracellular dopamine. However, the application of several other neurotransmitters did not cause any detectable fluorescence change, except for NE, which drove a moderate fluorescence increase. Despite the development of a variety of available fluorescence methods for detecting DA, specific and low-detection-limit detection of dopamine remains a huge challenge.

[0003] Imidazole is a five-membered aromatic heterocyclic compound containing two meta-nitrogen atoms in its molecular structure and has been proven to be very important in biological systems. It exists in amino acid tissues. The influence of its substituents on the basicity and acidity of imidazole is similar to that of aniline and benzoic acid. Electron-withdrawing groups such as nitro, halogen, and phenyl will reduce the basicity, while electron-donating groups such as alkyl will increase the basicity. More importantly, some imidazoles or their derivatives have attracted the interest of the scientific community due to their catalytic properties.

[0004] To overcome these deficiencies, an efficient in-situ fluorescence reaction system triggered by imidazole substances is proposed here for highly selective and extremely low-level detection of dopamine from different sources. This method is based on the specific reaction between imidazole substances and dopamine catalyzed by 1,5-dihydroxynaphthalene. In this sensing system, the interference of other neurotransmitters, especially norepinephrine, is completely eliminated, showing unique high specificity for dopamine. In addition, our sensing platform still has high sensitivity, with a high signal-to-noise ratio (about 300 times) and a low detection limit. The detection limit (LOD) is 33.3 pM in the range of 0-1 nM. Then, when the buffer is changed to Tris-HCl + 1% TW 80, the detection limit can be as low as 66.7 fM, which is due to the near-zero background in the catalytic reaction system. In addition, the method we developed has also been successfully applied to the detection of DA from different sources (mice, rabbits, dogs, humans, and horses), and the results are satisfactory. It is worth noting that the method we developed has also been successfully applied to the detection of DA in human urine. Therefore, the proposed sensor for highly specific and sensitive detection of DA will provide broad prospects for the precise diagnosis, treatment, and prevention of various neurological diseases in clinical practice. Summary of the Invention

[0005] The object of the present invention is to propose a preparation method of a dopamine detection kit for different species (mice, rabbits, dogs, humans, horses) using an efficient in-situ fluorescence reaction system triggered by 1-(3-aminopropyl)imidazole to overcome the deficiencies of the prior art. This system is based on the formation of a yellow azoheterocyclic fluorescent compound (AFC) by the catalysis of dopamine and 1,5-dihydroxy by 1-(3-aminopropyl)imidazole. This system realizes highly sensitive DA detection in the range of 0-200 μM, with a detection limit (LOD) of 33.3 pM. Then, when the buffer is changed to Tris-HCl + 1% TW 80, the detection of dopamine at a lower concentration is realized in the range of 0-100 pM, and the detection limit (LOD) can be as low as 66.7 fM, with good specificity. In addition, selectivity tests show that the system we developed can effectively distinguish several of the most common interfering substances (levodopa, 5-hydroxyamine, norepinephrine, epinephrine) at high concentrations (100 μM, 10 μM, and 100 nM), and even show significant selectivity for dopamine at a concentration as low as 10 nM. In addition, the system can detect dopamine from different sources (mice, rabbits, dogs, humans, and horses) and obtain satisfactory results.

[0006] To solve the technical problems of the present invention, the technical solution proposed is: A method for detecting dopamine from different species using an in-situ fluorescence reaction system triggered by imidazole substances, comprising the following steps:

[0007] An in-situ fluorescence reaction can occur between dopamine and phenolic compounds. In the presence of 1-(3-aminopropyl)imidazole, dopamine reacts to form a yellow azo fluorescent compound AFC. Based on the change in fluorescence intensity, dopamine can be detected. At room temperature, in a Tris-HCl buffer solution with a pH of 7.4, dopamine, 1,5-dihydroxynaphthalene, and 1-(3-aminopropyl)imidazole are added and mixed evenly to form the yellow fluorescent compound AFC. The fluorescence intensity of the synthesized AFC is detected using a fluorescence spectrometer. High-sensitivity and high-selectivity detection of dopamine are achieved based on the change in fluorescence intensity.

[0008] Synthesis conditions of AFC: The concentration of the Tris-HCl buffer solution is 10 mM, dopamine is added, the concentration of 1,5-dihydroxynaphthalene is 50 μM, the concentration of 1-(3-aminopropyl)imidazole is 100 mM, and the reaction time is 5 min.

[0009] Preferably, at room temperature, in a Tris-HCl buffer solution, in the presence of 1-(3-aminopropyl)imidazole, dopamine reacts with 1,5-dihydroxynaphthalene for 5 min to form AFC. The fluorescence intensity of the synthesized AFC is detected using a fluorescence spectrometer. High-sensitivity and high-selectivity detection of dopamine are achieved based on the change in fluorescence intensity.

[0010] Preferably, it includes the following steps:

[0011] First, in the presence of 1-(3-aminopropyl)imidazole, dopamine and 1,5-dihydroxynaphthalene with different source concentrations are added and mixed evenly, and the reaction proceeds for 5 min to form the yellow fluorescent compound AFC. The fluorescence intensity of the synthesized AFC is detected using a fluorescence spectrometer. The detection process for other dopamine with different sources is the same. The conditions of the sensing platform are as follows: The pH of the Tris-HCl buffer solution is 7.4, the concentration of Tris-HCl is 10 mM, dopamine with different source concentrations, the concentration of 1,5-dihydroxynaphthalene is 50 μM, the concentration of 1-(3-aminopropyl)imidazole is 100 mM, and the reaction time is 5 min. The fluorescence intensity of the synthesized AFC is detected using a fluorescence spectrometer to quantitatively detect dopamine.

[0012] Preferably, the different source dopamine includes human dopamine, horse dopamine, canine dopamine, rabbit dopamine, murine dopamine, or human urine.

[0013] Preferably, the fluorescence intensity of AFC is detected using a fluorescence spectrometer, with an excitation wavelength of 490 nm and an emission wavelength of 540 nm. The detection range of this method for dopamine is 0 - 200 μM, and the detection limit is 33.3 pM.

[0014] A method for detecting dopamine from different sources using an in-situ fluorescence reaction system triggered by imidazole substances. At room temperature, take 300 μL of Tris-HCl + 1% TW 80 buffer solution, add 5 nM of 1,5-dihydroxynaphthalene, 100 mM of 1-(3-aminopropyl)imidazole, and 0 - 100 pM of dopamine respectively, shake well, after reacting for 5 min, under the irradiation of an ultraviolet lamp, yellow fluorescence is observed, indicating the successful preparation of AFC. The fluorescence intensity of AFC is detected by a fluorescence spectrometer, with an excitation wavelength of 490 nm and an emission wavelength of 540 nm. The detection range of this method for dopamine is 0 - 100 pM, and the detection limit is 66.7 fM.

[0015] 1. A method based on an in-situ fluorescence reaction triggered by an imidazole substance, comprising the following steps: At room temperature, in a Tris-HCl buffer solution, add dopamine, 1,5-dihydroxynaphthalene, and 21 different imidazoles, such as imidazole, 2-methylimidazole, 2-propylimidazole, etc., mix well to generate a yellow fluorescent compound, and detect the fluorescence intensity of the synthesized yellow fluorescent compound by a fluorescence spectrometer.

[0016] When proposing a method based on an in-situ fluorescence reaction triggered by an imidazole substance, the pH of the Tris-HCl buffer solution is 7.4, the concentration of the Tris-HCl buffer solution is 10 mM, the concentration of 1,5-dihydroxynaphthalene is fixed at 50 μM, and the concentration of each different imidazole is 100 mM; take 300 μL of Tris-HCl buffer solution, add 50 μM of 1,5-dihydroxynaphthalene, 100 μM of dopamine, and 100 mM of 21 imidazoles respectively, shake well, after reacting for 5 min, measure its fluorescence intensity by a fluorescence spectrometer, and record the change in fluorescence intensity of each imidazole.

[0017] 2. Application of a method based on an in-situ fluorescence reaction triggered by an imidazole substance. At room temperature, in a Tris-HCl buffer solution, add dopamine, 1,5-dihydroxynaphthalene, and 21 different imidazoles, such as imidazole, 2-methylimidazole, 2-propylimidazole, etc., mix well to generate a yellow fluorescent compound, detect the fluorescence intensity of the synthesized yellow fluorescent compound by a fluorescence spectrometer, and the fluorescence intensity of the yellow fluorescent compound is closely related to the type of imidazole, and thus the different catalytic intensities of different imidazoles in a complex environmental system can be distinguished.

[0018] When detecting different imidazole catalytic intensities in a complex environmental system using an in-situ fluorescence reaction triggered by an imidazole substance, the pH of Tris-HCl is 7.4, the concentration of Tris-HCl is 10 mM, the concentration of 1,5-dihydroxynaphthalene is 50 μM, and the concentration of imidazole is 100 mM; take 300 μL of Tris-HCl, add 1% human urine, add 50 μM of 1,5-dihydroxynaphthalene, 100 μM of dopamine, and 100 mM of imidazole, shake well, after reacting for 5 min, measure the fluorescence intensity with a fluorescence spectrometer, and record the change in fluorescence intensity of each imidazole.

[0019] 3. A method for detecting dopamine based on an in-situ fluorescence reaction triggered by an imidazole substance, comprising the following steps: Under room temperature conditions, in a Tris-HCl buffer solution, add dopamine, 1,5-dihydroxynaphthalene, and 1-(3-aminopropyl)imidazole, mix evenly to generate a yellow fluorescent compound AFC, and detect the fluorescence intensity of the synthesized AFC with a fluorescence spectrometer.

[0020] The detection of dopamine is achieved by detecting the fluorescence intensity of the synthesized AFC with a fluorescence spectrometer, and the fluorescence intensity of AFC gradually increases with the increase in dopamine concentration, thereby realizing the quantitative detection of dopamine.

[0021] When a method for detecting dopamine based on an in-situ fluorescence reaction triggered by an imidazole substance is proposed, the pH of the Tris-HCl buffer solution is 7.4, the concentration of the Tris-HCl buffer solution is 10 mM, the concentration of 1,5-dihydroxynaphthalene is 50 μM, and the concentration of 1-(3-aminopropyl)imidazole is 100 mM; take 300 μL of the Tris-HCl buffer solution, add 50 μM of 1,5-dihydroxynaphthalene and different concentrations of dopamine from 0 to 200 μM, and 100 mM of 1-(3-aminopropyl)imidazole, shake well, react for 5 min, measure and plot the fluorescence intensity graph with a fluorescence spectrometer, and record the change in fluorescence intensity before and after adding dopamine.

[0022] 4. A method for detecting dopamine based on an in-situ fluorescence reaction triggered by an imidazole substance, comprising the following steps: Under room temperature conditions, in a Tris-HCl + 1% TW 80 buffer solution, add dopamine, 1,5-dihydroxynaphthalene, and 1-(3-aminopropyl)imidazole, mix evenly to generate a yellow fluorescent compound AFC, and perform real-time fluorescence intensity detection on the synthesized AFC with a fluorescence spectrometer.

[0023] The real-time detection of dopamine is achieved by performing real-time fluorescence intensity detection on the synthesized AFC with a fluorescence spectrometer, and the fluorescence intensity of AFC gradually increases with the increase in dopamine concentration, thereby realizing the quantitative detection of dopamine.

[0024] When a method for detecting dopamine based on an in-situ fluorescence reaction triggered by an imidazole substance is proposed, the buffer solution is Tris-HCl + 1% TW 80, the concentration of 1,5-dihydroxynaphthalene is 50 μM, and the concentration of 1-(3-aminopropyl)imidazole is 100 mM; take 300 μL of Tris-HCl + 1% TW 80 buffer solution, add 50 μM 1,5-dihydroxynaphthalene and different concentrations of dopamine from 0 to 100 pM, and 100 mM 1-(3-aminopropyl)imidazole respectively, shake well, react for 5 min, and measure and plot the real-time fluorescence intensity graph through a fluorescence spectrometer, and record the change in fluorescence intensity before and after adding dopamine.

[0025] 5. A method for detecting dopamine in a complex environmental system based on an in-situ fluorescence reaction triggered by an imidazole substance. At room temperature, add a certain amount of human urine to Tris-HCl, then add a certain amount of 1,5-dihydroxy, different concentrations of dopamine and a certain amount of 1-(3-aminopropyl)imidazole, and AFC will be generated. The fluorescence intensity of the synthesized AFC is detected by a fluorescence spectrometer to achieve the detection of dopamine, and the fluorescence intensity of the fluorescent compound gradually increases with the increase of dopamine concentration, thereby realizing the quantitative detection of dopamine in a complex environmental system.

[0026] When developing a method for detecting dopamine in a complex environmental system based on an in-situ fluorescence reaction triggered by an imidazole substance, the pH of Tris-HCl is 7.4, the concentration of Tris-HCl is 10 mM, the concentration of 1,5-dihydroxynaphthalene is 50 μM, and the concentration of 1-(3-aminopropyl)imidazole is 100 mM; take 300 μL of Tris-HCl, add 1% of human urine, then add 50 μM 1,5-dihydroxynaphthalene and different concentrations of dopamine, and 100 mM 1-(3-aminopropyl)imidazole, shake well, after reacting for 5 min, measure and plot the fluorescence intensity graph through a fluorescence spectrometer, and record the change in fluorescence intensity before and after adding dopamine.

[0027] 6. A preparation method of a kit for detecting different sources of dopamine based on an in-situ fluorescence reaction triggered by an imidazole substance. First, under the action of 1-(3-aminopropyl)imidazole, add different concentrations of dopamine and 1,5-dihydroxynaphthalene of one source, mix evenly to generate a yellow fluorescent compound AFC, detect the fluorescence intensity of the synthesized AFC by a fluorescence spectrometer, and the detection process of other different sources of dopamine is the same as this;

[0028] When preparing a kit for detecting dopamine from different sources based on an in-situ fluorescence reaction triggered by an imidazole substance, the pH of the Tris-HCl buffer solution is 7.4, the concentration of Tris-HCl is 10 mM, dopamine from different sources and at different concentrations, the concentration of 1,5-dihydroxynaphthalene is 50 μM, the concentration of 1-(3-aminopropyl)imidazole is 100 mM, the reaction time is 5 min, and the fluorescence intensity of the synthesized AFC is detected by a fluorescence spectrometer to achieve quantitative detection of dopamine.

[0029] 7. Application of the method for preparing a kit for detecting dopamine from different sources based on an in-situ fluorescence reaction triggered by an imidazole substance. First, add a certain amount of urine samples from different patients, then add a certain amount of 1,5-dihydroxynaphthalene and 1-(3-aminopropyl)imidazole. After reaction and mixing, AFC is generated, and the fluorescence intensity of AFC gradually increases with the increase of dopamine, thereby realizing quantitative detection of dopamine in the urine samples of patients.

[0030] When preparing a kit for detecting dopamine from different sources based on an in-situ fluorescence reaction triggered by an imidazole substance, the conditions of this fluorescence sensing platform are as follows: the pH of the Tris-HCl buffer solution is 7.4, the concentration of Tris-HCl is 10 mM, the concentration of 1,5-dihydroxynaphthalene is 50 μM, the concentration of 1-(3-aminopropyl)imidazole is 100 mM, the urine samples of patients are diluted to 1%, the reaction time is 5 min, and the fluorescence intensity of the synthesized AFC is detected by a fluorescence spectrometer to quantitatively detect the content of dopamine in the urine of patients.

[0031] Advantages of the present invention:

[0032] 1. As a Lewis base catalyst, 1-(3-aminopropyl)imidazole can quickly trigger the fluorescence reaction between dopamine and 1,5-dihydroxy to form a yellow fluorescent compound, and the fluorescence intensity of the synthesized fluorescent compound is detected by a fluorescence spectrometer.

[0033] 2. The in-situ fluorescence reaction triggered by an imidazole substance can quickly form a yellow fluorescent compound AFC. In addition, the fluorescence intensity of AFC gradually increases with the increase of dopamine concentration. Quantitative detection of dopamine can be achieved through the change of fluorescence intensity. The method for detecting dopamine by in-situ forming a fluorescent compound in the present invention has mild synthesis conditions, strong and stable fluorescence, and has the advantages of rapid response, high selectivity and high sensitivity for the detection of dopamine. These studies provide a new method for realizing high-sensitivity and high-selectivity detection of dopamine.

[0034] The established in-situ fluorescence reaction system has good analytical data and has broad application prospects in the detection of dopamine-related diseases.

[0035] 3. Using this in-situ fluorescence reaction, a preparation method for a detection kit for different sources of dopamine was constructed with dopamine from different sources as the carrier.

[0036] At room temperature, in a complex system containing human urine, dopamine and a certain amount of 1,5-dihydroxynaphthalene and 1-(3-aminopropyl)imidazole were added, mixed evenly, and reacted for 5 min to generate AFC. The fluorescence intensity of the fluorescent compound would increase with the increase in the dopamine concentration, which was used to detect dopamine in the complex system.

[0037] An application of an in-situ fluorescence reaction system initiated by an imidazole substance in the preparation method of a detection kit for different sources of dopamine. Under the action of 1-(3-aminopropyl)imidazole, urine samples from different patients and 1,5-dihydroxynaphthalene were added, mixed evenly, and then tested in a fluorescence spectrometer after 5 min; first, the dopamine content in the urine samples of different patients was measured using a human-derived dopamine standard kit, and then the samples were diluted according to the previously tested standard curve to make their concentrations within the curve range with a dilution factor of 10 times, and then the test was carried out according to the above steps; according to the fluorescence intensity of the formed AFC, it was used to detect dopamine in the urine samples of patients.

[0038] 4. In order to correctly evaluate the catalytic activities of imidazoles and imidazole derivatives, 21 different imidazoles were measured, such as imidazole, 2-methylimidazole, 2-propylimidazole, etc., and other catalysts (potassium permanganate, sodium hydroxide, HRP + H2O2, Cu 2 + ) were compared in the fluorescence reaction system of dopamine and 1,5-dihydroxynaphthalene. It was found that they all had catalytic activities, but the catalytic strengths were different. Then, 1-(3-aminopropyl)imidazole with the best catalytic effect was selected as the Lewis base to catalyze the fluorescence reaction.

[0039] At room temperature, in a Tris-HCl buffer solution, dopamine, a certain amount of 1,5-dihydroxynaphthalene and 21 different imidazoles were added, mixed evenly, and reacted for 5 min to generate a yellow fluorescent compound. The fluorescence intensity of the yellow fluorescent compound would be different with different added imidazoles, which was used to distinguish the catalytic strengths of imidazoles in a complex environmental system.

[0040] The synthesis conditions were as follows: the pH of the Tris-HCl buffer solution was 7.4, the concentration of the Tris-HCl buffer solution was 10 mM, the fixed concentration of dopamine was 100 μM, the concentration of 1,5-dihydroxynaphthalene was 50 μM, and the concentrations of 21 different imidazoles were all 100 mM, such as imidazole, 2-methylimidazole, 2-propylimidazole, etc. The three would react to generate a yellow fluorescent compound, and the fluorescence intensity of the synthesized yellow fluorescent compound was detected by a fluorescence spectrometer, and then the catalytic strengths of different imidazoles could be distinguished through the fluorescence intensity.

[0041] 5. The system can also be used for the detection of DA in clinical urine samples. The established in-situ fluorescence reaction system has good analytical data and broad application prospects in the detection of dopamine-related diseases.

[0042] 6. It can be seen from Example 9 that 1-(3-aminopropyl)imidazole as a catalyst can achieve high sensitivity for the detection of dopamine at low concentrations compared with the other four catalysts (potassium permanganate, sodium hydroxide, HRP+H2O2, Cu 2+ ). The detection range of this method for dopamine is 0-200 μM, and the detection limit is 33.3 pM. 1-(3-aminopropyl)imidazole is the best catalyst.

[0043] It can be seen from Example 11 that 1-(3-aminopropyl)imidazole as a catalyst has better specificity for the detection of dopamine at low concentrations compared with the other four catalysts (potassium permanganate, sodium hydroxide, HRP+H2O2, Cu 2+ ). For example Figure 14 , Figure 15 , Figure 16 , Figure 17 . In comparison, 1-(3-aminopropyl)imidazole as a catalyst is more specific for the detection of dopamine at low concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The embodiments of the present invention will be further described below in conjunction with the drawings.

[0045] Figure 1 Fluorescence intensity diagram of the fluorescent compound prepared in Example 1

[0046] Figure 2 Relative fluorescence intensity histogram of the fluorescent compound prepared in Example 1

[0047] Figure 3 Relative fluorescence intensity histogram of the fluorescent compound prepared in Example 2 after adding a reducing agent

[0048] Figure 4 Scanning time diagram of the fluorescence system with different concentrations of imidazole added in Example 3

[0049] Figure 5 Fluorescence emission spectrum diagram of AFC prepared in Example 4

[0050] Figure 6 Time scanning diagram of AFC prepared in Example 5

[0051] Figure 7 Fluorescence lifetime scanning diagram of AFC prepared in Example 6

[0052] Figure 8 AFC mechanism diagram obtained in Example 7

[0053] Figure 9 Fluorescence intensity diagram of AFC prepared with different concentrations of dopamine in Example 8

[0054] Figure 10 Fluorescence intensity diagram of AFC prepared with low concentration of dopamine in Example 8

[0055] Figure 11 Scatter plot of relative fluorescence intensity of AFC prepared with different concentrations of dopamine in Example 8

[0056] Figure 12 Standard curve diagram of AFC prepared with different concentrations of dopamine in Example 8

[0057] Figure 13 Real-time fluorescence intensity diagram of AFC prepared with extremely low dopamine in Example 9

[0058] Figure 14 Real-time fluorescence standard curve diagram of AFC prepared with extremely low concentration of dopamine in Example 9

[0059] Figure 15 Normalized fluorescence intensity bar chart of different dopamine interfering substances prepared with imidazole as catalyst in Example 12

[0060] Figure 16 Normalized fluorescence intensity bar chart of different dopamine interfering substances prepared with potassium permanganate as catalyst in Example 13

[0061] Figure 17 Normalized fluorescence intensity bar chart of different dopamine interfering substances prepared with sodium hydroxide as catalyst in Example 13

[0062] Figure 18 Normalized fluorescence intensity bar chart of different dopamine interfering substances prepared with HRP + H2O2 as catalyst in Example 13

[0063] Figure 19 For Example 13, Cu 2+ Normalized fluorescence intensity bar chart of different dopamine interfering substances prepared as catalyst

[0064] Figure 20 Schematic diagram for detecting dopamine of different sources

[0065] Figure 21 Fluorescence intensity diagram of AFC prepared for detecting human dopamine in Example 14

[0066] Figure 22Standard curve graph of AFC prepared for detecting human dopamine in Example 14

[0067] Figure 23 Fluorescence intensity graph of AFC prepared for detecting equine dopamine in Example 15

[0068] Figure 24 Standard curve graph of AFC prepared for detecting equine dopamine in Example 15

[0069] Figure 25 Fluorescence intensity graph of AFC prepared for detecting canine dopamine in Example 16

[0070] Figure 26 Standard curve graph of AFC prepared for detecting canine dopamine in Example 16

[0071] Figure 27 Fluorescence intensity graph of AFC prepared for detecting rabbit dopamine in Example 17

[0072] Figure 28 Standard curve graph of AFC prepared for detecting rabbit dopamine in Example 17

[0073] Figure 29 Fluorescence intensity graph of AFC prepared for detecting murine dopamine in Example 18

[0074] Figure 30 Standard curve graph of AFC prepared for detecting murine dopamine in Example 18

[0075] Figure 31 Schematic diagram of the present invention Detailed implementation manners

[0076] The following is a detailed description of the embodiments of the present invention with reference to the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and the detailed implementation manners and processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0077] Example 1: At room temperature, take 300 μL of 10 mM Tris-HCl buffer solution, and add 100 μM dopamine, 50 μM 1,5-dihydroxynaphthalene, and 100 mM of 21 different imidazoles, such as imidazole, 2-methylimidazole, 2-propylimidazole, etc., and shake well. After reacting for 5 minutes, under ultraviolet light irradiation, yellow fluorescence is observed, indicating the successful preparation of the yellow fluorescent azo compound. The fluorescence intensity of the fluorescent compound is detected by a fluorescence spectrometer, and a fluorescence intensity graph and a relative fluorescence intensity bar graph are drawn, such as Figure 1 , Figure 2 . And summarize the comparison of the fluorescence reaction system intensities of 21 imidazoles catalyzed and different catalysts catalyzing dopamine and 1,5-dihydroxynaphthalene, Table 1.

[0078] Table 1 Comparison of the fluorescence reaction system intensities of 21 imidazoles and different catalysts for dopamine and 1,5-dihydroxynaphthalene

[0079]

[0080] Example 2: At room temperature, take 300 μL of 10 mM Tris-HCl buffer solution with a pH of 7.4 in the Tris-HCl buffer solution, and add 100 μM dopamine, 50 μM 1,5-dihydroxynaphthalene, and 100 mM 1-(3-aminopropyl)imidazole respectively, and shake well. After reacting for 5 min, under ultraviolet light irradiation, yellow fluorescence was observed. Then, different reducing agents were added, and it was found that the fluorescence intensity decreased significantly, proving that this fluorescence reaction was caused by Lewis base catalysis. The fluorescence intensity of the fluorescent compound was detected by a fluorescence spectrometer, and a relative fluorescence intensity histogram was plotted, as Figure 3 。

[0081] Example 3: In order to select a suitable imidazole concentration to catalyze this in-situ fluorescence reaction, at room temperature, take 300 μL of 10 mM Tris-HCl buffer solution, and add 100 μM dopamine, 50 μM 1,5-dihydroxynaphthalene, and different concentrations of 1-(3-aminopropyl)imidazole (1 nM, 100 nM, 1 μM, 100 μM, 1 mM, 100 mM, 250 mM, 500 mM, 1 M, 2 M) respectively, and shake well. Time scanning was performed on the fluorescent compound by a fluorescence spectrometer with an excitation wavelength of 490 nm and an emission wavelength of 540 nm, and a time scan graph (lines from bottom to top) was plotted, as Figure 4 。It can be seen from the figure that the greater the concentration of imidazole added, the shorter the time to reach stability. Considering the actual analysis application, the concentration is 100 mM.

[0082] Example 4: At room temperature, take 300 μL of 10 mM Tris-HCl buffer solution, and add 100 μM dopamine, 50 μM 1,5-dihydroxynaphthalene, and 100 mM 1-(3-aminopropyl)imidazole respectively, and shake well. After reacting for 5 min, under ultraviolet light irradiation, yellow fluorescence was observed, indicating the successful preparation of the fluorescent azo compound AFC. The fluorescence intensity of AFC was detected by a fluorescence spectrometer, and a fluorescence emission spectrum graph was plotted, as Figure 5 。The excitation wavelength of this substance is 490 nm, and the emission wavelength is 540 nm.

[0083] Example 5: At room temperature, take 300 μL of 10 mM Tris-HCl buffer solution, and add 100 μM dopamine, 50 μM 1,5-dihydroxynaphthalene, and 100 mM 1-(3-aminopropyl)imidazole respectively. Shake well, and perform a time scan on AFC using a fluorescence spectrometer. The excitation wavelength is 490 nm, and the emission wavelength is 540 nm. Draw a time scan graph, as shown in Figure 6 . It shows that the fluorescence intensity system of the generated substance reaches stability at 5 min.

[0084] Example 6: At room temperature, take 300 μL of 10 mM Tris-HCl buffer solution, and add 100 μM dopamine, 50 μM 1,5-dihydroxynaphthalene, and 100 mM 1-(3-aminopropyl)imidazole respectively. Shake well. After reacting for 5 min, under ultraviolet light irradiation, yellow fluorescence is observed, indicating the successful preparation of AFC. The prepared yellow fluorescent compound is used to draw the fluorescence lifetime of the yellow fluorescent organic matter through lifetime detection. As shown in Figure 7 . The lifetime is 4.37 ns.

[0085] Example 7: At room temperature, take 300 μL of 10 mM Tris-HCl buffer solution, and add 100 μM dopamine, 50 μM 1,5-dihydroxynaphthalene, and 100 mM 1-(3-aminopropyl)imidazole respectively. Shake well. After reacting for 5 min, under ultraviolet light irradiation, yellow fluorescence is observed. This fluorescence reaction can be used to detect dopamine, and the reaction mechanism is as shown in Figure 8 .

[0086] Example 8: At room temperature, take 300 μL of 10 mM Tris-HCl buffer solution, and add 50 μM 1,5-dihydroxynaphthalene, 100 mM 1-(3-aminopropyl)imidazole, and 0 - 200 μM dopamine respectively. Shake well. After reacting for 5 min, under ultraviolet light irradiation, yellow fluorescence is observed, indicating the successful preparation of AFC. Perform fluorescence intensity detection on AFC using a fluorescence spectrometer. The excitation wavelength is 490 nm, the emission wavelength is 540 nm, and the slit is (5, 5). Draw a fluorescence intensity graph, a low-concentration fluorescence intensity graph, a relative fluorescence intensity scatter plot, and a standard curve graph, as shown in Figure 9 、 Figure 10 、 Figure 11 and Figure 12 . It shows that the detection range of this method for dopamine is 0 - 200 μM, and the detection limit is 33.3 pM.

[0087] Example 9: At room temperature, take 300 μL of 10 mM Tris-HCl + 1% TW 80 buffer solution, and add 5 nM of 1,5-dihydroxynaphthalene, 100 mM of 1-(3-aminopropyl)imidazole, and 0 - 100 pM of dopamine respectively, then shake well. After reacting for 5 min, under ultraviolet light irradiation, yellow fluorescence was observed, indicating the successful preparation of AFC. The real-time fluorescence intensity of AFC was detected by a fluorescence spectrometer, with an excitation wavelength of 490 nm, an emission wavelength of 540 nm, and slits (10, 20). The real-time fluorescence intensity graph and the relative fluorescence intensity scatter plot were drawn, as shown in Figure 13 , Figure 14 . It shows that the detection range of dopamine by this method is 0 - 100 pM, and the detection limit is 66.7 fM.

[0088] Example 10: At room temperature, take 300 μL of 10 mM different buffer solutions (Tris-HCl + 1% TW 80, Tris-HCl + 1% TW 20, PBS, TBST, Hepes), and add 5 nM of 1,5-dihydroxynaphthalene, 100 mM of 1-(3-aminopropyl)imidazole, and 0 - 100 pM of dopamine respectively, then shake well. The real-time fluorescence intensity of AFC was detected by a fluorescence spectrometer, with an excitation wavelength of 490 nm, an emission wavelength of 540 nm, and slits (10, 20). The detection limit comparison table was drawn, as shown in Table 2. It shows that the buffer solution Tris-HCl + 1% TW 80 has a lower detection limit for dopamine.

[0089] Table 2 shows the detection range and detection limit comparison of dopamine detection under different buffer solutions in Example 10

[0090]

[0091] Example 11: 1-(3-aminopropyl)imidazole as a catalyst can achieve high sensitivity for dopamine detection at low concentrations compared with the other four catalysts (potassium permanganate, sodium hydroxide, HRP + H2O2, Cu 2+ ). At room temperature, take 300 μL of 10 mM Tris-HCl buffer solution, and add 50 μM of 1,5-dihydroxynaphthalene, 100 mM of potassium permanganate, sodium hydroxide, Cu 2+ , and HRP (4 mU / mL) + H2O2 (10 mM) and 0 - 200 μM of dopamine respectively, then shake well. After reacting for 5 min, under ultraviolet light irradiation, yellow fluorescence was observed. The fluorescence intensity of AFC was detected by a fluorescence spectrometer, with an excitation wavelength of 490 nm and an emission wavelength of 540 nm. The detection limit comparison table was drawn, as shown in Table 2. It shows that 1-(3-aminopropyl)imidazole as a catalyst has high sensitivity for dopamine detection at low concentrations

[0092] Table 3 shows the comparison of the detection range and detection limit of dopamine under different catalysts in Example 11

[0093]

[0094] Example 12: At room temperature, take 35 portions of 300 μL of 10 mM Tris-HCl buffer solution, add 50 μM of 1,5-dihydroxynaphthalene and 100 mM of 1-(3-aminopropyl)imidazole respectively, shake well, and then add 100 μM, 10 μM, 100 nM, 10 nM of different substances: 1. epinephrine hydrochloride; 2. norepinephrine; 3. serotonin hydrochloride; 4. levodopa; 5. methyldopamine; 6. dopamine; 7. vanillylmandelic acid respectively, and shake well. After reacting for 5 min, the fluorescence intensity is detected by a fluorescence spectrometer, the excitation wavelength is 490 nm, and the emission wavelength is 540 nm. Draw a three-dimensional bar chart of the fluorescence normalized intensity, as Figure 15 . This method has specificity for the detection of dopamine.

[0095] Example 13: 1-(3-aminopropyl)imidazole as a catalyst has better specificity for the detection of dopamine at low concentrations than the other four catalysts (potassium permanganate, sodium hydroxide, HRP + H2O2, Cu 2+ ). Take 35 portions of 300 μL of 10 mM Tris-HCl buffer solution for each catalyst, add 50 μM of 1,5-dihydroxynaphthalene and 100 mM of three catalysts respectively, shake well, and then add 100 μM, 10 μM, 100 nM, 10 nM of different substances: 1. epinephrine hydrochloride; 2. norepinephrine; 3. serotonin hydrochloride; 4. levodopa; 5. methyldopamine; 6. dopamine; 7. vanillylmandelic acid respectively, and shake well. After reacting for 5 min, the fluorescence intensity is detected by a fluorescence spectrometer, the excitation wavelength is 490 nm, and the emission wavelength is 540 nm. Draw the three-dimensional bar charts of the fluorescence normalized intensity catalyzed by potassium permanganate, sodium hydroxide, and HRP + H2O2 respectively, as Figure 16 , Figure 17 , Figure 18 , Figure 19 . In comparison, 1-(3-aminopropyl)imidazole as a catalyst has more specificity for the detection of dopamine at low concentrations.

[0096] Example 14: At room temperature, take 300 μL of 10 mM Tris-HCl buffer solution, and add 0 - 120 nM of human dopamine, 50 μM of 1,5-dihydroxynaphthalene, and 100 mM of 1-(3-aminopropyl)imidazole respectively, then shake well. After reacting for 5 min, under the irradiation of an ultraviolet lamp, yellow fluorescence is observed, indicating the successful preparation of AFC. The fluorescence intensity of AFC is detected by a fluorescence spectrometer, and the fluorescence emission spectrum and standard curve are plotted, as shown in Figure 20 and Figure 21 . It shows that the detection range of this method for dopamine is 0 - 120 nM, and the detection limit is 0.17 nM.

[0097] Example 15: At room temperature, take 300 μL of 10 mM Tris-HCl buffer solution, and add 0 - 1.31 nM of horse dopamine, 50 μM of 1,5-dihydroxynaphthalene, and 100 mM of 1-(3-aminopropyl)imidazole respectively, then shake well. After reacting for 5 min, under the irradiation of an ultraviolet lamp, yellow fluorescence is observed, indicating the successful preparation of AFC. The fluorescence intensity of AFC is detected by a fluorescence spectrometer, and the fluorescence emission spectrum and standard curve are plotted, as shown in Figure 22 and Figure 23 . It shows that the detection range of this method for dopamine is 0 - 1.31 μM, and the detection limit is 0.001 nM.

[0098] Example 16: At room temperature, take 300 μL of 10 mM Tris-HCl buffer solution, and add 0 - 1.04 nM of canine dopamine, 50 μM of 1,5-dihydroxynaphthalene, and 100 mM of 1-(3-aminopropyl)imidazole respectively, then shake well. After reacting for 5 min, under the irradiation of an ultraviolet lamp, yellow fluorescence is observed, indicating the successful preparation of AFC. The fluorescence intensity of AFC is detected by a fluorescence spectrometer, and the fluorescence emission spectrum and standard curve are plotted, as shown in Figure 24 and Figure 25 . It shows that the detection range of this method for dopamine is 0 - 1.04 nM, and the detection limit is 0.002 nM.

[0099] Example 17: At room temperature, take 300 μL of 10 mM Tris-HCl buffer solution, and add 0 - 0.52 nM of rabbit dopamine, 50 μM of 1,5-dihydroxynaphthalene, and 100 mM of 1-(3-aminopropyl)imidazole respectively, then shake well. After reacting for 5 min, under the irradiation of an ultraviolet lamp, yellow fluorescence is observed, indicating the successful preparation of AFC. The fluorescence intensity of AFC is detected by a fluorescence spectrometer, and the fluorescence emission spectrum and standard curve are plotted, as shown in Figure 26 and Figure 27 . It shows that the detection range of this method for dopamine is 0 - 0.52 nM, and the detection limit is 0.002 nM.

[0100] Example 18: At room temperature, take 300 μL of 10 mM Tris-HCl buffer solution, add 0 - 100 nM murine dopamine, 50 μM 1,5-dihydroxynaphthalene, and 100 mM 1-(3-aminopropyl)imidazole respectively, and shake well. After reacting for 5 min, under ultraviolet light irradiation, yellow fluorescence was observed, indicating the successful preparation of AFC. The fluorescence intensity of AFC was detected by a fluorescence spectrometer, and the fluorescence emission spectrum and standard curve were plotted, as shown in Figure 28 and Figure 29 . It shows that the detection range of this method for dopamine is 0 - 100 nM, and the detection limit is 0.25 nM.

[0101] Example 19: Take 300 μL of 10 mM Tris-HCl buffer solution, add 1% human urine, then add 50 μM 1,5-dihydroxynaphthalene, 100 mM 1-(3-aminopropyl)imidazole, and different concentrations of dopamine (0.01 μM, 0.1 μM, 10 μM, 50 μM, and 150 μM), shake well. After reacting for 5 min, the fluorescence intensity of AFC was detected by a fluorescence spectrometer; the recovery rate of dopamine was calculated, as shown in Table 4. It shows that this method can also detect dopamine in a complex system.

[0102] Table 4 shows the recovery rate of dopamine in urine samples in Example 19

[0103]

[0104] Example 20: First, calculate the concentration of dopamine in each patient's urine sample using a standard kit. Then, according to the routine operation of this fluorescence immunoassay platform, add 1% of the patient's urine to 300 μL of 10 mM Tris-HCl buffer solution, then add 50 μM 1,5-dihydroxynaphthalene and 100 mM 1-(3-aminopropyl)imidazole, shake well. After reacting for 5 min and mixing evenly, fluorescence intensity measurement was carried out and the recovery rate of dopamine was calculated, and it was compared with that of the standard kit, as shown in Table 5. It shows that this method can be applied to actual patient samples.

[0105] Table 5 shows the comparison of the recovery rate of detecting dopamine in patient urine samples in Example 20 with that of the standard kit

[0106]

[0107] Example 21: The time and cost of testing with the fluorescence immunoassay platform and commercial kits are listed (Tables 5 - 7). Compared with the standard ELISA used for detecting samples, our fluorescence immunoassay platform shows advantages of being faster and cheaper. This fluorescence immunoassay platform only takes 13 minutes and costs 1.2495 yuan to test one sample. However, the traditional ELISA method takes 20 minutes and costs 12.968 yuan respectively, not including the time for preparing the ELISA kit. The results show that the system we developed has a reliable DA evaluation ability in clinical samples and can provide important indicators for the diagnosis of DA - related diseases.

[0108] Table 6 shows the comparison between this fluorescence method and the kit in Example 21

[0109]

[0110] Table 7 shows the cost of the kit used to detect dopamine in Example 21

[0111]

[0112] Table 8 shows the cost of the fluorescence method used to detect dopamine in Example 21

[0113]

[0114] The present invention is not limited to the specific technical solutions described in the above embodiments, and all technical solutions formed by equivalent substitution are within the protection scope required by the present invention.

Claims

1. A method for detecting dopamine from different sources using an in-situ fluorescence reaction system initiated by imidazole-based substances, characterized in that: It includes the following steps: An in-situ fluorescence reaction can occur between dopamine and phenolic compounds. Under the action of 1-(3-aminopropyl)imidazole, 1,5-dihydroxynaphthalene reacts with dopamine to generate a yellow azo fluorescent compound AFC. Based on the change in fluorescence intensity, dopamine is detected. At room temperature, in a Tris-HCl buffer solution with a pH of 7.4, different concentrations of dopamine ranging from 0 to 200 μM, dopamine, 1,5-dihydroxynaphthalene, and 1-(3-aminopropyl)imidazole are added and mixed evenly to generate the yellow fluorescent compound AFC. The fluorescence intensity of the synthesized AFC is detected by a fluorescence spectrometer. High-sensitivity and high-selectivity quantitative detection of dopamine is achieved based on the change in fluorescence intensity. Synthesis conditions of AFC: The concentration of the Tris-HCl buffer solution is 10 mM, dopamine is added, the concentration of 1,5-dihydroxynaphthalene is 50 μM, the concentration of 1-(3-aminopropyl)imidazole is 100 mM, and the reaction time is 5 min.

2. The method for detecting dopamine of different sources by using the in-situ fluorescence reaction system triggered by imidazole substances according to claim 1, wherein: At room temperature, in a Tris-HCl buffer solution, under the action of 1-(3-aminopropyl)imidazole, different sources of dopamine react with 1,5-dihydroxynaphthalene for 5 min to generate AFC. The fluorescence intensity of the synthesized AFC is detected by a fluorescence spectrometer. High-sensitivity and high-selectivity detection of dopamine is achieved based on the change in fluorescence intensity.

3. The method for detecting dopamine of different sources by using the in-situ fluorescence reaction system triggered by imidazole substances according to claim 1, characterized in that: It includes the following steps: First, under the action of 1-(3-aminopropyl)imidazole, different concentrations of dopamine from one source and 1,5-dihydroxynaphthalene are added and mixed evenly, and the reaction lasts for 5 min to generate the yellow fluorescent compound AFC. The fluorescence intensity of the synthesized AFC is detected by a fluorescence spectrometer. The detection processes of other different sources of dopamine are the same as this one. The conditions of the sensing platform are as follows: The pH of the Tris-HCl buffer solution is 7.4, the concentration of Tris-HCl is 10 mM, different concentrations of dopamine from different sources, the concentration of 1,5-dihydroxynaphthalene is 50 μM, the concentration of 1-(3-aminopropyl)imidazole is 100 mM, and the reaction time is 5 min. The fluorescence intensity of the synthesized AFC is detected by a fluorescence spectrometer to quantitatively detect dopamine.

4. The method for detecting dopamine of different sources by using the in-situ fluorescence reaction system initiated by imidazole substances according to claim 1, characterized in that: The different sources of dopamine include human dopamine, horse dopamine, canine dopamine, rabbit dopamine, murine dopamine, or human urine.

5. The method for detecting different sources of dopamine using the in-situ fluorescence reaction system triggered by imidazole substances according to claim 1, wherein: The fluorescence intensity of AFC is detected by a fluorescence spectrometer, with an excitation wavelength of 490 nm and an emission wavelength of 540 nm. The detection range of this method for dopamine is 0 - 200 μM, and the detection limit is 33.3 pM.

6. A method for detecting dopamine from different sources using an in-situ fluorescence reaction system initiated by imidazole-based substances, characterized in that: At room temperature, take 300 μL of Tris-HCl + 1% TW 80 buffer solution, and add 5 nM of 1,5-dihydroxynaphthalene, 100 mM of 1-(3-aminopropyl)imidazole, and 0 - 100 pM of dopamine respectively. Shake well. After reacting for 5 min, under the irradiation of an ultraviolet lamp, yellow fluorescence is observed, indicating the successful preparation of AFC. The fluorescence intensity of AFC is detected by a fluorescence spectrometer. The excitation wavelength is 490 nm and the emission wavelength is 540 nm. The detection range of this method for dopamine is 0 - 100 pM, and the detection limit is 66.7 fM.

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