A Ratiometric Dopamine Fluorescent Probe and Its Application for the Kinetic and Highly Selective Determination of Dopamine

Abstract

The present invention discloses a ratio-type dopamine fluorescent probe and its application for the kinetic highly selective determination of dopamine. The method for determining dopamine is as follows: After adjusting the pH value of a 3,5-dihydroxybenzoic acid solution with a base, it is used as a fluorescent probe. After reacting with the test solution, the fluorescence emission spectra are measured respectively at excitation wavelengths of 340 ± 10 nm and 375 ± 10 nm. The dopamine concentration is calculated through the ratio equation of the intensity ratio between the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine and the strongest peak of 3,5-dihydroxybenzoic acid in the fluorescence emission spectrum. The 3,5-dihydroxybenzoic acid probe prepared by the present invention has excellent selectivity, very high stability, high sensitivity based on the ratio-type probe, extremely low detection limit and good biocompatibility, and can be used for the detection of dopamine. At present, it has been successfully applied to the selective detection of dopamine in human urine samples and PC12 cell lysates.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and particularly relates to a ratiometric dopamine fluorescent probe and its application for highly selective determination of dopamine by kinetics. Background Art

[0002] Dopamine (DA), as an important neurotransmitter in the human body, affects human behaviors such as thinking, working, and movement. Abnormal content changes of dopamine indicate the occurrence of mental diseases. Therefore, sensitive and selective detection of dopamine is of great significance. Currently, the detection methods for dopamine mainly include electrochemical analysis, high-performance liquid chromatography, capillary electrophoresis, fluorescence spectroscopy, etc. Although high-performance liquid chromatography has obvious advantages in separation, it has high analysis costs and long analysis times. Capillary electrophoresis has short separation times and low costs, but has weak separation capabilities and poor reproducibility. Electrochemical analysis methods are easy to operate and highly sensitive, but have low selectivity, poor repeatability, and stability. The advantages of fluorescence spectroscopy, such as specificity, biocompatibility, and high spatiotemporal resolution, make up for the limitations of other methods, such as low spatiotemporal resolution and expensive instruments. Dopamine and resorcinol and its derivatives will undergo a specific fluorescence reaction and be converted into a bright blue strong fluorescent substance, as shown in formula (1). However, there are some difficult problems in simply using the reaction between resorcinol and dopamine to detect dopamine. One is poor selectivity and the inability to eliminate the interference of catecholamine neurotransmitter analogs. The other is low sensitivity.

[0003] Summary of the Invention

[0004] The object of the present invention is to provide a ratiometric dopamine fluorescent probe and its application for highly selective determination of dopamine by kinetics.

[0005] The structural formula of the ratiometric dopamine fluorescent probe is as follows:

[0006]

[0007] The method for determining dopamine is as follows: Add alkali to a 3,5-dihydroxybenzoic acid solution with a concentration of 0.2 - 1 mM to adjust the pH value to 8 - 11, then add the test solution. The volume ratio of the 3,5-dihydroxybenzoic acid solution to the test solution is 1 - 10:1. Stir and react at room temperature for 120 ± 5 s, and then divide it into two samples. Measure the fluorescence emission spectra at an excitation wavelength of 340 ± 10 nm and an excitation wavelength of 375 ± 10 nm respectively. The ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid in the fluorescence emission spectrum obtained at an excitation wavelength of 340 ± 10 nm is substituted into the high-concentration dopamine ratio equation for calculation. If the obtained concentration is greater than 100 nM, it is the dopamine concentration in the test solution. If the calculated concentration is less than 100 nM, then use the ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid in the fluorescence emission spectrum obtained at an excitation wavelength of 375 ± 10 nm, and substitute it into the low-concentration dopamine ratio equation for calculation. The obtained concentration is the dopamine concentration in the test solution.

[0008] The alkali described above is sodium carbonate and / or sodium hydroxide.

[0009] The method for obtaining the low-concentration dopamine ratio equation is as follows: The same as the above method for determining dopamine, replace the test solution with dopamine solutions with different concentrations within the range of 0 - 100 nM. Measure the fluorescence emission spectra at the same excitation wavelength within the range of 375 ± 10 nm. Plot a standard curve of the ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid in the obtained fluorescence emission spectrum against the dopamine solution concentration, and then the low-concentration dopamine ratio equation F1 = α1C + β1 can be obtained. F1 is the ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid, C is the concentration of the dopamine solution, with the unit of nM, α1 is the slope, and β1 is the intercept.

[0010] The method for obtaining the high-concentration dopamine ratio equation is as follows: The same as the above method for determining dopamine, replace the test solution with dopamine solutions with different concentrations within the range of 0.1 - 50 μM. Measure the fluorescence emission spectra at the same excitation wavelength within the range of 340 ± 10 nm. Plot a standard curve of the ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid in the obtained fluorescence emission spectrum against the dopamine solution concentration, and then the high-concentration dopamine ratio equation F2 = α2C + β2 can be obtained. F2 is the ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid, C is the concentration of the dopamine solution, with the unit of μM, α2 is the slope, and β2 is the intercept.

[0011] The present invention utilizes the fluorescence of 3,5-dihydroxybenzoic acid molecules themselves at 310 nm and the change in fluorescence emission caused by the structures of dopamine and resorcinol, generating a new blue fluorescence emission on top of the original benzoic acid fluorescence emission, presenting a phenomenon of ratiometric fluorescence, thereby enhancing the sensitivity of the fluorescence probe for detecting dopamine, with a detection limit as low as 0.0274 nM. The carboxyl group of 3,5-dihydroxybenzoic acid changes the original electron distribution of resorcinol, making its reaction rate with dopamine faster, dozens of times that of other catecholamine neurotransmitters. Therefore, dopamine can be selectively detected by controlling the reaction time, as Figure 1 shown. The 3,5-dihydroxybenzoic acid probe prepared by the present invention has excellent selectivity, very high stability, high sensitivity based on the ratiometric probe, extremely low detection limit, and good biocompatibility, and can be used for the detection of dopamine. It has currently been successfully applied to the selective detection of dopamine in human urine samples and PC12 cell lysates. Brief Description of the Drawings

[0012] Figure 1 Fluorescence kinetics of the reaction of the 3,5-dihydroxybenzoic acid molecule with different substituents of the fluorescence probe and different neurotransmitters in Example 1.

[0013] Figure 2 Fluorescence spectra of the 3,5-dihydroxybenzoic acid molecule with different substituents of the fluorescence probe and dopamine under 340 nm excitation and the optimal excitation wavelength in Example 1.

[0014] Figure 3 Ratiometric probe images and the source of the ratiometric probe of the fluorescence probe 3,5-dihydroxybenzoic acid at 340 nm and 375 nm.

[0015] Figure 4 Infrared spectrum and mass spectrum of the reaction product of the fluorescence probe 3,5-dihydroxybenzoic acid and dopamine.

[0016] Figure 5 Fluorescence spectra of the fluorescence probe 3,5-dihydroxybenzoic acid at different pH values.

[0017] Figure 6 Temperature optimization of the reaction for the fluorescence probe 3,5-dihydroxybenzoic acid to detect dopamine.

[0018] Figure 7 Fitting curve graphs of the fluorescence probe 3,5-dihydroxybenzoic acid for detecting low-concentration and high-concentration dopamine.

[0019] Figure 8 Selectivity and interference tests of the fluorescence probe 3,5-dihydroxybenzoic acid.

[0020] Figure 9Stability test for the reaction of the fluorescent probe 3,5-dihydroxybenzoic acid to detect dopamine. Detailed implementation mode

[0021] To illustrate the present invention more clearly, the following examples are listed, but the protection scope of the present invention is not limited to the following examples.

[0022] The structure of the reaction product of the monobenzene ring fluorescent probe 3,5-dihydroxybenzoic acid and dopamine is as follows:

[0023]

[0024] Example 1

[0025] 1. Test the fluorescence properties of 3,5-dihydroxybenzoic acid: Prepare 3,5-dihydroxybenzoic acid solution, resorcinol solution, and 3,5-dihydroxybenzyl alcohol solution with a concentration of 400 μM respectively, add Na2CO3 to adjust the pH to 10, and then add dopamine solutions with concentrations of 0 μM and 30 μM respectively. The volume ratio of the fluorescent probe solution to the dopamine solution is 1:1. Stir at room temperature for 120 s to obtain the product solution, and then use a fluorescence spectrophotometer to measure the fluorescence emission spectrum at an excitation wavelength of 340 nm. The results are as Figure 2 shown. It can be seen from the results that there are obvious optical differences between the reaction products of 3,5-dihydroxybenzoic acid and dopamine and the reaction products of resorcinol, 3,5-dihydroxybenzyl alcohol and dopamine, indicating the feasibility of using 3,5-dihydroxybenzoic acid as a fluorescent probe.

[0026] 2. Obtain the ratio equation for low-concentration dopamine: Add Na2CO3 to the 3,5-dihydroxybenzoic acid solution with a concentration of 400 μM to adjust the pH value to 10, and then add dopamine solutions with concentrations of 0 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, and 100 nM respectively. The volume ratio of the 3,5-dihydroxybenzoic acid solution to the dopamine solution is 1:1. After stirring at room temperature for 120 s, measure the fluorescence emission spectrum at an excitation wavelength of 375 nm. Plot the standard curve of the ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid in the obtained fluorescence emission spectrum against the dopamine solution concentration. The results are as Figure 7 (a,b) shown, that is, the ratio equation for low-concentration dopamine F l = 0.0526C (μM) + 0.4163.

[0027] 3. Obtaining the high-concentration dopamine ratio equation: Add Na2CO3 to a 3,5-dihydroxybenzoic acid solution with a concentration of 400 μM to adjust the pH value to 10, and then add dopamine solutions with concentrations of 0.1 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM respectively. The volume ratio of the 3,5-dihydroxybenzoic acid solution to the dopamine solution is 1:1. After stirring and reacting at room temperature for 120 s, measure the fluorescence emission spectrum using an excitation wavelength of 340 nm. Plot a standard curve of the ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid in the obtained fluorescence emission spectrum against the dopamine solution concentration. The results are as Figure 7 (c, d) shown, and the high-concentration dopamine ratio equation F2 = 0.1532C (nM) + 0.3612 is obtained.

[0028] 4. Detection of dopamine in human urine: Add Na2CO3 to a 3,5-dihydroxybenzoic acid solution with a concentration of 400 μM to adjust the pH value to 10, and then add a human urine sample diluted 100 times. The volume ratio of the 3,5-dihydroxybenzoic acid solution to the diluted human urine solution is 1:1. After stirring and reacting at room temperature for 120 s, divide the sample into two parts, and measure the fluorescence emission spectrum using an excitation wavelength of 340 nm and 375 nm respectively. Obtain the ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid in the two fluorescence emission spectra. First, substitute the fluorescence intensity ratio obtained at an excitation wavelength of 340 nm into the high-concentration dopamine ratio equation for calculation. If the obtained concentration is less than 100 nM, then substitute the fluorescence intensity ratio obtained at an excitation wavelength of 375 nm into the low-concentration dopamine ratio equation for calculation. The obtained concentration is the concentration of dopamine in the test solution. The detection results obtained by this method are basically the same as those obtained by high-performance liquid chromatography. This experiment is repeated three times, and finally the RSD is calculated to be 0.0325.

[0029] Example 2

[0030] Detection of dopamine in PC12 cells: First, divide PC12 cells into four groups for culture. The first group is used as the blank group and only the culture medium is added; the second group contains the culture medium and PC12 cells; then in the third group, incubate PC12 cells with K + (4 mM) in the culture medium for 10 minutes; in the fourth group, incubate PC12 cells with K +(4 mM) was incubated in the culture medium for 30 minutes. Then, the above four samples were centrifuged for 5 minutes to obtain the supernatant, and the centrifugation speed was 500 rpm. Na2CO3 was added to the 3,5-dihydroxybenzoic acid solution with a concentration of 400 μM to adjust the pH value to 10. The solution was divided into four portions, and then each portion was added with the supernatant after centrifugation diluted 100 times. The volume ratio of the 3,5-dihydroxybenzoic acid solution to the diluted PC12 cell lysate was 1:1. After stirring and reacting at room temperature for 120 s, the solution was divided into two samples. The fluorescence emission spectra were measured at the excitation wavelengths of 340 nm and 375 nm respectively, and the ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid in the two fluorescence emission spectra was obtained. For each sample, the ratio of the fluorescence intensities obtained at the excitation wavelength of 340 nm was first substituted into the high-concentration dopamine ratio equation for calculation. It was found that the obtained concentration was less than 100 nM. Then, the ratio of the fluorescence intensities obtained at the excitation wavelength of 375 nm was substituted into the low-concentration dopamine ratio equation for calculation, and the obtained concentration was the dopamine concentration in the test solution. The results showed that: the first group had no fluorescence, and the fluorescence of the second group, the third group, and the fourth group increased successively. Moreover, the detection results obtained by this method were basically the same as those obtained by high-performance liquid chromatography, indicating that the monobenzene ring fluorescent probe 3,5-dihydroxybenzoic acid prepared by the present invention has good potential for intracellular dopamine detection. This experiment was repeated three times to finally calculate the RSD. The above data are shown in the following table:

[0031]

[0032] Example 3

[0033] Selectivity test of the fluorescent probe 3,5-dihydroxybenzoic acid for detecting dopamine: Na2CO3 was added to the 3,5-dihydroxybenzoic acid solution with a concentration of 400 μM to adjust the pH value to 10. Multiple portions of this fluorescent probe solution were prepared, and then K with a concentration of 1 mM was added to the probe solution respectively + , Ca 2+ , Mg 2+ , Zn 2+, solutions of L-alanine (Ala), L-aspartic acid (Asp), arginine (Arg), serine (Ser), glycine (Gly), glutathione (GSH) and glucose (glucose) and various neurotransmitter analogs (DOPA, DOPAM, LNAA), and the volume ratio of the 3,5-dihydroxybenzoic acid solution to the above solution is 1:1. After stirring and reacting at room temperature for 120 s, the mixture is divided into two samples. Respectively, at excitation wavelengths of 340 nm and 375 nm, using a fluorescence spectrophotometer, the change in the ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid is measured under the condition of having / not having dopamine (30 μM). The results are as Figure 8 shown. Other metal ions, organic biological small molecules and other catecholamine neurotransmitters do not interfere with the detection results, indicating that the monobenzene ring fluorescent probe 3,5-dihydroxybenzoic acid prepared by the present invention has high selectivity for dopamine.

[0034] Example 4

[0035] Stability test of the fluorescent probe 3,5-dihydroxybenzoic acid: Add Na2CO3 to a 3,5-dihydroxybenzoic acid solution with a concentration of 400 μM to adjust the pH value to 10, and prepare two groups of such identical probe solutions, with 16 portions of 3,5-dihydroxybenzoic acid probe solution in each group. In the first group of 16 samples, an aqueous dopamine solution with the same concentration is added to 30 nM, and in the second group of samples, an aqueous dopamine solution with the same concentration is added to 20 μM. The volume ratio of the 3,5-dihydroxybenzoic acid solution to the dopamine solution is 1:1. After stirring and reacting at room temperature for 120 s, the fluorescence intensity is measured under an F7000 fluorescence instrument. The first group measures the fluorescence spectrum at an excitation wavelength of 375 nm; the second group measures the fluorescence spectrum at an excitation wavelength of 340 nm. The data of the ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid in the two groups of fluorescence emission spectra are 6.30 and 3.13 respectively; calculate the RSD of the two groups of data, which are 0.0257 and 0.0151 respectively. As Figure 9 shown, it can be seen that the experiment has excellent repeatability.

Claims

1. A method for determining dopamine, characterized in that, The specific operation of the method is as follows: Add alkali to a 3,5-dihydroxybenzoic acid solution with a concentration of 0.2 - 1 mM to adjust the pH value to 8 - 11, then add the test solution. The volume ratio of the 3,5-dihydroxybenzoic acid solution to the test solution is 1 - 10:

1. Stir and react at room temperature for 120 ± 5 s, and then divide it into two samples. Measure the fluorescence emission spectra at excitation wavelengths of 340 ± 10 nm and 375 ± 10 nm respectively. The ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid in the fluorescence emission spectrum obtained at the excitation wavelength of 340 ± 10 nm is substituted into the high-concentration dopamine ratio equation for calculation. If the obtained concentration is greater than 100 nM, it is the concentration of dopamine in the test solution. If the calculated concentration is less than 100 nM, then use the ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid in the fluorescence emission spectrum obtained at the excitation wavelength of 375 ± 10 nm, and substitute it into the low-concentration dopamine ratio equation for calculation. The obtained concentration is the concentration of dopamine in the test solution.

2. The method according to claim 1, wherein The alkali described is sodium carbonate and / or sodium hydroxide.

3. The method according to claim 1, characterized in that, The method for obtaining the low-concentration dopamine ratio equation is as follows: The same as the above method for measuring dopamine, replace the test solution with dopamine solutions with different concentrations in the range of 0 - 100 nM. Measure the fluorescence emission spectra at the same excitation wavelength within the range of 375 ± 10 nm. Plot the standard curve of the ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid in the obtained fluorescence emission spectrum against the concentration of the dopamine solution, and then the low-concentration dopamine ratio equation F1 = α1C + β1 can be obtained. F1 is the ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid, C is the concentration of the dopamine solution, with the unit of nM, α1 is the slope, and β1 is the intercept.

4. The method according to claim 1, wherein The method for obtaining the high-concentration dopamine ratio equation is as follows: The same as the above method for measuring dopamine, replace the test solution with dopamine solutions with different concentrations in the range of 0.1 - 50 μM. Measure the fluorescence emission spectra at the same excitation wavelength within the range of 340 ± 10 nm. Plot the standard curve of the ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid in the obtained fluorescence emission spectrum against the concentration of the dopamine solution, and then the high-concentration dopamine ratio equation F2 = α2C + β2 can be obtained. F2 is the ratio of the intensity of the strongest peak of the reaction product of 3,5-dihydroxybenzoic acid and dopamine to the intensity of the strongest peak of 3,5-dihydroxybenzoic acid, C is the concentration of the dopamine solution, with the unit of μM, α2 is the slope, and β2 is the intercept.

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