Ratio fluorescence sensor for detecting dopamine as well as preparation method and application of ratio fluorescence sensor

Abstract

The invention discloses a ratiometric fluorescence sensor for detecting dopamine as well as a preparation method and application of the ratiometric fluorescence sensor, the raw materials comprise FeCl36H2O and H2BDC, and the molar ratio of FeCl36H2O to H2BDC is 1: (1-1.5). Due to the existence of the specific cyclization reaction between the 1, 3-binaphthol and the dopamine, the sensing system disclosed by the invention has very good specificity. In addition, the reliability of the whole system can be improved through a ratio fluorescence and ultraviolet double-signal output mode, and the ratio fluorescence can better shield the strong biological matrix effect in blood and urine samples. Furthermore, the prepared MIL-101 (Fe) has double mimic enzyme activity of peroxidase and oxidase, and the cyclization reaction can be accelerated due to the existence of multiple free radicals, so that the detection limit of dopamine is further reduced, and the sensitivity of a detection system is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and in particular relates to a ratio fluorescence sensor for detecting dopamine and a preparation method and application thereof. Background Art

[0002] Catecholamines (CAs) are a class of monoamine compounds containing catechol (i.e., catechol) structures, which are converted from L-tyrosine as a precursor. Catecholamines and their metabolites often act as a neurotransmitter or neurohormone in the body and participate in many important physiological processes, such as neurotransmission, cardiovascular regulation, immune response, etc., and are of great significance to the regulation of human physiological functions and the treatment of diseases. Active catecholamines in the human body mainly include epinephrine (E), norepinephrine (NE), and dopamine (DA). Among them, dopamine is an important typical adrenergic receptor agonist, which is involved in functions such as emotion, motivation, and motor control. In addition, changes in dopamine content are also involved in the development of some diseases, such as Parkinson's disease and schizophrenia, and are a very important disease marker. In addition, dopamine can also be used in clinical practice to assist in the diagnosis of endocrine-related diseases such as hypertension, hyperthyroidism, pheochromocytoma, and neuroblastoma. Therefore, accurate monitoring of dopamine levels in the human body can provide guidance for disease development and clinical medication.

[0003] Colorimetry is a classic dopamine detection method. Its principle is that dopamine can increase the electron transfer efficiency of certain metal ions. In the presence of 3,3',5,5'-tetramethylbenzidine, blue oxidized 3,3',5,5'-tetramethylbenzidine is produced. The dopamine content is detected colorimetrically by observing the depth of the color. RGB colorimetry is another method for detecting dopamine content. Its principle is to use dopamine and 3,3',5,5'-tetramethylbenzidine as dual substrates. Under the catalysis of pseudo-oxidase active substances, the former appears orange in the solution and the latter appears blue in the solution. In this way, a simple mixture of the two colors can be used to produce a variety of different color systems. The color comparison can then be observed with the naked eye, or the absorbance can be detected with a spectrophotometer or an enzyme reader. Compared with the change in the depth of the previous single color system, this RGB colorimetric method has higher sensitivity. The principle and reagents used in the colorimetric method are simple, and do not require complex and expensive analytical instruments, but the overall sensitivity is low.

[0004] Electrochemical detection techniques usually have relatively high sensitivity, but the detection process is relatively complicated and requires dedicated detection instruments. The general electrochemical method is to couple the aptamer with the corresponding sensor and then specifically capture dopamine. Therefore, a simple and sensitive electrochemical aptasensor was developed for the detection of dopamine by redox cycle signal amplification using tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent. After the captured dopamine is electrochemically oxidized to dopamine quinones (DAQ), it is immediately regenerated by TCEP, resulting in an enhanced electrochemical signal.

[0005] Fluorescence analysis has a high sensitivity comparable to that of electrochemical methods. It can be used for signal detection with widely used analytical instruments such as microplate readers and fluorescence spectrophotometers. It is a powerful tool for the detection of biological macromolecules including dopamine. Enzyme-linked immunosorbent assay (ELISA) is a classic method for macromolecular immunoassay, but its operation process is extremely cumbersome. First, the analyte is coated in a microplate, and after binding and elution with the primary antibody, an enzyme-labeled secondary antibody is added and eluted again. Finally, a colorimetric substrate is added, and the fluorescence signal of the microplate is detected by a microplate reader to reflect the dopamine content in the sample. Therefore, ELISA is not suitable for sensitive and rapid detection of alkaline phosphatase activity. In recent years, new fluorescent sensing systems based on technologies such as fluorescence resonance energy transfer, fluorescence switch, fluorescence inner filter effect, and photoinduced electron transfer have emerged, providing reference and reference for the development of rapid, simple, and sensitive detection technology for dopamine.

[0006] Therefore, the present invention establishes a dual-mode sensing system based on ratiometric fluorescence and ultraviolet to detect dopamine. Summary of the invention

[0007] Technical problem solved: In response to the above technical problem, the present invention provides a ratio fluorescence sensor for detecting dopamine and a preparation method and application thereof, which can effectively solve the shortcomings of the above method such as complex detection process, high cost and low sensitivity.

[0008] Technical solution: In the first aspect, the present invention provides a ratio fluorescence sensor for detecting dopamine, the raw materials include: FeCl 3 •6H 2 O and H 2 BDC, FeCl 3 •6H 2 O and H 2 The molar ratio of BDC is 1:1 to 1.5.

[0009] In a second aspect, the present invention provides a method for preparing a ratiometric fluorescence sensor for detecting dopamine according to the first aspect, comprising the following steps: Preparation of S1, MIL-101 (Fe): FeCl 3 •6H 2 O and H 2 BDC was added to a round-bottom flask in proportion, and then 50 mL of dimethylformamide was slowly added. The resulting mixture was ultrasonically treated at 37°C for 10-15 min, then transferred to a Teflon reactor, heated at 100-110°C for 20 h, and finally centrifuged at 6000 rpm for 5-10 min to obtain a reddish-brown precipitate. The reddish-brown precipitate was repeatedly washed three times in ethanol to remove unreacted ligand H 2 BDC and metal ions until the supernatant solution is clear and transparent to obtain MIL-101 (Fe), which is then stored at 4°C; S2. Add 1 mL of 1,3-dinaphthol and 10 mL of H 2 O 2 , and finally a ratiometric fluorescence sensor was obtained.

[0010] Preferably, the temperature of the ethanol is 30° C., and the time for each washing in ethanol is 5 min.

[0011] In a third aspect, the present invention provides the use of the ratiometric fluorescence sensor for detecting dopamine described in the first aspect or the ratiometric fluorescence sensor for detecting dopamine prepared by the preparation method described in the second aspect in detecting the dopamine content in a biological sample.

[0012] Preferably, the application comprises the following steps: 1) Place the sample to be tested in a constant temperature oscillator for incubation. After incubation, transfer it to a transparent 96-well plate and a black 96-well plate respectively. Set the UV scanning wavelength to 462 nm and the fluorescence detection wavelength to 440 nm and 480 nm. Perform the test under the test conditions and detect the absorbance and fluorescence intensity at the same time. 2) Calculate the dopamine content in the sample to be tested based on the standard curve.

[0013] Furthermore, the incubation conditions are: pH 6-7, temperature 25-37°C, and time 45-60 min.

[0014] Beneficial effects: The present invention establishes a dual-mode sensing system based on ratio fluorescence and ultraviolet for detecting dopamine. The sensing system has very good specificity due to the presence of a specific cyclization reaction between 1,3-dinaphthol (NR) and dopamine. In addition, the output mode of ratio fluorescence and ultraviolet dual signals can improve the reliability of the entire system, among which ratio fluorescence can better shield the strong biological matrix effect in blood and urine samples. Furthermore, the MIL-101 (Fe) prepared by the present invention has dual mimetic enzyme activity of peroxidase and oxidase, and the presence of multiple free radicals can accelerate the cyclization reaction, thereby further reducing the detection limit of dopamine and improving the sensitivity of the detection system. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the transmission electron microscope characterization image of MIL-101 (Fe); Figure 2 It is the UV spectrum of MIL-101 (Fe); Figure 3 is the fluorescence spectrum of MIL-101 (Fe); Figure 4 This is a standard curve diagram for detecting dopamine in serum using fluorescence spectroscopy; Figure 5 This is a standard curve chart for detecting dopamine in urine using fluorescence spectroscopy. DETAILED DESCRIPTION

[0016] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments: Example 1: Preparation of MIL-101 (Fe) Preparation of MIL-101 (Fe) by hydrothermal method: 0.675 g of FeCl 3 •6H 2 O and 0.206 g of H 2 BDC (2-hydroxyterephthalic acid) was placed in a round-bottom flask, and then 15 mL of DMF (dimethylformamide) was slowly added. The resulting mixture was ultrasonically treated at 37 °C for 10 min, then transferred to a Teflon reactor, heated at 110 °C for 20 h, and then centrifuged at 6000 rpm for 5 min to obtain a reddish-brown precipitate. The reddish-brown precipitate was repeatedly washed three times in hot ethanol (70 °C, 2 h) to remove unreacted ligand H 2 BDC and metal ions were added until the supernatant solution became clear and transparent and finally stored at 4°C.

[0017] The scanning electron microscope images of MIL-101(Fe) at different scales were studied, such as Figure 1As shown: The distance between two adjacent vertices was measured using Image J, and the average size of MIL-101 (Fe) was found to be 600 nm, which is consistent with the measurement results of transmission electron microscopy. In addition, the MIL-101 (Fe) prepared by this method has very good dispersion and crystallinity, and both present a uniform octahedral configuration. The dynamic light scattering measurement results show that the distribution range is very narrow, indicating that the particle size distribution is uniform.

[0018] In order to eliminate the influence of the reaction system on the final detection performance, the UV and fluorescence spectra of MIL-101 (Fe) and its ligand 2-hydroxyterephthalic acid were studied, such as Figure 2 As shown: It can be observed that the significant absorption peak of terephthalic acid itself at 240 nm is greatly reduced after the formation of MIL-101 (Fe), red-shifted to 250 nm, showing a wide range of peaks, and there is a certain overlap with the ultraviolet absorption of 1,3-dinaphthol (NR) at 280 nm. Therefore, there may be an inner filter effect between the two that affects the detection. Therefore, the fluorescence spectra of NR and MIL-101 (Fe) were further studied, as shown in Figure 3 As shown: It can be found that the fluorescence of NR can be quenched by MIL-101 (Fe) to a certain extent, but the fluctuation range is very small. Therefore, under ideal conditions, the concentration of MIL-101 (Fe) can be optimized to achieve the best detection signal. In addition, it is worth noting that the fluorescence intensity of 2-hydroxyterephthalic acid decreases significantly after forming MIL-101 (Fe).

[0019] Example 2: Optimization of conditions for detecting dopamine In order to achieve the best detection performance of the ratio fluorescence sensor, this embodiment investigates various influencing factors in the dopamine detection system. The enzyme activity of the nanomaterial is strongly restricted by the pH value. Under different pH conditions, the fluorescence intensity and peak shape produced by the reaction driven by the pseudo-enzyme activity are very different. Taking into account the degree of separation and fluorescence intensity of the fluorescence peaks, pH = 6 is finally selected as the final detection condition, and the activity of most nanozymes will be greatly weakened under this pH condition. In addition, as the chemical reaction proceeds, its final product will gradually reach saturation with the extension of time. Therefore, this embodiment optimizes the detection time, and the results show that the cyclization reaction of dopamine and NR will reach a plateau at around 60 min. Therefore, 60 min is finally selected as the optimal detection time. It is worth noting that the reaction temperature also has a huge impact on the progress of the chemical reaction. By recording the fluorescence intensity of the fluorescent product at different temperatures, 37 ° C is finally selected as the optimal reaction temperature. Further, H 2 O 2 It is also a key variable in the reaction process. Excess H 2 O 2It will directly oxidize NR, causing its peak at 440 nm to disappear, obscuring the stoichiometric relationship between NR and dopamine, affecting the final quantification of dopamine. 2 O 2 The fluorescence spectrum of the detection system was obtained by 10 mM H 2 O 2 As shown in Example 1, there is an inner filter effect between MIL-101 (Fe) and NR, so it is crucial to optimize the concentration of MIL-101 (Fe). According to the fluorescence spectrum, MIL-101 (Fe) diluted 1000 times was finally selected as the final reaction concentration. The specific optimization process is as follows: 800 μL of acetic acid-sodium acetate buffer (0.1 M, pH = 4, 5, 6) and PBS buffer (0.1 M, pH = 7, 8) were taken into 1.5 mL ep tubes, and then 50 μL of dopamine solution (1 mM), 50 μL of NR solution (15 mM), 50 μL of MIL-101(Fe) (×1000) and 50 μL of H 2 O 2 (10 mM), and then placed in a constant temperature shaking incubator at 37°C and 800 rpm for 30 min. Finally, the fluorescence spectrum of the fluorescent product was measured at 320 nm as the excitation wavelength, and it was found that the optimal pH was 6.

[0020] 800 μL of acetic acid-sodium acetate buffer (0.1 M, pH = 6) was placed in a 1.5 mL epoxide tube, followed by the addition of 50 μL of dopamine solution (350 μM), 50 μL of NR solution (5 mM), 50 μL of MIL-101(Fe) (×1000), and 50 μL of H 2 O 2 (10mM), and then placed in a constant temperature oscillating incubator at different temperatures and 800 rpm for different times. Finally, the fluorescence intensity of the fluorescent product FP at 480nm was measured with an excitation wavelength of 320nm. It was found that the optimal reaction temperature was 37℃ and the optimal reaction time was 60min.

[0021] 800 μL of acetic acid-sodium acetate buffer (0.1 M, pH = 6) was placed in a 1.5 mL ep tube, followed by the addition of 50 μL of dopamine solution (1 mM), 50 μL of NR solution (15 mM), 50 μL of MIL-101(Fe) (×1000), and 50 μL of H 2 O 2(5, 10, 50, 100, 500 and 1000 mM), and then placed in a constant temperature shaking incubator at 37 °C and 800 rpm for 60 min. Finally, the fluorescence spectrum of the fluorescent product FP was measured at 320 nm as the excitation wavelength. 2 O 2 The optimal concentration is 10 mM.

[0022] 800 μL of acetic acid-sodium acetate buffer (0.1 M, pH = 6) was placed in a 1.5 mL eppendorf tube, followed by the addition of 50 μL of dopamine solution (1 mM), 50 μL of NR solution (5 mM), 50 μL of different concentrations of MIL-101(Fe) (×10, ×50, ×100, ×500, ×1000, and ×5000), and 50 μL of H 2 O 2 (10 mM), and then placed in a constant temperature shaking incubator at 37°C and 800 rpm for 60 min. Finally, the fluorescence spectrum of the fluorescent product FP was measured at 320 nm as the excitation wavelength. It was found that the optimal concentration of MIL-101(Fe) was 1000×.

[0023] Example 3: Using the ratiometric fluorescence sensor prepared in Example 1 to detect dopamine content in different samples under optimal detection conditions 800 μL of acetic acid-sodium acetate buffer (0.1 M, pH = 6) was taken into a 1.5 mL eppendorf tube, and then 50 μL of dopamine solution of different concentrations (5, 10, 50, 100, 200, 400 and 700 μM, each concentration was repeated 3 times) was added, and then 50 μL of NR solution (5 mM), 50 μL of MIL-101(Fe) (×1000) and 50 μL of H 2 O 2 (10 mM), and then placed in a constant temperature shaking incubator at 37°C and 800 rpm for 60 minutes to obtain a solution sample; according to the above preparation method, the acetic acid-sodium acetate buffer was replaced with serum and urine, respectively, to obtain a blood sample and a urine sample, and finally the fluorescence spectra of the fluorescent product FP at 440 nm and 480 nm were measured at an excitation wavelength of 320 nm, and the ultraviolet spectrum at 462 nm was detected, and two standard curves with fluorescence and ultraviolet as the ordinate were drawn respectively. At the same time, three concentration points of low, medium and high (60, 300 and 600 μM, each concentration was repeated 6 times) were set in the solution sample, blood sample and urine sample, and their fluorescence spectra and ultraviolet spectra were measured after the reaction, and the bioanalysis recovery rates were calculated respectively.

[0024] The results are as follows Figure 4As shown in Figure 2, there is a good linear relationship between the ratio fluorescence signal and dopamine concentration in the range of 0.25-35 μM (Y=0.1427[DA]+0.3211, R 2 =0.999), the detection limit was calculated to be 34.5 ng / mL using the 3σ principle; similarly, Figure 5 As shown: there is a good linear relationship between the UV signal and the dopamine concentration (Y=0.0112[DA]+0.0636, R 2 =0.999), the detection limit was calculated to be 34.5 ng / mL based on the 3σ principle. At the same time, three concentration points of low, medium and high were set in solution samples, blood samples and urine samples. The recovery rate of the ratio fluorescence method in solution samples was 99.67%~101.6%, the precision (RSD%) was 2.7%~5.7%, the recovery rate of the UV method was 90.98%~99.17%, and the precision was 0.9%~8.7%; the recovery rate of the ratio fluorescence method in blood samples was 100.2%~104.4%, and the precision was 1.58 %~3.18%, the recovery rate of the UV method was 96.36%~110.0%, and the precision was 3.3%~9.2%; the recovery rate of the ratio fluorescence method in urine samples was 95.23%~107.1%, and the precision was 1.7%~4.7%, and the recovery rate of the UV method was 88.86%~113.0%, and the precision was 1.4%~8.5%; the test results of serum samples and urine samples showed that the quantitative method of the present invention was not affected by the serum and urine matrix effects. In summary, the above results are in line with the guiding principles for the validation of quantitative analysis methods and can be used as a reliable quantitative method for measuring dopamine detection.

[0025] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A ratiometric fluorescence sensor for detecting dopamine, characterized in that: Ingredients include: FeCl3•6H2O and H2BDC, the molar ratio of FeCl3•6H2O to H2BDC is 1:1~1.

5.

2. A method for preparing a ratiometric fluorescence sensor for detecting dopamine according to claim 1, characterized in that: The following steps are involved: S1. Preparation of MIL-101 (Fe): FeCl3•6H2O and H2BDC were added to a round-bottom flask in proportion, and then 50 mL of dimethylformamide was slowly added. The resulting mixture was ultrasonically treated at 37°C for 10-15 min, then transferred to a Teflon reactor, heated at 100-110°C for 20 h, and finally centrifuged at 6000 rpm for 5-10 min to obtain a reddish-brown precipitate; the reddish-brown precipitate was repeatedly washed three times in ethanol to remove unreacted ligand H2BDC and metal ions until the supernatant solution was clear and transparent to obtain MIL-101 (Fe), which was stored at 4°C; S2. Add 1 mL of 1,3-dinaphthol and 10 mL of H2O2 to MIL-101 (Fe) to finally obtain a ratiometric fluorescence sensor.

3. The preparation method according to claim 2, characterized in that: The temperature of the ethanol is 30° C., and each cleaning time in the ethanol is 5 min.

4. Use of the ratiometric fluorescence sensor for detecting dopamine according to claim 1 or the ratiometric fluorescence sensor for detecting dopamine prepared by the preparation method according to claim 2 or 3 in detecting the dopamine content in a biological sample.

5. The use according to claim 4, characterized in that: The following steps are involved: 1) Place the sample to be tested in a constant temperature oscillator for incubation. After incubation, transfer it to a transparent 96-well plate and a black 96-well plate respectively. Set the UV scanning wavelength to 462 nm and the fluorescence detection wavelength to 440 nm and 480 nm. Perform the test under the test conditions and detect the absorbance and fluorescence intensity at the same time. 2) Calculate the dopamine content in the sample to be tested based on the standard curve.

6. The use according to claim 5, characterized in that The incubation conditions are: pH 6-7, temperature 25-37°C, and time 45-60 min.

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