Preparation Method and Application of a Ratiometric Fluorescent Sensor for Detecting Dopamine

Abstract

The present invention discloses a ratio fluorescence sensor for detecting dopamine and its preparation method and application. The raw materials include: FeCl <subgt;3< / subgt;•6H<subgt;2< / subgt;O和H<subgt;2< / subgt;BDC,FeCl<subgt;3< / subgt;•6H<subgt;2< / subgt;O与H<subgt;2< / subgt;BDC的摩尔份数比1:1~1.5。本发明传感体系由于1,3‑二萘酚和多巴胺间的特异性环化反应的存在,具有非常良好的特异性。另外,比率荧光和紫外双信号的输出模式可以提高整个系统的可靠性,其中比率荧光能更好的屏蔽血液和尿液样本中强烈的生物基质效应。更进一步来说,本发明所制备的MIL‑101 (Fe) 具有过氧化酶和氧化酶的双重模拟酶活性,多种自由基的存在能够加速环化反应的进行,从而进一步降低多巴胺的检测限,提高检测体系的灵敏度。

Description

Technical Field

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

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

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

[0004] Electrochemical detection techniques typically offer high sensitivity, but the detection process is complex and requires specialized instrumentation. Typical electrochemical methods involve coupling an aptamer to a corresponding sensor, followed by the specific capture of dopamine. Therefore, a simple and sensitive electrochemical aptasensor was developed for dopamine detection using redox cycling signal amplification using tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent. The captured dopamine is electrochemically oxidized to dopamine quinones (DAQ), which are then immediately regenerated by TCEP, resulting in an enhanced electrochemical signal.

[0005] Fluorescence analysis offers high sensitivity comparable to electrochemical methods. Signal detection can be performed using widely used analytical instruments such as microplate readers and fluorescence spectrophotometers, making it a powerful tool for detecting biomacromolecules, including dopamine. The enzyme-linked immunosorbent assay (ELISA) is a classic method for macromolecular immunoassays, but its operation is extremely cumbersome. First, the analyte is coated in a microplate, bound to a primary antibody and eluted, followed by the addition of an enzyme-labeled secondary antibody and further elution. Finally, a chromogenic substrate is added, and the fluorescence signal from the microplate is detected using a microplate reader, reflecting 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 switching, the fluorescent inner filter effect, and photoinduced electron transfer have emerged, providing insights and references for the development of rapid, simple, and sensitive detection technologies 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 problems solved: In response to the above technical problems, the present invention provides a ratiometric fluorescence sensor for detecting dopamine, and its preparation method and application, 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 including: FeCl3•6H2O and H2BDC, the molar ratio of FeCl3•6H2O to H2BDC 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:

[0010] 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 sonicated at 37°C for 10-15 min, then transferred to a Teflon reactor and heated at 100-110°C for 20 h. Finally, the mixture was centrifuged at 6000 rpm for 5-10 min to obtain a reddish-brown precipitate. The reddish-brown precipitate was 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 then stored at 4°C.

[0011] S2. Add 1 mL of 1,3-dinaphthol and 10 mL of H2O2 to MIL-101 (Fe) to obtain a ratiometric fluorescence sensor.

[0012] Preferably, the temperature of the ethanol is 30° C., and the time for each washing in ethanol is 5 minutes.

[0013] In a third aspect, the present invention provides 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 dopamine content in a biological sample.

[0014] Preferably, the application comprises the following steps:

[0015] 1) Place the sample to be tested in a constant temperature shaker 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 wavelengths to 440 nm and 480 nm. Perform the test under the detection conditions and measure the absorbance and fluorescence intensity simultaneously.

[0016] 2) Calculate the dopamine content in the sample to be tested based on the standard curve.

[0017] Furthermore, the incubation conditions are: pH 6-7, temperature 25-37°C, and time 45-60 min.

[0018] Beneficial effects: The present invention establishes a dual-mode sensing system based on ratio fluorescence and ultraviolet for detecting dopamine. Due to the specific cyclization reaction between 1,3-dinaphthol (NR) and dopamine, this sensing system has very good specificity. In addition, the output mode of dual signals of ratio fluorescence and ultraviolet can improve the reliability of the whole system, and the ratio fluorescence can better shield the strong biomatrix effect in blood and urine samples. Furthermore, the prepared MIL-101(Fe) of the present invention has dual mimic enzyme activities of peroxidase and oxidase, and the presence of various free radicals can accelerate the progress of the cyclization reaction, thereby further reducing the detection limit of dopamine and improving the sensitivity of the detection system. Description of the Drawings

[0019] Figure 1 is the transmission electron microscope characterization diagram of MIL-101(Fe);

[0020] Figure 2 is the ultraviolet spectrum diagram of MIL-101(Fe);

[0021] Figure 3 is the fluorescence spectrum diagram of MIL-101(Fe);

[0022] Figure 4 is the standard curve diagram for detecting dopamine in serum by fluorescence spectrometry;

[0023] Figure 5 is the standard curve diagram for detecting dopamine in urine by fluorescence spectrometry. Detailed Embodiments

[0024] The present invention will be described in detail below with reference to the drawings and specific embodiments:

[0025] Example 1: Preparation of MIL-101(Fe)

[0026] MIL-101(Fe) was prepared by a hydrothermal method: First, 0.675 g of FeCl3•6H2O and 0.206 g of H2BDC (2-hydroxyterephthalic acid) were weighed into a round-bottom flask, and then 15 mL of DMF (dimethylformamide) was slowly added. The obtained mixture was ultrasonically treated at 37 °C for 10 min, and then transferred to a Teflon reaction kettle, 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 the unreacted ligand H2BDC and metal ions until the supernatant solution was clear and transparent, and finally stored at 4 °C.

[0027] The electron microscope images of MIL-101(Fe) at different scales were studied by scanning electron microscopy, as Figure 1As shown: The distance between two adjacent vertices was measured using Image J, and the average size of MIL-101(Fe) was measured to be 600 nm, which was consistent with the measurement results of transmission electron microscopy. Moreover, the dispersion and crystallinity of MIL-101(Fe) prepared by this method were very good, and they were all in a uniform regular octahedron configuration. The dynamic light scattering measurement results showed that the distribution range was very narrow, indicating a uniform particle size distribution.

[0028] To exclude the influence of the reaction system on the final detection performance, the ultraviolet and fluorescence spectra of MIL-101(Fe) and its ligand 2-hydroxyterephthalic acid were studied, as Figure 2 shown: It can be observed that the significant absorption peak of terephthalic acid itself at 240 nm decreased significantly after the formation of MIL-101(Fe), red-shifted to 250 nm, presenting a wide peak over a large range, and there was a certain overlap with the ultraviolet absorption of 1,3-dinaphthol (NR) at 280 nm. Therefore, an inner filter effect may occur between the two, affecting the detection. So, the fluorescence spectra of NR and MIL-101(Fe) were further studied, as Figure 3 shown: It can be found that the fluorescence of NR can indeed be quenched to a certain extent by MIL-101(Fe), but the fluctuation range is very small. Therefore, ideally, the detection signal can be optimized by optimizing the concentration of MIL-101(Fe). In addition, it is worth noting that the fluorescence intensity of 2-hydroxyterephthalic acid decreased significantly after the formation of MIL-101(Fe).

[0029] Example 2: Optimization of the conditions for detecting dopamine

[0030] To achieve optimal detection performance for the ratiometric fluorescence sensor, this example investigated various influencing factors in the dopamine detection system. The enzymatic activity of nanomaterials is strongly constrained by pH. Under different pH conditions, the fluorescence intensity and peak shape produced by the reaction driven by the mimicking enzyme activity vary significantly. Taking into account the degree of separation and fluorescence intensity of the fluorescence peaks, pH = 6 was ultimately selected as the final detection condition. However, the activity of most nanozymes will be greatly weakened under this pH condition. Furthermore, as the chemical reaction proceeds, its final product will gradually reach saturation over time. Therefore, this example optimized the detection time. The results showed that the cyclization reaction of dopamine and NR will reach a plateau at around 60 minutes. Therefore, 60 minutes was ultimately selected as the optimal detection time. It is worth noting that the reaction temperature also has a significant impact on the progress of the chemical reaction. By recording the fluorescence intensity of the fluorescent product at different temperatures, 37°C was ultimately selected as the optimal reaction temperature. Furthermore, H2O2 is also a key variable in the reaction process. Excessive H2O2 will directly oxidize NR, causing its peak at 440 nm to disappear, obscuring the stoichiometric relationship between NR and dopamine and affecting the final quantification of dopamine. By scanning the fluorescence spectrum of the detection system under different concentrations of H2O2, 10 mM H2O2 is preferentially selected as the final detection concentration. As shown in Example 1: MIL-101 (Fe) and NR have an inner filter effect, so optimizing the concentration of MIL-101 (Fe) is crucial. 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:

[0031] 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 respectively, and then 50 μL of dopamine solution (1 mM), 50 μL of NR solution (15 mM), 50 μL of IL-101 (Fe) (×1000) and 50 μL of H2O2 (10 mM) were added. Then, the tubes were 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 an excitation wavelength of 320 nm, and the optimal pH was 6.

[0032] Take 800 μL of acetic acid-sodium acetate buffer (0.1 M, pH = 6) in a 1.5 mL ep tube, and then add 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 H2O2 (10 mM). Then place it in a thermostatic shaking incubator and react at different temperatures at 800 rpm for different times. Finally, measure the fluorescence intensity of the fluorescent product FP at 480 nm with an excitation wavelength of 320 nm. It can be known that the optimal reaction temperature is 37 °C and the optimal reaction time is 60 min.

[0033] Take 800 μL of acetic acid-sodium acetate buffer (0.1 M, pH = 6) in a 1.5 mL ep tube, and then add 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 H2O2 with different concentrations (5, 10, 50, 100, 500, and 1000 mM). Then place it in a thermostatic shaking incubator and react at 37 °C and 800 rpm for 60 min. Finally, measure the fluorescence spectrum of the fluorescent product FP with an excitation wavelength of 320 nm. It can be known that the optimal concentration of H2O2 is 10 mM.

[0034] Take 800 μL of acetic acid-sodium acetate buffer (0.1 M, pH = 6) in a 1.5 mL ep tube, and then add 50 μL of dopamine solution (1 mM), 50 μL of NR solution (5 mM), 50 μL of MIL-101(Fe) with different concentrations (×10, ×50, ×100, ×500, ×1000, and ×5000), and 50 μL of H2O2 (10 mM). Then place it in a thermostatic shaking incubator and react at 37 °C and 800 rpm for 60 min. Finally, measure the fluorescence spectrum of the fluorescent product FP with an excitation wavelength of 320 nm. It can be known that the optimal concentration of MIL-101(Fe) is 1000×.

[0035] Example 3: Detect the dopamine content in different samples using the ratiometric fluorescence sensor prepared in Example 1 under the optimal detection conditions.

[0036] 800 μL of acetic acid-sodium acetate buffer (0.1 M, pH = 6) was taken into a 1.5 mL ep 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 H2O2 (10 mM) were added, and then placed in a constant temperature shaking incubator at 37 ° C and 800 rpm for 60 min to obtain solution samples; according to the above preparation method, the acetic acid-sodium acetate buffer was replaced with serum and urine, respectively, to obtain blood samples and urine samples. 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 462 The UV spectra at 100 nm were plotted, with fluorescence and UV as the vertical axes. Three concentration points (60, 300, and 600 μM, with six replicates for each concentration) were set in solution, blood, and urine samples. After the reaction, fluorescence and UV spectra were measured, and bioanalytical recoveries were calculated.

[0037] The results are as follows Figure 4 As 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), and the detection limit calculated by the 3σ principle was 34.5 ng / mL. At the same time, low, medium, and high concentration points were set in solution samples, blood samples, and urine samples respectively. The recovery rates of the ratio fluorescence method in solution samples were 99.67% - 101.6%, and the precision (RSD%) was 2.7% - 5.7%. The recovery rates of the ultraviolet method were 90.98% - 99.17%, and the precision was 0.9% - 8.7%. The recovery rates of the ratio fluorescence method in blood samples were 100.2% - 104.4%, and the precision was 1.58% - 3.18%. The recovery rates of the ultraviolet method were 96.36% - 110.0%, and the precision was 3.3% - 9.2%. The recovery rates of the ratio fluorescence method in urine samples were 95.23% - 107.1%, and the precision was 1.7% - 4.7%. The recovery rates of the ultraviolet method were 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 matrix effects of serum and urine. In summary, the above results all met the guiding principles for the verification of quantitative analysis methods and could be used as a reliable quantitative method for measuring dopamine detection.

[0038] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a ratiometric fluorescence sensor for detecting dopamine, characterized in that, The raw materials of the ratiometric fluorescence sensor for detecting dopamine include: FeCl3•6H2O and H2BDC, and the preparation method comprises the following steps: S1. Preparation of MIL-101(Fe): Add 0.675 g of FeCl3•6H2O and 0.206 g of H2BDC into a round-bottom flask, then slowly add 15 mL of dimethylformamide. The obtained mixture is ultrasonically treated at 37 °C for 10 - 15 min, and 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 is repeatedly washed three times with hot ethanol to remove the unreacted ligand H2BDC and metal ions until the washing liquid is clear and transparent, obtaining MIL-101(Fe), stored at 4 °C, and the washing liquid is hot ethanol; S2. Add 1 mL of 1,3-dinaphthol and 10 mL of H2O2 to MIL-101(Fe) to finally obtain the ratiometric fluorescence sensor, wherein the concentration of H2O2 is 10 mM.

2. The preparation method according to claim 1, characterized in that: The temperature of the hot ethanol is 70 °C, and the time for each washing in ethanol is 5 min.

3. Application of the ratiometric fluorescence sensor for detecting dopamine prepared by the preparation method according to claim 2 in detecting the dopamine content in biological samples.

4. The application according to claim 3, characterized in that Comprises the following steps: 1) Place the sample to be tested in a thermostatic shaker for incubation. After incubation is completed, transfer it to a transparent 96-well plate and a black 96-well plate respectively. Set the ultraviolet scanning wavelength to 462 nm, the fluorescence detection wavelengths to 440 nm and 480 nm, and perform detection under the detection conditions, while detecting the absorbance and fluorescence intensity; 2) Calculate the dopamine content in the sample to be tested according to the standard curve.

5. The application according to claim 4, wherein The incubation conditions are: pH is 6 - 7, temperature is 25 - 37 °C, and time is 45 - 60 min.

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