CN113773313B - Novel fluorescent probe for simultaneously and quantitatively detecting Cys, Hcy and GSH in blood plasma as well as preparation method and application thereof - Google Patents
Novel fluorescent probe for simultaneously and quantitatively detecting Cys, Hcy and GSH in blood plasma as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a novel fluorescent probe capable of simultaneously and quantitatively detecting Cys, Hcy and GSH in blood plasma, a preparation method and application thereof, wherein the preparation steps comprise: preparing 7- (diethylamino) coumarin-3-nitro; preparing 7- (diethylamino) coumarin-3-amino; preparing 7- (diethylamino) coumarin-3-hydroxyl; and then adding 7- (diethylamino) coumarin-3-hydroxy and 4-chloro-7-nitrobenz-2-oxa-1, 3-diazole into the acetonitrile solution, then adding potassium carbonate, stirring at room temperature for reaction, removing the solvent after the reaction is finished, and purifying to obtain the novel fluorescent probe for quantitatively distinguishing and detecting Cys, Hcy and GSH. The invention develops a novel fluorescent probe which can simultaneously quantitatively distinguish and detect the content of Cys, Hcy and GSH in blood plasma under the conditions of different pH values and different fluorescent channels.
Description
Technical Field
The invention relates to the technical field of fluorescence detection, in particular to a novel fluorescent probe for simultaneously quantitatively detecting and distinguishing Cys, Hcy and GSH in blood plasma, and a preparation method and application thereof.
Background
Cysteine (Cys), homocysteine (Hcy) and Glutathione (GSH) are the most abundant small molecule biological thiols known in the human body at present, and play a key role in many human metabolism and homeostasis. Although the structures and activities of Cys, Hcy and GSH are very similar, they have different physiological and pathological roles. Cys is involved in enzyme catalysis, detoxification, protein synthesis and metabolism. Aberrant levels of Cys can cause a variety of diseases, such as growth retardation, skin damage, edema, lethargy, and liver damage; elevated levels of Hcy in plasma are considered to be high risk factors for cardiovascular disease, thrombosis, alzheimer's disease, renal dysfunction and osteoporosis. Glutathione is capable of maintaining redox homeostasis in biological systems. The glutathione level imbalance is related to liver injury, AIDS, cancer, aging, etc. Biological fluids such as plasma are the best non-invasive substrate for tracking human biological thiol levels. Therefore, quantitative detection of Cys, Hcy and GSH levels in biological fluids provides a rapid method for screening and diagnosing diseases associated with biological thiols and dissecting their physiological and pathological processes.
At present, various analysis techniques for detecting biological thiol have been reported, and compared with traditional methods such as High Performance Liquid Chromatography (HPLC), mass spectrometry, capillary electrophoresis, electrochemistry and the like, the fluorescent probe has high selectivity and high sensitivity, and has advantages in qualitative and quantitative detection of biological thiol in vitro and/or in vivo. To date, great progress has been made in the construction of biological thiol fluorescent probes. These probes are capable of distinguishing biological thiols from other amino acids. However, since the structures and activities of the three biological thiols are very similar, most of the reported fluorescent probes cannot distinguish between Cys, Hcy and GSH, which hinders further studies on their role in physiological and pathological events. To date, a few fluorescent probes have been developed that specifically detect Cys, GSH and Cys/Hcy. In addition, few fluorescent probes have been reported that can simultaneously recognize Cys and GSH and Cys/Hcy and GSH in different fluorescent channels. However, the development of fluorescent probes that can simultaneously recognize three kinds of biological thiols remains a great challenge.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a novel fluorescent probe for simultaneously and quantitatively detecting biological mercaptan in blood plasma as well as a preparation method and application thereof. The probe can simultaneously identify Cys, Hcy and GSH in different fluorescence channels under different pH conditions, and has been successfully applied to simultaneously and quantitatively detect the content of Cys, Hcy and GSH in a blood plasma sample, and the probe not only has good selectivity and high sensitivity, but also has a simple preparation method. The invention is realized by the following technical scheme:
a fluorescent probe for simultaneously and quantitatively detecting Cys, Hcy and GSH in blood plasma has the following structure:
the preparation method of the fluorescent probe for simultaneously and quantitatively detecting Cys, Hcy and GSH in blood plasma comprises the following steps:
And 4, adding the 7- (diethylamino) coumarin-3-hydroxy and 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole obtained in the step 3 into an acetonitrile solution, then adding potassium carbonate, stirring at room temperature for reaction, removing redundant solvents after the reaction is finished, and purifying residues through column chromatography to obtain the novel fluorescent probe for quantitatively distinguishing and detecting Cys, Hcy and GSH.
In the step 1, the dosage ratio of the 4- (diethylamino) salicylaldehyde to the n-butyl alcohol to the ethyl nitroacetate to the piperidine to the acetic acid is 1 g-2 g: 10 mL-20 mL: 0.5 mL-1 mL: 70-80 μ L: 0.2mL to 0.3 mL.
In the step 1, the temperature of the heating reflux stirring reaction is 100-105 ℃, and the time is 10-12 h.
In the step 2, the dosage ratio of the stannous chloride, the concentrated hydrochloric acid and the 7- (diethylamino) coumarin-3-nitro is 0.6 g-0.7 g: 2 mL-4 mL: 0.2g to 0.3 g.
In the step 2, the stirring reaction time at the normal temperature is 1-2 h, and the temperature for recrystallization is 90 ℃. The solvent removal method is rotary evaporation.
In the step 3, the dosage ratio of the 7- (diethylamino) coumarin-3-amino to the hydrochloric acid is 0.4 g-0.5 g: 5mL to 6mL, wherein the concentration of hydrochloric acid is 1M.
In the step 3, the heating and stirring reaction temperature is 110-120 ℃, and the reaction temperature time is 2-4 h. The solvent removal method is rotary evaporation.
In the step 4, the dosage ratio of the 7- (diethylamino) coumarin-3-hydroxy, the 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole, the potassium carbonate and the acetonitrile is 0.3 g-0.4 g: 0.4 g-0.5 g: 0.2 g-0.3 g: 4 mL-5 mL.
In the step 4, the room temperature reaction time is 4-5 h. The solvent removal method is rotary evaporation.
The novel fluorescent probe is used for simultaneously quantitatively distinguishing and detecting Cys, Hcy and GSH in blood plasma.
The fluorescent probe provided by the invention can be used for simultaneously and quantitatively detecting the content of Cys, Hcy and GSH in blood plasma, and the specific method comprises the following steps:
(1) venous blood of healthy volunteers was taken at 3.0 mL. The blood samples were placed in pre-cooled ethylenediaminetetraacetic acid (EDTA) vacuum tubes and immediately centrifuged at 2500r/min for 10min at 4 ℃. The supernatant was taken as human plasma for analysis. Probe YF (1X 10) was prepared using DMSO-3M) of the mother liquor. Several 5mL volumetric flasks were taken, and 25. mu.L of the stock solution of the fluorescent probe and 1mL of DMSO were measured in each 5mL volumetric flask. Then, 100. mu.L of supernatant of the plasma extract sample and different volumes of 1X 10 were added to each flask- 2Cys solution of M (0, 2.5, 5, 7.5, 10, 15, 20. mu.L). Then, the volume of the flask containing the mixture was adjusted to 5mL with 20mM phosphate buffer (pH 7.4 or 5.6), and the mixture was shaken and left at room temperature for 10 min. Finally mixing the mixtureTransferring the solution into a quartz cuvette for later use;
(2) when the pH value of the sample obtained in the step (1) is 7.4 and the excitation wavelength is 350nm, the total concentration of Cys, Hcy and GSH can be detected at the position of an emission wavelength of 508 nm;
(3) when the pH of the sample obtained in the step (1) is still 7.4 and the excitation wavelength is changed to 475nm, the total concentration of Cys and Hcy can be detected at the position of an emission wavelength of 550 nm;
(4) when the pH value of the sample obtained in the step (1) is changed to 5.6, the excitation wavelength is still 475nm, and the concentration of Cys can be detected at the position of an emission wavelength of 550 nm.
Then the content of Hcy and GSH in the plasma is calculated by mathematics.
The invention has the following beneficial effects:
(1) the invention provides a brand-new fluorescent probe for quantitative analysis and detection of Cys, Hcy and GSH, the synthetic method of the fluorescent probe is simple, the fluorescent probe has good selectivity on Cys, Hcy and GSH, Lys, Arg, Ser, Leu, Phe, Ala, Gly, Glu, Val, Gln, K+,Na+,Mg2+,Zn2+,H2O2The relevant interferents have no influence on the detection; when the pH value of the detection solution is 7.4, the fluorescence intensity is 508nm (lambda ex is 350nm), and the probe YF can detect the total concentration (C) of Cys, Hcy and GSHC+H+G). Cys and Hcy (C) can be achieved by fluorescence centered at 550nm (λ ex ═ 475nm) at pH 7.4C+H) And (4) detecting the total concentration. Cys, Hcy and GSH are low molecular weight biological thiols with the most abundant content in biological fluid, and the fluorescence intensity of the probe YF and the concentrations of the three thiols have a good linear relation under the conditions, so that the concentrations of Cys, Hcy and GSH in blood plasma can be simultaneously and quantitatively detected through simple mathematical calculation.
(2) After the synthesis method is improved, the side reaction can be effectively prevented, impurities which are difficult to separate in the reaction process are greatly reduced, and a high-purity target product can be obtained.
(3) The invention develops a novel fluorescent probe for quantitative analysis and detection of Cys, Hcy and GSH. The fluorescent probe is applied to simultaneously and quantitatively distinguish and detect Cys, Hcy and GSH in plasma, and the probe YF and a fluorescence spectrometer are applied for the first time to carry out fluorescence intensity so as to simultaneously and quantitatively distinguish and detect Cys, Hcy and GSH in plasma. The cysteine, homocysteine and glutathione in biological liquid such as blood plasma are quantitatively detected by a fluorescent probe. Provides a quick and cheap method for screening and diagnosing biological thiol related diseases and analyzing physiological and pathological processes.
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FIG. 1 is a synthesis scheme of a novel fluorescent probe for quantitative determination of Cys, Hcy and GSH in plasma in example 1 of the present invention;
FIG. 2 is a graph of the fluorescence titration for the fluorescent probe provided in example 1; the abscissa is the condition of different emission wavelengths, and the ordinate is the fluorescence intensity value;
FIG. 3 is a bar graph of the selectivity of the fluorescent probe provided in example 1; the abscissa is different ion or molecule addition conditions, and the ordinate is a fluorescence intensity value;
FIG. 4 is a graph showing the influence of pH of the solution on the fluorescence intensity values before and after the interaction of the fluorescent probe with Cys, Hcy and GSH in example 1 of the present invention; the abscissa is pH, and the ordinate is fluorescence intensity value;
FIG. 5 is a kinetic study of the fluorescent probe obtained in example 1; the abscissa is time, and the ordinate is fluorescence intensity;
FIGS. 6 a-c are graphs of the spectral data obtained in example 1 for the detection of biological thiols in plasma with fluorescent probes; the abscissa is the emission wavelength and the ordinate is the fluorescence intensity; d-f in FIG. 6 is a linear plot of fluorescence response versus Cys volume constructed from the spectroscopic data obtained in example 1 for the quantitative determination of Cys, Hcy and GSH in plasma using the fluorescent probe; the abscissa is the volume of Cys; the ordinate represents the fluorescence intensity value (I-I)0)/I0In which I0And I is fluorescence intensity in the probe YF single detection liquid and 1X 10 different volumes in the plasma detection liquid respectively-2Fluorescence intensity of M Cys.
FIG. 7 is a schematic diagram of simultaneous quantitative differential detection of Cys, Hcy and GSH by probe YF.
Detailed Description
The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.
Example 1:
the synthesis of the fluorescent probe comprises the following specific steps:
(1) adding 1.1g of 4- (diethylamino) salicylaldehyde into a 50mL flask, dissolving the mixture by using 13mL of n-butanol, adding 0.6mL of ethyl nitroacetate, uniformly stirring the mixture, adding 74 mu L of piperidine, adding 0.26mL of acetic acid, and heating, refluxing and stirring the mixed solution at 102 ℃ for reacting for 11 hours; after the reaction is finished, the reaction product is cooled to room temperature, the precipitate is filtered to obtain a filter cake, the precipitate is washed and purified by n-butanol and petroleum ether, and the precipitate is dried in vacuum to obtain 1.1g of 7- (diethylamino) coumarin-3-nitro orange solid with the yield of 81 percent.
(2) Adding 3mL of hydrochloric acid solution into a 50mL flask, then adding 0.65g of stannous chloride in an ice bath environment, then adding 0.21g of 7- (diethylamino) coumarin-3-nitro prepared in the step 1, stirring at normal temperature, reacting for 1.5h, adding sodium hydroxide in an ice bath to neutralize until the pH value reaches 7, performing extraction and liquid separation on the mixed solution by using distilled water and ethyl acetate, recovering an organic layer, performing reduced pressure distillation to remove excessive solvent, and then recrystallizing by using absolute ethyl alcohol at 90 ℃ to obtain 76mg of 7- (diethylamino) coumarin-3-amino, wherein the yield is 86%.
(3) Weighing 0.42g of 7- (diethylamino) coumarin-3-amino obtained in the step 2, dissolving in 5.2mL of hydrochloric acid, heating the mixed solution at 114 ℃, and carrying out reflux stirring reaction for 3 hours; after the reaction, ammonia water was added to neutralize the reaction mixture to adjust the pH to 7, and the mixture was extracted with distilled water and methylene chloride to separate the liquid, and the organic layer was recovered, and the organic solvent was removed under reduced pressure. Purification by column chromatography (petroleum ether/ethyl acetate 3:1, v/v) gave 210mg of 7- (diethylamino) coumarin-3-hydroxy yellow solid in 70% yield.
(4) And (2) adding 0.33g of 7- (diethylamino) coumarin-3-hydroxy obtained in the step (3) and 0.41g of 4-chloro-7-nitrobenz-2-oxa-1, 3-diazole into 4.8mL of acetonitrile solution, then adding 0.26g of potassium carbonate, stirring at room temperature for reaction for 4.5h, removing redundant solvent by using a rotary evaporator after the reaction is finished, and purifying by column chromatography (petroleum ether/ethyl acetate is 3:1, v/v), so that 350mg of the novel fluorescent probe for quantitatively distinguishing and detecting Cys, Hcy and GSH can be obtained, wherein the yield is 68%.
Example 2:
the fluorescence spectra response of the fluorescent probes obtained in example 1 to the detection of Cys, Hcy and GSH fluorescence.
A5. mu.M fluorescent probe test solution was prepared with 20mM potassium phosphate buffer/DMSO (4:1v/v, pH 7.4 or 5.6) and was ready for use. Preparing Cys, Hcy and GSH solutions with concentration of 1 × 10 with deionized water-2And M. The fluorescence emission spectra were measured by adding Cys, Hcy and GSH solutions of different concentrations to 5 μ M fluorescent probe test solutions with excitation wavelengths of 350nm and 475nm, respectively, and the results are shown in FIG. 2.
From the results of FIG. 2, it was found that the fluorescence intensity at the emission wavelength of 508nm at pH 7.4 and an excitation wavelength of 350nm had a good linear relationship with Cys, Hcy and GSH in the concentration range of 0 to 60. mu.M, respectively. YF probe can detect the total concentration of Cys, Hcy and GSH (C)C+H+G). When the pH is still 7.4 and the excitation wavelength is changed to 475nm, the fluorescence intensity at the position of the emission wavelength of 550nm is in a linear relation with Cys or Hcy within the concentration range of 0-60 mu M respectively. YF probe can detect total concentration of Cys and Hcy (C)C+H). When the pH was changed to 5.6, the excitation wavelength was still 475nm and the Cys concentration was detectable at an emission wavelength of 550 nm. Respectively form a linear relation with Cys in the concentration range of 0-300 mu m; YF probe can detect the total concentration of Cys (C)C). The results show that: the fluorescent probe has good fluorescent response to Cys, Hcy and GSH.
Example 3:
the selectivity of the fluorescent probes obtained in example 1 for Cys, Hcy and GSH fluorescence detection.
Test solutions were prepared as in example 2, and various analytes (Lys, Arg, Ser, Leu, Phe, Ala, Gly, Glu, Val, Gln, K) were prepared with deionized water+,Na+,Mg2+,Zn2+,H2O2) The concentration of the solution was 1X 10-2And M. Adding 12 equivalents of other possible interferents into 5 μ M fluorescent probe molecule test solution, including blank, Lys, Arg, Ser, Leu, Phe, Ala, Gly, Glu, Val, Gln, K+,Na+,Mg2+,Zn2+,H2O2To these solutions, 12 equivalents of Cys, Hcy and GSH were added, respectively. After mixing for 10 minutes, fluorescence spectra of each group of solutions were obtained by performing fluorescence spectrum measurement under the same conditions with excitation wavelengths of 350nm and 475nm, respectively.
From the results shown in FIG. 3, it was found that when Lys, Arg, Ser, Leu, Phe, Ala, Gly, Glu, Val, Gln, K was added to the system+,Na+,Mg2+,Zn2+,H2O2After the potential interferents are detected, under the detection solution with the pH of 7.4 and the excitation wavelength of 350nm, the obvious fluorescence enhancement can be obtained only by adding cysteine, homocysteine and glutathione. Whereas, at an excitation wavelength of 475nm under the same pH conditions, only the enhancement of Cys and Hcy fluorescence was observed. When the pH value of the detection solution is 5.6 and the excitation wavelength is 475nm, the probe YF can selectively respond to Cys. The results show that: the highly selective fluorescent response shows that the probe YF can be used for detecting Cys, Hcy and GSH in a complex biological environment and is not interfered by other coexisting ions or molecules.
Example 4:
the effect of pH on Cys, Hcy and GSH was measured with the fluorescent probe obtained in example 1.
As the pH value of the detection solution is one of the key factors for quantitatively identifying Cys, Hcy and GSH, the response of the probe molecules to Cys, Hcy and GSH under different pH conditions is researched, and phosphate buffer solutions with different pH values (2-12) are prepared respectively. The fluorescence spectrum of the fluorescent probe obtained in example 1 was measured sequentially at pH from 2 to 12 systems, as shown in FIG. 4. It can be seen that the fluorescence of the probe YF itself is hardly affected by the change in pH; after Cys, Hcy and GSH are introduced, the fluorescence of the probe YF is obviously enhanced at 508nm (lambda ex is 350nm) within the pH range of 4.0-10.0. Cys, Hcy and GSH (C) can be detectedC+H+G) (ii) total concentration of (d); then, the excitation wavelength is changed to 475nm, and after Cys and Hcy treatment, the fluorescence of the probe YF is enhanced at 550nm when the pH value is 5.0-11.0 and 5.7-11.0 respectively. In addition, there was little fluorescence enhancement at 550nm after probe YF treatment of GSH. Therefore, Cys and Hcy (C) were detected in the pH 7.4 detection solutionC+H) Cys (C) was detected in a test solution at pH 5.6C) The concentration of (c).
Example 5:
kinetic study of the fluorescent probe obtained in example 1.
The kinetic distribution of probe YF to Cys, Hcy and GSH was studied. The pH value of the detection solution is kept at 7.4, the excitation wavelength is 350nm, and the fluorescence intensity of the probe YF at 508nm is not obviously changed. Fluorescence at 508nm (λ ex 350nm) increased significantly within 5 minutes with the addition of Cys, Hcy or GSH treatments, respectively, indicating a rapid response to the three biological thiols. Furthermore, only Cys and Hcy showed rapid fluorescence enhancement at pH 7.4 and excitation wavelength 475nm, whereas GSH showed little fluorescence. In addition, when the pH value of the detection solution is 5.6 and the excitation wavelength is still kept at 475nm, only Cys generates a rapid fluorescence response at 550 nm. These results indicate that probe YF reacts rapidly to Cys, Hcy, or GSH, thereby enabling real-time detection of Cys, Hcy, or GSH.
Example 6:
practical application of the fluorescent probe obtained in example 1.
(1) The fluorescent probe obtained in example 1 detects thiols in plasma.
Venous blood of healthy volunteers was taken at 3.0 mL. The blood samples were placed in pre-cooled ethylenediaminetetraacetic acid (EDTA) vacuum tubes and immediately centrifuged at 2500r/min for 10min at 4 ℃. The supernatant was analyzed as human plasma. 0.025mL of fluorescent probe stock solution (1X 10)-3M) and 1mL DMSO in a 5mL volumetric flask, human plasma samples (0 μ L,50 μ L,100 μ L,150 μ L,200 μ L,250 μ L or 300 μ L) were added, respectively, and then the volume was adjusted to 5mL with 20mM phosphate buffer (pH 7.4 or pH 5.6), respectively, shaken, left at room temperature for 10min, then the mixed solution was transferred to a quartz cuvette, and after excitation at 350nm or 475nm, the fluorescence spectrum of the solution was recorded with a fluorescence spectrophotometer.
To determine the utility of probe YF in biological fluids, probe YF was first used to monitor Cys, Hcy, and GSH levels in human plasma. As shown in a-c of FIG. 6, from left to right, it can be seen that when different volumes of plasma samples (0. mu.L, 50. mu.L, 100. mu.L, 150. mu.L, 200. mu.L, 250. mu.L or 300. mu.L) were added, the pH of the measurement solution was 7.4 or 5.6, the fluorescence intensity was gradually increased at the excitation wavelength of 350nm or 475nm, and the above results indicate that the fluorescent probe of the present invention can detect Cys, Hcy and GSH in plasma.
(2) The fluorescent probe obtained in example 1 quantitatively detects Cys, Hcy and GSH in plasma at the same time.
Several 5mL volumetric flasks were each charged with 0.025mL of fluorescent probe molecule stock solution (1X 10)-3M) and 100. mu.L plasma samples, respectively, and then added to different volumes of 1X 10-2M Cys solutions (0, 2.5, 5, 7.5, 10, 15, 20 μ L) were then separately diluted to 5mL with 20mM phosphate buffer (pH 7.4 or pH 5.6), shaken well, left at room temperature for 10min, and then the fluorescence spectra were recorded with a fluorescence spectrophotometer at a fluorescence excitation wavelength of 350nm or 475nm, respectively.
Unknown levels of cysteine (Cys), homocysteine (Hcy) and Glutathione (GSH) in plasma samples were detected using standard labeling methods with cysteine (Cys) as the standard, as shown in d-f in fig. 6. The total concentration of Cys, Hcy and GSH in the plasma samples was found to be 147.6. mu.M by calculation; cys and Hcy were detected at a total concentration of 126.7. mu.M; cys concentration was 118.3. mu.M. From this, the concentrations of Cys, Hcy and GSH were calculated to be 118.3. mu.M, 8.4. mu.M and 20.9. mu.M, respectively, which fit the concentration ranges of Cys, Hcy and GSH in healthy human plasma. Therefore, the probe YF can be used for quantitatively detecting Cys, Hcy and GSH in human plasma.
In conclusion, we rationally designed and synthesized a novel fluorescent probe that can detect Cys, Hcy and GSH in different pH and different fluorescence channels. The results show that at pH 7.4 and excitation wavelength 350nm, the total concentration of Cys, Hcy and GSH can be detected at emission wavelength 508 nm; when the pH was still 7.4 and the excitation wavelength was changed to 475nm, the total concentration of Cys and Hcy could be detected at an emission wavelength of 550 nm; when the pH was changed to 5.6, the excitation wavelength was still 475nm and the Cys concentration was detectable at an emission wavelength of 550 nm. Then, through simple mathematical calculation, the content of Cys, Hcy and GSH in the plasma can be quantitatively distinguished and detected simultaneously.
Claims (5)
2. the method for preparing the fluorescent probe for simultaneously and quantitatively detecting Cys, Hcy and GSH in the plasma as claimed in claim 1, wherein the steps are as follows:
step 1, preparing 7- (diethylamino) coumarin-3-hydroxy for later use;
step 2, adding the 7- (diethylamino) coumarin-3-hydroxy and 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole obtained in the step 1 into an acetonitrile solution, then adding potassium carbonate, stirring at room temperature for reaction for 4-5 hours, removing redundant solvent after the reaction is finished, and purifying the residue through column chromatography to obtain a fluorescent probe for quantitatively detecting Cys, Hcy and GSH at the same time;
wherein the dosage ratio of the 7- (diethylamino) coumarin-3-hydroxy, the 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole, the potassium carbonate and the acetonitrile is 0.3 g-0.4 g: 0.4-0.5 g: 0.2 g-0.3 g: 4 mL-5 mL.
3. The method according to claim 2, wherein the solvent removal method in step 2 is rotary evaporation.
4. Use of the fluorescent probe of claim 1 for the preparation of a reagent for simultaneous quantitative differential detection of Cys, Hcy and GSH in plasma.
5. The use according to claim 4, wherein the detecting step is:
(1) plasma pretreatment;
(2) when the pH value of the sample obtained in the step (1) is 7.4 and the excitation wavelength is 350nm, the total concentration of Cys, Hcy and GSH can be detected at the position of the emission wavelength of 508 nm;
(3) when the pH of the sample obtained in the step (1) is still 7.4 and the excitation wavelength is changed to 475nm, the total concentration of Cys and Hcy can be detected at the position where the emission wavelength is 550 nm;
(4) when the pH value of the sample obtained in the step (1) is changed to 5.6, the excitation wavelength is 475nm, and the concentration of Cys can be detected at the position of the emission wavelength of 550 nm; then, the content of Hcy and GSH in plasma was calculated.
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