Method and kit for determining sulfydryl content

Abstract

The invention provides a method and a kit for determining the content of sulfydryl. The method comprises the following steps: S1, reacting a dithio compound sulfydryl probe with sulfydryl of a standard substance and a sample to release a sulfydryl-containing compound, and labeling the sulfydryl-containing compound with a fluorescent probe; and S2, measuring the fluorescence intensity of the sulfydryl standard substance and the sample solution after the reaction. And S3, establishing a standard curvilinear equation according to the fluorescence intensity of the sulfydryl standard substance after the reaction, which is measured in the S2, and calculating the sulfydryl content of the sample to be detected by adopting the standard curvilinear equation according to the measured fluorescence intensity of the sample to be detected after the reaction. Compared with a traditional Ellman reagent method, the sulfydryl content detection method has the advantages that the sensitivity is improved by 29 times, and the problem that the sulfydryl content of a sample is underestimated due to the fact that the sulfydryl standard substance and the sulfydryl of the sample are directly marked by directly using a fluorescent probe at present can be avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of thiol detection, and in particular to a method for determining thiol content, in particular a method for highly sensitively detecting thiol content using a fluorescent probe, and a method for preparing a related kit. Background Art

[0002] The form in which the thiol groups of cysteine ​​in protein molecules exist has a great influence on the structural stability of proteins. The thiol groups on cysteine ​​residues at specific positions in protein molecules often form disulfide bonds, which play an important role in maintaining the three-dimensional structure of proteins. Although the thiol groups on specific cysteine ​​residues of some protein molecules do not form disulfide bonds with the thiol groups of other cysteine ​​residues, they do not necessarily exist in the form of free thiol groups, but may form mixed disulfide bonds with other small molecules of thiol-containing compounds. For example, studies have found that although the thiol group of the cysteine ​​residue (Cys34) at the 34th residue position on serum albumin in patients with hyperlipidemia and the thiol group of the cysteine ​​residue at the Cys34 position on serum albumin in normal people will not form disulfide bonds with the thiol groups on cysteine ​​residues at other positions on the protein molecule, the thiol group of the cysteine ​​residue at this residue position on serum albumin in patients with hyperlipidemia is thiolated by small molecule thiol compounds (such as cysteine, homocysteine, etc.) to form a mixed disulfide bond at a significantly higher rate than the thiol group of the cysteine ​​residue at the corresponding residue position on normal human serum albumin. In addition, a higher proportion of the thiol groups of some cysteine ​​residues in serum albumin in patients with hyperlipidemia that can form intramolecular disulfide bonds are thiolated by small molecule thiol compounds, thereby affecting the formation of normal intramolecular disulfide bonds of the protein or causing the rupture of these intramolecular disulfide bonds. Under some pathological conditions, the oxidized serum albumin (i.e., serum albumin with oxidized Cys34 thiol) will increase. Therefore, in clinical practice, the determination of the thiol content of specific proteins in the human body has important clinical significance.

[0003] Antibody Drug Conjugates (ADCs) are an important area of ​​anti-tumor drug development in recent years. ADCs are composed of monoclonal antibodies and toxins that can effectively kill tumor cells, thus combining the high tumor target specificity and long half-life of monoclonal antibodies with the strong toxicity of toxins against tumor cells. Because the toxins of ADCs are only released in tumor cells and not in normal cells under the guidance of monoclonal antibodies, the amount of toxins that ADCs can carry can be greatly increased without causing drug toxicity compared to using toxins alone. In many ADCs, toxins are coupled to monoclonal antibody molecules through thiol groups on antibody molecules. Before the coupling reaction, the content of antibody thiol groups needs to be measured to confirm that the content of antibody thiol groups is at the expected level, and after the coupling reaction, the content of antibody thiol groups needs to be measured to confirm the high efficiency of the coupling reaction. Establishing a quantitative determination method for thiol groups with strong specificity and sensitivity can reduce the detection amount of ADCs. The research and application of these methods are of great significance to promote the development of ADCs. In addition to protein sulfhydryls, the concentration levels of small molecule sulfhydryl compounds in the blood, such as cysteine, homoamino acids, and glutathione, are also related to a person's health status. Determination of these small molecule sulfhydryls in plasma or serum has important clinical significance.

[0004] The most classic method for quantitative determination of free thiol groups is the Ellman reagent method. Ellman reagent was developed by George L. Ellman, and its chemical name is 5,5′-dithiobis-(2-nitrobenzoic acid) (5,5′-dithiobis-(2-nitrobenzoic acid, abbreviated as DTNB). When DTNB reacts with thiol groups, the disulfide bond in the molecule breaks, and under slightly alkaline conditions, 2-nitro-5-mercaptobenzoic acid (TNB) is generated in an amount equal to the thiol groups involved in the reaction. TNB is yellow in alkaline solution, and the depth of yellow is related to its concentration. The extinction coefficient at 412nm is 14150M -1 cm -1 DTNB has strong specificity and is easy to use. However, the sensitivity of this method is low. When the sample amount is extremely small or the thiol content in the sample is very low, the absorption intensity of TNB released by the reaction of DTNB with thiol groups at a wavelength of 412nm is not sufficient to quantitatively determine the thiol content of the sample.

[0005] In view of the low sensitivity of the classic Ellman reagent method, in order to improve the sensitivity of thiol detection, some laboratories in academia and industry have developed some fluorescent probes for thiol groups. Among them, ThioGlo-1 ((10-methyl-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-9-methoxy-3-oxo-3H-benzo[f]-chromene-2-carboxylic acid) is a thiol-specific probe with good performance and convenient use. However, when ThioGlo-1 is used alone to determine the thiol content of bovine serum albumin (BSA), the measured thiol content of BSA deviates too much from the actual content. There are two possible reasons for this problem: (1) fluorescent probes such as ThioGlo-1 are not easy to approach the thiol groups of BSA molecules, resulting in low reaction efficiency; (2) the fluorescence intensity unit of the fluorescent complex formed by the reaction of cysteine ​​used as a thiol standard with ThioGlo-1 is higher than the fluorescence intensity unit of the fluorescent complex formed by the reaction of BSA thiol with Thioglo-1. For example, Hoff et al. (2013) found that when equimolar amounts of cysteine, homocysteine, ethyl cysteine, N-acetylcysteine, and glutathione were labeled with the ThioGlo-1 fluorescent probe, the fluorescence intensity units of the fluorescent complexes formed by equimolar amounts of these thiol-containing small molecules and the fluorescent probe ThioGlo-1 were different (see Figure 1 ). The results show that the fluorescence intensity unit of the thiol compound-fluorescent probe complex formed by the reaction of the thiol group on the thiol compound with the fluorescent probe is affected by the structure of the adjacent group of the fluorescent probe in the complex. The results suggest that if the fluorescent probe is used to directly react with the thiol standard and the sample containing thiol substances to determine the thiol content of the sample, the following two situations may lead to underestimation or overestimation of the thiol content of the sample: there are thiol compounds with different structures in the sample or the structure of the thiol-containing substance in the sample is different from that of the thiol standard used for quantitative determination. In addition to the above two situations, when determining the thiol content of protein, if the fluorescent probe cannot reach the site where the thiol group is located due to large molecules or other physical and chemical reasons, it will also lead to underestimation of the protein thiol content.

[0006] Therefore, in order to establish a highly sensitive and accurate method for determining thiol groups by using fluorescent probes, the above-mentioned problem must be solved in such a way that the thiol groups from the standard and the sample can obtain the same fluorescence intensity unit measurement. If the thiol content of the protein is measured, it must also be ensured that the thiol probe can reach the specific epitope of the protein where the protein thiol group is located. Summary of the invention

[0007] In view of the above-mentioned problem of underestimation or overestimation of protein thiol groups by using fluorescent probes, the present invention proposes a method for determining the thiol content and a method for preparing a kit corresponding to the method. The thiol detection method of the present invention utilizes the principle that disulfide compound thiol probes such as DTNB can react with thiol groups and release thiol-bearing compounds equivalent to the thiol groups involved in the reaction during the reaction, and the latter can be labeled with thiol-specific fluorescent probes, thereby solving the above-mentioned problem.

[0008] The present invention takes DTNB as the thiol probe of the first step reaction of the method and ThioGlo-1 as the thiol fluorescent probe of the second step as an example, and the new method and its principle are explained as follows: the first step of the new method is to use DTNB to fully react with the thiol standard and the sample to be tested, so that DTNB releases TNB equivalent to the thiol participating in the reaction. Since each TNB molecule carries a thiol group, through this reaction, the thiol groups of the standard and the sample can be represented by the thiol groups of the TNB molecules produced by the reaction, and the thiol groups on TNB can react with ThioGlo-1 to form a ThioGlo-1-TNB complex with strong fluorescence. Therefore, a fluorescence intensity / thiol concentration standard curve can be established by measuring the fluorescence intensity of the standard with different concentrations after the above two-step reaction, and finally the thiol content of the sample is calculated by the standard curve according to the fluorescence intensity of the sample after the above two-step reaction. Through these two-step reaction, the content of the thiol groups of the thiol standard and the thiol groups of the sample are measured by the same fluorescent complex. In this way, the above-mentioned problem of seriously underestimating or overestimating the sample sulfhydryl content due to the difference in structure between the sulfhydryl content of the sulfhydryl standard and the sample sulfhydryl content meter can be solved. In addition, compared with some fluorescent probes, many disulfide compound sulfhydryl probes that can be used to measure the sulfhydryl content are smaller in molecules and more easily accessible to sulfhydryl groups located at different epitopes of the detected protein. Therefore, the above-mentioned possibility of underestimating or overestimating the sample sulfhydryl content can be avoided or reduced. Figure 2 is a graphic illustration of the new method.

[0009] The present invention provides a method for determining the content of thiol groups, comprising the following steps: S1: preparing a plurality of thiol standard solutions of different concentrations with a buffer solution and diluting the sample to be tested with the same buffer solution, adding a disulfide compound thiol probe to the thiol standard solution and the sample solution to be tested, and incubating the disulfide compound thiol probe with the thiol groups in the standard and the sample, and the disulfide compound releases TNB equivalent to the thiol groups involved in the reaction; S2: adding a fluorescent probe solution to the thiol standard solution incubated with the disulfide compound thiol probe in S1 and the sample to be tested, and incubating the fluorescent probe with the TNB in ​​S1 to form a fluorescent complex that emits strong fluorescence; S3: measuring the fluorescence intensity of the thiol standard solution and the sample to be tested after incubation in S2; S4: Establish a fluorescence intensity / thiol concentration standard curve equation based on the fluorescence intensity value corresponding to the thiol standard solution measured in S3, and calculate the thiol content of the sample using the standard curve equation based on the fluorescence intensity value corresponding to the sample to be tested.

[0010] The disulfide compound thiol probe is any one or more of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), 5-(2-aminoethyl)dithio-2-nitrobenzoic acid (ADNB), and n-octyldithionitrobenzoic acid (ODNB).

[0011] Since disulfide compound thiol probes have different physical characteristics such as molecular size, electrostatic charge and hydrophobicity, the efficiency of each of them approaching the thiol groups on specific compounds may be different. When determining the thiol content of proteins, the ease with which each of these disulfide compound thiol probes can approach the thiol groups in specific regions of proteins may be significantly different. Therefore, in certain circumstances, the use of a mixture of two or more disulfide compound thiol probes may improve the detection rate of thiol groups, preferably any one or more of DTNB, ADNB and ODNB.

[0012] The method of the present invention can be used to determine the content of protein thiol groups, and can also be used to determine the total amount of thiol groups in other samples containing different thiol compounds.

[0013] In some embodiments, the thiol standard solution in S1 is any one of a cysteine ​​standard solution, a homocysteine ​​standard solution, an N-acetylcysteine ​​standard solution, a cysteine ​​ethyl ester standard solution, and a glutathione standard solution.

[0014] In some embodiments, the working concentration of the disulfide compound thiol probe described in S1 is 5 to 100 times the maximum concentration of the thiol standard solution; preferably 10 to 200 μM; The working concentration of the fluorescent probe described in S2 is 2.5 to 20 times the maximum concentration of the thiol standard solution; preferably 5 to 40 μM.

[0015] In some embodiments, the fluorescent probe includes but is not limited to the ThioGlo-1 fluorescent probe.

[0016] The present invention also provides a thiol content detection kit, comprising the following components: A first storage tube, used for storing thiol standards; A second storage tube, used for storing the disulfide compound thiol probe; The third storage tube is used to store the fluorescent probe or other fluorescent probes.

[0017] In some embodiments, the thiol content detection kit further includes a disulfide compound thiol probe reaction buffer, a pH adjuster, and a fluorescent probe buffer.

[0018] In some embodiments, the pH of the disulfide compound thiol probe reaction buffer is 7.8-9.5.

[0019] In some embodiments, the pH adjuster is any one of a phosphate solution, a phosphate buffer solution, a citric acid solution, a citric acid buffer solution, an acetic acid solution or an acetic acid buffer solution, preferably a 1 M sodium dihydrogen phosphate solution.

[0020] The fluorescent probe buffer includes but is not limited to 100 mM sodium phosphate buffer, pH 7.0.

[0021] The present invention also provides a thiol content detection system, comprising: A standard curve preparation module: comprising thiol standard solutions of multiple concentrations, wherein the thiol standard solutions contain a disulfide compound thiol probe and a fluorescent probe; Test sample solution module: a test sample solution containing a disulfide compound thiol probe and a fluorescent probe; Sulfhydryl-related fluorescence signal detection module: measuring the fluorescence intensity of the sulfhydryl standard solution and the sample solution to be tested; Calculation module: obtain the standard curve equation according to the fluorescence intensity of the thiol standard solution, and calculate the thiol content of the sample solution to be tested according to the fluorescence intensity of the sample corresponding to the sample solution to be tested.

[0022] In summary, compared with the prior art, the present invention achieves the following technical effects: (1) The present invention discloses a method for detecting thiol content. Compared with the traditional Ellman reagent method, the sensitivity is increased by 29 times, the detection limit can be as low as 0.009 μM, and the quantitative limit can be as low as 0.029 μM. In addition, the detection method of the present invention avoids the problem of underestimating the thiol content in the sample by using a fluorescent probe to directly react with the thiol groups of the standard and the sample, thereby ensuring the accuracy of the detection result.

[0023] (2) The method for detecting thiol content of the present invention is relatively simple. The establishment of a thiol quantitative determination method with strong specificity and high sensitivity can greatly reduce the amount of sample used for detection, which is of great significance for the development of related scientific research and industrial fields that require high-sensitivity thiol detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, some diagrams that are helpful for understanding the principles of the new method and illustrating the experimental results in the embodiments are shown below. It should be understood that the following drawings only show certain descriptions and implementation examples related to the present invention, and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can also be obtained based on these drawings without creative work.

[0025] Figure 1 The standard curve is a fluorescence intensity of the fluorescent complex formed by the reaction of small molecule sulfhydryl compounds such as cysteine, homocysteine, ethyl cysteine, N-acetylcysteine, glutathione, etc. with ThioGlo-1 against the concentration of the sulfhydryl compound.

[0026] Figure 2 The figure is an illustration of the principle of the thiol content detection method of the present invention; in the figure, RSH represents a substance with a thiol group, including a thiol standard substance and a thiol-containing substance in a sample to be tested.

[0027] Figure 3 This is a standard curve diagram of the DTNB / ThioGlo-1 method of Example 1 of the present invention.

[0028] Figure 4 This is a standard curve diagram of the ADNB / ThioGlo-1 method of Example 2 of the present invention.

[0029] Figure 5 This is a standard curve diagram of the Ellman reagent method of comparative experimental example 1 of the present invention.

[0030] Figure 6 This is a standard curve diagram of the method of using ThioGlo-1 fluorescent probe to directly react with the thiol group of a standard or a sample in comparative experimental example 2 of the present invention. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0032] In the prior art, the method of using thiol-specific fluorescent probe to determine the thiol content is to use the fluorescent probe to directly react with the thiol standard and the sample, respectively, to form two types of strong fluorescent complexes, "thiol standard molecule-fluorescent probe molecule complex (ThiolStd-FP)" and "thiol-containing sample molecule-fluorescent probe molecule complex (SampleThiol-FP)", and to establish a standard curve with the fluorescence intensity of ThiolStd-FP obtained from thiol standards of different concentrations, and then calculate the content of the sample according to the fluorescence intensity of SampleThiol-FP of the sample. Although this method seems to improve the sensitivity of thiol content determination, it often seriously underestimates or overestimates the thiol content of the sample compared with the Ellman reagent method. The possible reasons are as follows: Possibility 1: The fluorescence intensity units of ThioStd-FP and SampleThiol-FP are different; Possibility 2: There are thiol compounds with different structures in the sample, and the fluorescence intensity units of the complexes formed by these compounds and the thiol fluorescent probe are different. For example, the fluorescence intensity units of the complexes formed by cysteine ​​and homocysteine ​​and the fluorescent probe are different, and the fluorescence intensity units of the complexes formed by the thiol groups of cysteine ​​residues at different positions on the polypeptide chain of protein molecules and the fluorescent probe are different.

[0033] Note: The definition of "Fluorescent Intensity Unit (FIU)" here is: 1FIU is equal to the number of fluorescent photons emitted by 1 mole of fluorescent substance in 1 second.

[0034] Possibility 3: The fluorescent probe cannot access the thiol groups of the sample. For example, the thiol groups located in a certain part of the protein molecule may be more easily accessible to DTNB than to the fluorescent probe. Therefore, the thiol content measured by the fluorescent probe is lower than the protein thiol content measured by DTNB.

[0035] The present invention provides a method for determining the thiol content of a sample (including the thiol content of protein), which can avoid all the above problems. The method first uses a disulfide compound thiol probe (such as DTNB) to react with the thiol groups on the thiol standard and the thiol groups of the sample to be tested, respectively, and the reaction causes the disulfide compound thiol probe to release a thiol-carrying compound (such as TNB) equivalent to the thiol groups participating in the reaction. In this way, the number of thiol groups in both the thiol standard and the sample can be represented by the number of thiol groups of the thiol-containing compound (such as TNB) released from the disulfide compound thiol probe. These released thiol-containing compounds can be labeled with a thiol-specific fluorescent probe to form a fluorescent complex. Through these two steps of reaction, the thiol groups on both the thiol standard and the sample can be measured by the fluorescence of the fluorescent complex with the same structure. Therefore, the new method is not only highly sensitive, but also avoids the above three problems that lead to underestimation of the protein thiol content. For convenience, the method is further described here by taking DTNB as a disulfide compound thiol probe and ThioGlo-1 as a thiol-specific fluorescent probe as an example. The process of the method is as follows: (1) Add equal volumes of thiol standards and test samples of different concentrations dissolved in slightly alkaline buffer to different test tubes.

[0036] (2) Add the same volume of DTNB solution dissolved in slightly alkaline buffer to each test tube in (1). DTNB can react specifically with sulfhydryl groups. After a certain incubation period, DTNB reacts fully with sulfhydryl groups to release TNB equivalent to the sulfhydryl groups of the standard or sample. Through this reaction, the sulfhydryl groups of the sulfhydryl standard and the sample are converted into sulfhydryl groups on the same quality of TNB (other disulfide compound sulfhydryl probes can also be used for this step of reaction. The disulfide compound sulfhydryl probe used for this reaction must be able to react specifically with sulfhydryl groups and release a compound with a sulfhydryl group equivalent to the sulfhydryl groups involved in the reaction after the reaction).

[0037] (3) After the above reaction is completed, add the same volume of pH adjustment solution to the standard and test protein reaction tubes respectively, and adjust the pH of the reaction mixture to pH 7.0-7.2. Then add an equal volume of ThioGlo-1 solution to each reaction tube and incubate for a certain period of time to allow ThioGlo-1 to fully react with the TNB produced in each standard tube and sample tube. In this step of the reaction, since the thiol groups in the thiol standard and sample are converted into thiol groups on TNB in ​​the previous step of the reaction, the ThioGlo-1-TNB complexes formed in the thiol standard reaction tube and the sample reaction tube in this step of the reaction are homogeneous and have the same unit fluorescence intensity. Other thiol-specific fluorescent probes can also be used in this step of the reaction.

[0038] (4) Measure the fluorescence intensity of the solution in each test tube after the above reaction.

[0039] (5) Establishing a fluorescence intensity / thiol concentration standard curve equation (hereinafter referred to as "fluorescence standard curve equation") based on the measured fluorescence intensity of each set concentration of the thiol standard after the above reaction.

[0040] (6) Substitute the fluorescence intensity of the sample after the above reaction into the fluorescence standard curve equation to calculate the thiol content of the sample.

[0041] In order to verify that the new method can avoid the problem of underestimating the sample thiol content caused by using the fluorescent probe alone, we used the new method to measure the thiol content of BSA (Examples 1 and 2) and compared it with the BSA thiol content measured by the Ellman reagent method (Comparative Experimental Example 1). The results showed that whether DTNB or ADNB was used as the disulfide compound thiol probe in the first step reaction, the BSA thiol content measured by the detection method of the present application was very close to the result measured by the Ellman reagent method, which proved that the new method for measuring the thiol content can avoid the problem of underestimating the BSA thiol content when using the fluorescent probe alone (i.e., the fluorescent probe is directly reacted with the thiol standard and the sample to be tested). In addition, the sensitivity of the detection method of the present application is 29 times higher than that of the Ellman reagent method.

[0042] Reagents and preparation methods used in the experiment: Buffer 1: 100 mM sodium phosphate buffer containing 1 mM ethylenediaminetetraacetic acid disodium salt (EDTA), pH 8.0; Buffer 2: 200 mM sodium phosphate buffer (containing 1 mM EDTA), pH 7.0.

[0043] pH adjustment solution: 1 M sodium dihydrogen phosphate (containing 1 mM EDTA).

[0044] Cysteine ​​(Cys) stock solution: 200 mM, dissolved in pure water.

[0045] ThioGlo-1 stock solution, at a concentration of 1 mM, is dissolved in dimethyl sulfoxide (DMSO).

[0046] BSA (bovine serum albumin) stock solution: concentration is 100 mM, dissolved in pure water.

[0047] Example 1 In this example, DTNB is used as the disulfide compound thiol probe in the first step of the new method to determine the thiol content of BSA. The detection method includes two steps. The first step is to use DTNB to react with the thiol groups of the thiol standard and the sample to be tested, respectively, to release TNB with a thiol group equivalent to the thiol groups involved in the reaction. The second step is to use the fluorescent probe ThioGlo-1 to react with the thiol groups of TNB released in the first step. The specific detection steps are as follows: (1) Dissolve DTNB in ​​buffer 1 to prepare a DTNB stock solution with a concentration of 5 mM.

[0048] (2) Dilute the DTNB stock solution, Cys standard stock solution, and BSA stock solution with buffer 1. Dilute the DTNB stock solution to a 60 μM solution; dilute the Cys stock solution to prepare 0.2-, 0.4-, 0.6-, 0.8-, and 1 μM Cys standard solutions; and dilute the BSA stock solution to 0.1 mg / mL.

[0049] (3) Dilute the ThioGlo-1 stock solution (1 mM) to 20 μM using buffer 2.

[0050] (4) Take six 1.5 mL centrifuge tubes, add 400 μL of buffer 1 (used as background) to one of them, and add 400 μL of 0.2-, 0.4-, 0.6-, 0.8-, and 1 μM cysteine ​​standard solutions to the remaining five tubes, respectively. Then, add 400 μL of 60 μM DTNB solution to each of the above centrifuge tubes, vortex for 3 seconds to mix, and centrifuge in a micro desktop centrifuge for 5 seconds to allow the liquid attached to the tube wall to merge with the liquid at the bottom of the centrifuge tube.

[0051] (5) Take three 1.5 mL centrifuge tubes and add 400 μL of diluted BSA solution (0.1 mg / mL) and 400 μL of 60 μM DTNB solution to each tube. Vortex for 3 seconds to mix well. Centrifuge in a micro-tabletop centrifuge for 5 seconds to allow the liquid attached to the tube wall to merge with the liquid at the bottom of the tube.

[0052] (6) After the three reaction mixtures, i.e., DTNB / buffer 1 (background), DTNB / Cys (standard), and DTNB / BSA (sample protein) were incubated at room temperature (about 25°C) for 15 minutes, DTNB reacted with sulfhydryl groups to release TNB. 120 μL of pH adjustment solution (1 M NaH2PO4 solution) was added to adjust the pH of the mixture to 7.0-7.2.

[0053] (7) Then, 400 μL of 20 μM ThioGlo-1 solution was added to each of the above test tubes, vortexed for 3 seconds to mix, and centrifuged in a micro-tabletop centrifuge for 5 seconds to allow the liquid attached to the tube wall to merge with the liquid at the bottom of the centrifuge tube. Incubated at room temperature (about 25°C) for 40 min to allow ThioGlo-1 to fully react with TNB generated in the previous step. In the reaction mixture prepared according to the above method, the concentrations of cysteine ​​standard and BSA were diluted 3.3 times compared with the solutions of these two substances used to prepare the mixture. Therefore, in the five cysteine ​​reaction mixtures used to prepare the standard curve, the concentrations of cysteine ​​were: 0.0, 0.0303, 0.0606, 0.1212, 0.1818, 0.2424, 0.3030 μM. In the BSA reaction mixture, the concentration of BSA was 0.0303 mg / mL (0.4545 μM).

[0054] (8) Each of the above reaction mixtures was sampled 3 times, and 200 μL was added to 3 different wells of a black 96-well microplate. After the three types of mixtures were added to the black 96-well microplate in a repeated manner, the 96-well microplate was placed in a fluorescence microplate reader to measure the fluorescence value (excitation wavelength 384 nm, emission wavelength 513 nm).

[0055] (9) The fluorescence readings of the background, the standard substance at each concentration, and the BSA reaction mixture are shown in Table 1. A standard curve was constructed based on the data in Table 1 ( Figure 3 ).

[0056] (10) To calculate the standard deviation of background, the background samples were divided into 10 groups, with three replicates of 200 μL in each group. The samples were added to a 96-well microplate and the fluorescence values ​​were read on a microplate reader (excitation wavelength: 384 nm, emission wavelength: 513 nm). The average of the three readings of each background group was taken (a total of 10 background averages were obtained).

[0057] (11) Detection results: Substitute the net fluorescence value of the BSA reaction mixture into the regression equation y = 18861611 x -26119 (see Figure 3 ) was calculated to be 0.2449 μM in the BSA solution (0.0303 mg / mL). Since the micromolar concentration of BSA at a concentration of 0.3030 mg / mL is 0.4545 μM, the measured BSA thiol content was 0.539 mol / mol.

[0058] Table 1: Fluorescence values ​​of background, thiol standard, and BSA after adding ThioGlo-1 to the DTNB / ThioGlo-1 combined detection method

[0059] (12) Calculation of detection limit and quantitation limit: The detection limit (LOD) and quantitation limit (LOQ) representing the sensitivity of the Ellman method were calculated according to Formula 1 and Formula 2: LOD = 3A / S Formula 1; LOQ = 10A / S Formula 2; In formula-1 and formula-2, A is the standard deviation of the background, S is the standard curve y = 18861611 x The slope of -26119 is (18861611) and the standard deviation is calculated using Formula 3: Formula 3; In formula 3, N is the number of samples. According to the above method, N = 10; x i For the number i Background fluorescence readings. It is the average of 10 background fluorescence values ​​(each background fluorescence value is the average of 3 repeated background value readings).

[0060] The average values ​​of 10 groups of backgrounds were calculated based on the average values ​​of each group of backgrounds listed in Table 2, the standard deviations of the 10 groups of backgrounds were calculated using Formula 3, and then the detection limit and quantification limit of the method were calculated using Formulas 1 and 2.

[0061] Table 2: Fluorescence readings of 10 backgrounds and their average and standard deviations for the DTNB / ThioGlo-1 combined detection method

[0062] We obtained: LOD = (3 × 57598) / 18861611 = 0.009 μM; LOQ = (10 × 57598) / 18861611 = 0.031 μM.

[0063] That is, the detection limit of the BSA thiol content detected by the method of this embodiment is 0.009 μM, and the quantitative limit is 0.031 μM.

[0064] Example 2 In this example, ADNB was used as the disulfide compound thiol probe in the first step reaction to determine the thiol content of BSA. The specific detection steps are as follows: (1) Dissolve ADNB in ​​1 M hydrochloric acid to prepare an ADNB stock solution with a concentration of 10 mM.

[0065] (2) Dilute the ADNB stock solution, Cys standard stock solution, and BSA stock solution with buffer 1. Dilute the ADNB stock solution to a 40 μM solution; dilute the Cys stock solution to prepare 0.2-, 0.4-, 0.6-, 0.8-, and 1 μM Cys standard solutions; and dilute the BSA stock solution to 0.1 mg / mL.

[0066] (3) Dilute the ThioGlo-1 stock solution (1 mM) to 20 μM using buffer 2.

[0067] (4) Take six 1.5 mL centrifuge tubes, add 400 μL of buffer 1 (used as background) to one of them, and add 400 μL of 0.2, 0.4, 0.6, 0.8, and 1 μM cysteine ​​standard solutions to the other five tubes, respectively. Then, add 400 μL of 40 μM ADNB solution to each of the above centrifuge tubes, vortex for 3 seconds to mix, and centrifuge in a micro desktop centrifuge for 5 seconds to allow the liquid attached to the tube wall to merge with the liquid at the bottom of the centrifuge tube.

[0068] (5) Take three 1.5 mL centrifuge tubes, add 400 μL of diluted BSA solution (0.1 mg / mL) and 400 μL of 40 μM ADNB solution, vortex for 3 seconds to mix, and centrifuge in a micro-tabletop centrifuge for 5 seconds to allow the liquid attached to the tube wall to merge with the liquid at the bottom of the centrifuge tube.

[0069] (6) After the three reaction mixtures, i.e., ADNB / buffer 1 (background), ADNB / Cys (standard), and ADNB / BSA (sample protein) were incubated at room temperature (about 25°C) for 15 min, 120 μL of NaH2PO4 solution (1 M) was added to adjust the pH of the reaction mixture to 6.8-7.2.

[0070] (7) Add 400 μL of 20 μM ThioGlo-1 solution to each of the above test tubes, vortex for 3 seconds to mix, centrifuge for 5 seconds in a micro-tabletop centrifuge to allow the liquid attached to the tube wall to merge with the liquid at the bottom of the centrifuge tube, and incubate at room temperature (about 25°C) for 35 min to allow ThioGlo-1 to fully react with TNB generated in the previous step. In the reaction mixture prepared according to the above method, the concentrations of cysteine ​​standard and BSA are 3.3 times diluted compared to the solutions of these two substances used to prepare the mixture. Therefore, in the 7 cysteine ​​reaction mixtures used to prepare the standard curve, the concentrations of cysteine ​​are: 0.0, 0.0303, 0.0606, 0.1212, 0.1818, 0.2424, 0.3030 μM. In the BSA reaction mixture, the concentration of BSA is 0.0303 mg / mL (0.4545 μM).

[0071] (8) Repeat the above reaction mixture three times and take 200 μL of samples, respectively, and add them to three different wells of a black 96-well microplate. Place the 96-well microplate after adding the samples into a fluorescence microplate reader to measure the fluorescence value (excitation wavelength: 384 nm, emission wavelength: 513 nm).

[0072] (9) The fluorescence readings of the background, the standard substance at various concentrations, and the BSA reaction mixture are shown in Table 3. A standard curve was made based on the data in the table below. Figure 3 shown.

[0073] Table 3: Fluorescence values ​​of background, cysteine ​​and BSA after adding ThioGlo-1 to the ADNB / ThioGlo-1 combined detection method

[0074] (10) Detection results: Substitute the net fluorescence value of the BSA reaction mixture 4576667 into the regression equation y = 21887667 x -327626 (see Figure 4 ) calculated that the thiol concentration of BSA solution (0.0303 mg / mL) was 0.2241 μM. Since the micromolar concentration of BSA at a concentration of 0.3030 mg / mL is 0.4545 μM, the measured BSA thiol content was 0.493 mol / mol. This result is very different from the result (0.539 mol / mol) obtained by using DTNB as the first step of the disulfide compound thiol probe reaction.

[0075] (11) To calculate the standard deviation of the background, the background samples were divided into 10 groups, with three replicates of 200 μL in each group, and added to a 96-well microplate. The fluorescence values ​​were read on a microplate reader (excitation wavelength: 384 nm, emission wavelength: 513 nm).

[0076] (12) Calculation of limit of detection and limit of quantification: The LOD and LOQ of the ADNB / ThioGlo-1 combined detection method were calculated based on the background fluorescence values ​​of 10 replicates of the method listed in Table 4 (each background fluorescence value is the average of 3 replicate readings).

[0077] Table 4: Fluorescence readings of 10 backgrounds and their mean and standard deviations for the ADNB / ThioGlo-1 combined detection method

[0078] Referring to the calculation method of Example 1, we obtain: LOD = (3 × 63585) / 21887667 = 0.009 μM; LOQ = (10 × 63585) / 21887667 = 0.029 μM.

[0079] That is, the detection limit of the BSA thiol content detected by the above method is 0.009 μM, and the quantification limit is 0.029 μM.

[0080] Comparative Experimental Example 1: Determination of BSA sulfhydryl content using the classic sulfhydryl determination method (Ellman's reagent method) In this comparative experiment, the classic method for determining thiol groups (Ellman's reagent method) was used to determine the thiol content of BSA. The specific steps are as follows: (1) DTNB was dissolved in buffer 1 to prepare a DTNB stock solution with a concentration of 5 mM.

[0081] (2) Dilute the DTNB stock solution, Cys standard stock solution, and BSA stock solution with buffer 1. Dilute the DTNB stock solution to a 200 μM solution; dilute the Cys stock solution to prepare 2, 4, 6, 8, and 10 μM Cys standard solutions; and dilute the BSA stock solution to 1 mg / mL.

[0082] (3) Take 6 1.5 mL centrifuge tubes, add 400 μL of buffer 1 to one of them, and add 2, 4, 6, 8, and 10 μM cysteine ​​standard solutions (400 μL each) to the other 5 tubes, then add 400 μL of 200 μM DTNB solution to each of the above centrifuge tubes, vortex for 3 seconds to mix, and centrifuge in a micro-tabletop centrifuge for 5 seconds to allow the liquid attached to the tube wall to merge with the liquid at the bottom of the centrifuge tube. In these reaction mixtures, the final concentrations of cysteine ​​are 0, 1, 2, 3, 4, and 5 μM, respectively.

[0083] (4) Take three 1.5 mL centrifuge tubes, add 400 μL of diluted BSA solution (1 mg / mL) and 400 μL of 200 μM DNB solution, vortex for 3 seconds to mix, and centrifuge in a micro-tabletop centrifuge for 5 seconds to allow the liquid attached to the tube wall to merge with the liquid at the bottom of the centrifuge tube. The final BSA concentration of the mixture is 0.5 mg / mL.

[0084] (5) Incubate the three types of reaction mixtures, i.e., DTNB / buffer 1 (background), DTNB / Cys (standard), and DTNB / BSA (sample protein) at room temperature (about 25°C) for 15 min.

[0085] (6) Each of the above reaction mixtures was sampled 3 times, and 200 μL was added to 3 different wells of a transparent 96-well microplate. After the above three types of mixtures were added to the 96-well microplate in a repeated manner, the microplate was placed on a microplate reader to read the 412 nm light absorption value.

[0086] (7) To calculate the standard deviation of background, the background samples were divided into 10 groups, with three replicates of 200 μL in each group, added to a 96-well microplate, and the absorbance at 412 nm was read on a microplate reader.

[0087] (8) The average absorbance readings at 412 nm for the background, standards at various concentrations, and the BSA reaction mixture are shown in Table 5. A standard curve was constructed based on the data in Table 5. Figure 4 ).

[0088] Table 5 Average absorbance values ​​of the reaction between DTNB and cysteine

[0089] (9) Test results: Substitute the net absorbance at 412 nm of the BSA reaction mixture (minus the background) into the regression equation y = 0.007730 × + 0.000590 (see Figure 5 ), the thiol concentration of the reaction mixture was calculated to be 3.906 μM. Since the micromolar concentration of BSA at a concentration of 0.5 mg / mL is 7.5 μM, the thiol content of BSA is 0.521 mol / mol.

[0090] (10) Calculation of LOD and LOQ: In order to calculate the detection sensitivity of the Ellman method, 10 background groups (containing only DTNB) were prepared as described above, with three replicates in each group. 200 μL of each sample was added to a 96-well microplate and the absorbance at 412 nm was read. The average background absorbance and standard deviation were calculated based on the values ​​shown in Table 6.

[0091] Table 6 Ellman method background sample light absorption data

[0092] Referring to the calculation method of Example 1, we obtain: LOD = (3 × 0.000674928) / 0.00773 = 0.262 μM; LOQ = (10 × 0.000674928) / 0.00773 =0.873 μM.

[0093] That is, the detection limit of the protein thiol content detected by the method of Comparative Experimental Example 1 is 0.262 μM, and the quantitative limit is 0.873 μM.

[0094] Comparative Experimental Example 2: Determination of BSA thiol content by using fluorescent probes to directly react with thiol standards and samples This comparative experiment example uses ThioGlo-1 fluorescent probe alone to determine the thiol content of BSA. The purpose is to test whether the direct reaction of the thiol-specific fluorescent probe with the thiol standard BSA will lead to an underestimation of the thiol content of BSA. The specific steps are as follows: (1) Dissolve ThioGlo-1 and thiol standard Cys in buffer 2 (200 mM sodium phosphate buffer, pH 7.0) respectively. The concentration of ThioGlo-1 stock solution is 1 mM, and the concentration of Cys stock solution is 200 mM. Dissolve BSA in pure water to prepare a BSA stock solution with a concentration of 100 mg / mL.

[0095] (2) Dilute ThioGlo-1 stock solution, Cys stock solution, and BSA stock solution with buffer 2. ThioGlo-1 was diluted to 20 μM; Cys stock solution was diluted to solutions of different concentrations of 0.2, 0.4, 0.6, 0.8, and 1 μM; and BSA stock solution was diluted to 0.1 mg / mL sample.

[0096] (3) Take six 1.5 mL centrifuge tubes, add 400 μL of buffer 2 (background control) to one of them, and add 400 μL of 0.2, 0.4, 0.6, 0.8, and 1 μM Cys standard solutions to the other five tubes, respectively. Then, add 400 μL of 20 μM ThioGlo-1 solution and 520 μL of buffer 2 to each of the above centrifuge tubes, vortex for 3 seconds to mix, and centrifuge in a micro desktop centrifuge for 5 seconds to allow the liquid attached to the tube wall to merge with the liquid at the bottom of the centrifuge tube. In these reaction mixtures, the final concentrations of Cys were 0.0000, 0.0606, 0.1212, 0.1818, 0.2424, and 0.3030 μM, respectively.

[0097] (4) Take three 1.5 mL centrifuge tubes, add 400 μL of diluted BSA solution (0.1 mg / mL), 400 μL of 20 μM ThioGlo-1 solution, and 520 μL of buffer 2, vortex for 3 seconds to mix, and centrifuge in a micro-tabletop centrifuge for 5 seconds to allow the liquid attached to the tube wall to merge with the liquid at the bottom of the centrifuge tube; the final BSA concentration of the mixture is 0.03030 mg / mL (i.e., 0.4545 μM).

[0098] (5) Incubate the above ThioGlo-1 / Buffer 1 (background), ThioGlo-1 / Cys (standard), and ThioGlo-1 / BSA (sample protein) reaction mixtures at room temperature (about 25°C) for 35 minutes.

[0099] (6) Each of the above reaction mixtures was sampled 3 times, and 200 μL was added to 3 different wells of a black 96-well microplate. After the above three types of mixtures were added to the 96-well microplate in a repeated manner, the microplate was placed on a microplate reader to measure the fluorescence value (excitation wavelength 384 nm, emission wavelength 513 nm).

[0100] (7) To calculate the standard deviation of the background, the background samples were divided into 10 groups, with three replicates of 200 μL in each group. The samples were added to a 96-well microplate and the fluorescence values ​​were read on a microplate reader (excitation wavelength: 384 nm, emission wavelength: 513 nm).

[0101] (8) The fluorescence readings of the background, various concentrations of standard substances, and the BSA reaction mixture are shown in Table 7. Based on the data in the table below, a linear regression standard curve of the net fluorescence value (fluorescence value after removing the background fluorescence) versus the cysteine ​​concentration was drawn, as shown in the following table: Figure 5 shown.

[0102] Table 7 Fluorescence values ​​of the reaction products of ThioGlo-1 with cysteine ​​and BSA

[0103] (9) Test results: Substitute the net fluorescence value of the BSA reaction mixture (0.0303 mg / mL) 1161163 into the regression equation y = 15916701.32 x – 593278.7 (see Figure 6 ), the thiol concentration of the reaction mixture was calculated to be 0.1102 μM. Since the BSA micromolar concentration of the 0.03030 mg / mL BSA solution is 0.4545 μM, the thiol content of BSA is 0.242 mol / mol. The BSA thiol content measured by this method is significantly lower than the result measured by the Ellman method (0.521 mol / mol), so this method significantly underestimates the BSA thiol content.

[0104] (10) Calculation of detection limit and quantification limit: Calculate the LOD and LOQ of this method based on the background standard deviation listed in Table 8: Table 8 Fluorescence readings of 10 backgrounds using the ThioGlo-1 fluorescent probe method alone

[0105] Referring to the calculation method of Example 1, we obtain: LOD = (3 × 64520) / 15916701.32 = 0.012 μM; LOQ = (10 × 64520) / 15916701.32 =0.041 μM.

[0106] That is, the detection limit of the thiol content detected by the method of Comparative Experimental Example 2 is 0.012 μM, and the quantitative limit is 0.041 μM.

[0107] According to the experimental results listed here, if the BSA thiol content is determined by the fluorescent probe ThioGlo-1 using the currently commonly used method, the measured BSA thiol content is 0.242 mol / mol, which is much lower than the BSA thiol content measured by the Ellman reagent method (0.521 mol / mol, see comparative experimental example 1). The BSA thiol content measured by the new method is 0.539 mol / mol (using DTNB as the thiol probe of the first step reaction) or 0.493 mol / mol (using ADNB as the thiol probe of the first step reaction), which is very different from the BSA content (0.521 mol / mol) measured by the classic thiol determination method (Ellman reagent method).

[0108] In summary, the present invention provides a new thiol detection method, which first uses a disulfide compound thiol probe (such as DTNB or ADNB, or a mixture of the two) to react with a thiol standard and a sample respectively. During the reaction, the disulfide compound thiol probe releases a thiol-containing product (such as TNB) equivalent to the thiol participating in the reaction, and the reaction product (such as TNB) reacts with a thiol-specific fluorescent probe (such as ThioGlo-1) to form a complex. In this process, the thiol groups of both the thiol standard and the sample are equivalently converted into the same thiol-containing compound (such as TNB), and then the thiol-specific fluorescent probe is reacted with the product, so that the problem of underestimation or overestimation of the sample thiol content caused by the difference in unit fluorescence intensity between the ThioStd-FP complex (thiol standard-fluorescent probe complex) and the SampleThiol-FP (sample-fluorescent probe complex) can be avoided, because in this method, the substance for measuring the fluorescent signals of the thiol groups of the thiol standard and the thiol groups of the sample is the same.

[0109] 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, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for measuring thiol content, characterized in that The steps include: S1: preparing a plurality of thiol standard solutions of different concentrations with a buffer solution and diluting the sample to be tested with the same buffer solution, adding a disulfide compound thiol probe to the thiol standard solution and the sample solution to be tested, and incubating the disulfide compound thiol probe with the thiol groups in the standard and the sample, and the disulfide compound releases TNB equivalent to the thiol groups involved in the reaction; S2: adding a fluorescent probe solution to the thiol standard solution incubated with the disulfide compound thiol probe in S1 and the sample to be tested, and incubating the fluorescent probe with the TNB in ​​S1 to form a fluorescent complex that emits strong fluorescence; S3: measuring the fluorescence intensity of the thiol standard solution and the sample to be tested after incubation in S2; S4: establishing a fluorescence intensity / thiol concentration standard curve equation based on the fluorescence intensity value corresponding to the thiol standard solution measured in S3, and calculating the thiol content of the sample using the standard curve equation based on the fluorescence intensity value corresponding to the sample to be tested; The disulfide compound thiol probe is any one or more of 5,5′-disulfide bis(2-nitrobenzoic acid), 5-(2-aminoethyl)disulfide-2-nitrobenzoic acid, and n-octyl disulfide nitrobenzoic acid.

2. The method according to claim 1, characterized in that: S1 The thiol standard solution is any one of a cysteine ​​standard solution, a homocysteine ​​standard solution, an N-acetylcysteine ​​standard solution, a cysteine ​​ethyl ester standard solution, and a glutathione standard solution.

3. The method according to claim 1, characterized in that The working concentration of the disulfide compound thiol probe described in S1 is 5 to 100 times the highest concentration of the thiol standard solution; The working concentration of the fluorescent probe described in S2 is 2.5 to 20 times the maximum concentration of the thiol standard solution.

4. The method according to any one of claims 1 to 3, characterized in that The fluorescent probe includes but is not limited to ThioGlo-1 fluorescent probe.

5. A thiol content detection kit, characterized in that Includes the following components: A first storage tube, used for storing thiol standards; A second storage tube, used for storing the disulfide compound thiol probe; The third storage tube is used to store the fluorescent probe.

6. The sulfhydryl content detection kit according to claim 5, characterized in that: Also included are a disulfide compound thiol probe reaction buffer, a pH adjuster, and a fluorescent probe buffer.

7. The sulfhydryl content detection kit according to claim 6, characterized in that: The pH of the disulfide compound thiol probe reaction buffer is 7.8-9.

5.

8. The sulfhydryl content detection kit according to claim 6, characterized in that: The pH regulator is any one of a phosphate solution, a phosphate buffer solution, a citric acid solution, a citric acid buffer solution, an acetic acid solution or an acetic acid buffer solution.

9. A thiol content detection system, characterized in that: include: A standard curve preparation module: comprising thiol standard solutions of multiple concentrations, wherein the thiol standard solutions contain a disulfide compound thiol probe and a fluorescent probe; Test sample solution module: a test sample solution containing a disulfide compound thiol probe and a fluorescent probe; Sulfhydryl-related fluorescence signal detection module: measuring the fluorescence intensity of the sulfhydryl standard solution and the sample solution to be tested; Calculation module: obtain the standard curve equation according to the fluorescence intensity of the thiol standard solution, and calculate the thiol content of the sample solution to be tested according to the fluorescence intensity of the sample corresponding to the sample solution to be tested.

10. Use of the method according to any one of claims 1 to 4, the thiol content detection kit according to any one of claims 5 to 8, or the thiol content detection system according to claim 9 in detecting thiol content.

Images