阅读说明：本技术 一种高灵敏度的单组分tmb显色液及其制备方法 (High-sensitivity single-component TMB color developing solution and preparation method thereof ) 是由 周志鹏 于 2020-10-23 设计创作，主要内容包括：本发明公开了一种高灵敏度的单组分TMB显色液,包括以下组分：聚乙烯醇0.001g/L-20g/L,木糖0.16g/L-30g/L,柠檬酸1mM-100mM,乙二胺四乙酸二钠1mM-20mM,30％过氧化氢水溶液1mM-10mM,TMB 1mM-3mM,DMSO 20ml/L-30ml/L,Proclin300 0.04％,磷酸氢二钠1mM-25mM,超纯水定容至1L。一种高灵敏度的单组分TMB显色液的制备方法,包括以下步骤：步骤一：试剂母液配制：1.1：50g/L低粘度聚乙烯醇1788配制：准确称取5g 1788低粘度型聚乙烯醇于烧杯中,加入适量超纯水,置于65℃恒温水浴锅中,通过玻璃棒持续搅拌使颗粒分散均匀,溶解后继续保温2小时,超纯水定容至100ml,使用0.45μm滤膜过滤。本发明具有检测本底低的特点,有效提高了低浓度样本的检出率,不需要进行传统A液、B液的混合,为用户的操作提供了便利,使得本发明整体上具有突出性的进步。(The invention discloses a high-sensitivity single-component TMB color developing solution, which comprises the following components: 0.001-20 g/L of polyvinyl alcohol, 0.16-30 g/L of xylose, 1-100 mM of citric acid, 1-20 mM of ethylene diamine tetraacetic acid, 1-10 mM of 30% aqueous hydrogen peroxide, 1-3 mM of TMB, 20-30 ml/L of DMSO, 3000.04% of proclin, 1-25 mM of disodium hydrogen phosphate and the volume of ultrapure water is up to 1L. A preparation method of high-sensitivity single-component TMB color developing solution comprises the following steps: the method comprises the following steps: preparing a reagent mother solution: 1.1: 50g/L of low-viscosity polyvinyl alcohol 1788: accurately weighing 5g of 1788 low-viscosity polyvinyl alcohol in a beaker, adding a proper amount of ultrapure water, placing in a constant-temperature water bath kettle at 65 ℃, continuously stirring through a glass rod to uniformly disperse particles, continuously preserving heat for 2 hours after dissolution, fixing the volume of the ultrapure water to 100ml, and filtering by using a 0.45 mu m filter membrane. The invention has the characteristic of low detection background, effectively improves the detectable rate of low-concentration samples, does not need to mix the traditional liquid A and the traditional liquid B, provides convenience for the operation of users, and makes the invention have prominent progress on the whole.)

1. The high-sensitivity single-component TMB color developing solution is characterized by comprising the following components: 0.001-20 g/L of polyvinyl alcohol, 0.16-30 g/L of xylose, 1-100 mM of citric acid, 1-20 mM of ethylene diamine tetraacetic acid, 1-10 mM of 30% aqueous hydrogen peroxide, 1-3 mM of TMB, 20ml/L-30ml/L of DMSO, 3000.04% of proclin, 1-25 mM of disodium hydrogen phosphate and the volume of ultrapure water is up to 1L.

2. The high-sensitivity single-component TMB color developing solution according to claim 1, wherein a buffer solution of citric acid and disodium hydrogen phosphate is used as the single-component TMB color developing solution, and the pH value of the buffer solution is 3.1-3.6.

3. The high-sensitivity single-component TMB color developing solution according to claim 1, wherein the polyvinyl alcohol is 1788 low-viscosity type polyvinyl alcohol, and the degree of polymerization and alcoholysis of the polyvinyl alcohol is 1700 and 88%.

4. The method for preparing a high-sensitivity one-component TMB color developing solution according to any one of claims 1 to 3, comprising the steps of:

the method comprises the following steps: preparing a reagent mother solution:

1.1: 50g/L of polyvinyl alcohol: accurately weighing 5g of 1788 low-viscosity polyvinyl alcohol in a beaker, adding a proper amount of ultrapure water, placing the beaker in a constant-temperature water bath kettle at 65 ℃, continuously stirring the beaker by a glass rod to uniformly disperse particles, continuously preserving heat for 2 hours after dissolution, and fixing the volume of the ultrapure water to 100ml and filtering the mixture by using a 0.45 mu m filter membrane;

1.2: preparing TMB mother liquor: accurately weighing TMB powder in a centrifuge tube, adding a proper amount of DMSO, performing vortex oscillation to fully dissolve the TMB powder, and keeping out of the sun for later use;

1.3: other reagents are prepared according to the concentration requirements of the formula respectively;

step two: preparing a TMB system:

2.1: firstly, adding a proper amount of ultrapure water into a beaker, adding 1788 low-viscosity polyvinyl alcohol mother liquor, then uniformly stirring on a magnetic stirrer, sequentially adding xylose, citric acid and disodium ethylene diamine tetraacetate, and uniformly stirring;

2.2: then, adding 30% aqueous hydrogen peroxide and stirring uniformly;

2.3: then, wrapping the beaker by using tin foil paper, adding TMB mother solution and uniformly stirring;

2.4: then adding Proclin300, stirring and mixing uniformly, adjusting the pH value of the system to 3.1-3.6 by using sodium dihydrogen phosphate solution, and fixing the volume to 1L by using ultrapure water;

2.5: finally, the solution was filtered through a 0.22 μm nylon filter to obtain a homogeneous solution, which was stored in a brown plastic bottle and sealed and protected from light at 2-8 ℃.

5. The high-sensitivity single-component TMB color developing solution and the preparation method thereof according to claim 4, wherein each component is prepared by adopting a mother solution preparation method in the TMB color developing solution preparation process, and xylose is excluded, and then the components are mixed in sequence.

6. The high-sensitivity single-component TMB color developing solution and the preparation method thereof according to claim 1, wherein beakers, glass rods and ultrapure water used in the preparation process of the TMB color developing solution are subjected to high-temperature high-pressure sterilization treatment.

7. The high-sensitivity single-component TMB color developing solution and the preparation method thereof according to claim 1, wherein the former component prepared by the TMB system is mixed uniformly and then the latter component is added.

8. The high-sensitivity single-component TMB color developing solution and the preparation method thereof according to claim 1, wherein the nylon filter membrane is organic solvent-tolerant.

Technical Field

The invention relates to the technical field of TMB color developing solutions, in particular to a high-sensitivity single-component TMB color developing solution and a preparation method thereof.

Background

In enzyme-linked immunosorbent assay (ELISA), Horse Radish Peroxidase (HRP) catalyzes the decomposition of peroxide to release oxygen, so that a color developing solution substrate is oxidized, and the solution turns blue. The reaction is stopped after adding strong acid, the blue solution turns yellow, the maximum absorption peak is at 450nm, and the content of the detected object can be quantitatively calculated by measuring the absorbance value and combining the color development condition of a standard curve.

The commonly used color developing liquid substrates comprise o-phenylenediamine (OPD), 2,2' -dinitro-bis-3-ethylbenzthiazoline-6-sulfonic Acid (ABTS) and 3,3',5,5' -tetramethyl diphenyldiamine (TMB), and research shows that the OPD has potential carcinogenicity, the ABTS has poor color developing sensitivity, and the TMB is used as a novel safe chromogen reagent and becomes the mainstream color developing agent for ELISA detection by the characteristics of high color developing efficiency, no toxicity, no carcinogenesis, good stability and the like.

Because TMB is easily oxidized by oxygen in the air environment and peroxide in the solution, the production of the TMB color developing solution is limited to a certain extent; the TMB color developing solution adopted by the EILSA kit sold in the market at present is mostly bi-component, which is divided into solution A and solution B, so that the contact of the TMB and peroxide is reduced to the maximum extent, and the stability of the color developing solution is ensured. The commercially available ELISA kit also adopts a single-component TMB color developing solution, but the existing single-component TMB color developing solution has the problems of short storage time, high color developing background and the like; when a sample with low target protein expression abundance is detected, the high color development background directly causes the sample to be difficult to detect, and partial omission condition is caused.

In summary, a high-sensitivity single-component TMB color developing solution and a preparation method thereof are needed to solve the disadvantages of the prior art.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a high-sensitivity single-component TMB color developing solution and a preparation method thereof, aiming at solving the problems of short storage time, high color developing background, difficult detection of samples, partial omission and the like of the TMB color developing solution.

In order to achieve the purpose, the invention provides the following technical scheme: a high-sensitivity single-component TMB color developing solution comprises the following components: 0.001-20 g/L of polyvinyl alcohol, 0.16-30 g/L of xylose, 1-100 mM of citric acid, 1-20 mM of ethylene diamine tetraacetic acid, 1-10 mM of 30% aqueous hydrogen peroxide, 1-3 mM of TMB, 20-30 ml/L of DMSO, 3000.04% of proclin, 1-25 mM of disodium hydrogen phosphate and the volume of ultrapure water is up to 1L.

Further, the single-component TMB color developing solution adopts citric acid and disodium hydrogen phosphate buffer solution, and the pH value of the buffer solution is 3.1-3.6.

Further, the polyvinyl alcohol is 1788 low-viscosity polyvinyl alcohol, the polymerization degree of the polyvinyl alcohol is 1700, and the alcoholysis degree of the polyvinyl alcohol is 88%.

A preparation method of high-sensitivity single-component TMB color developing solution comprises the following steps:

the method comprises the following steps: preparing a reagent mother solution:

1.1: 50g/L of polyvinyl alcohol: accurately weighing 5g of 1788 low-viscosity polyvinyl alcohol in a beaker, adding a proper amount of ultrapure water, placing the beaker in a constant-temperature water bath kettle at 65 ℃, continuously stirring the beaker by a glass rod to uniformly disperse particles, continuously preserving heat for 2 hours after dissolution, and fixing the volume of the ultrapure water to 100ml and filtering the mixture by using a 0.45 mu m filter membrane;

1.2: preparing TMB mother liquor: accurately weighing TMB powder in a centrifuge tube, adding a proper amount of DMSO, performing vortex oscillation to fully dissolve the TMB powder, and keeping out of the sun for later use;

1.3: other reagents are prepared according to the concentration requirements of the formula respectively;

step two: preparing a TMB system:

2.1: firstly, adding a proper amount of ultrapure water into a beaker, adding 1788 low-viscosity polyvinyl alcohol mother liquor, then uniformly stirring on a magnetic stirrer, sequentially adding xylose, citric acid and disodium ethylene diamine tetraacetate, and uniformly stirring;

2.2: then, adding 30% aqueous hydrogen peroxide and stirring uniformly;

2.3: then, wrapping the beaker by using tin foil paper, adding TMB mother solution and uniformly stirring;

2.4: then adding Proclin300, stirring and mixing uniformly, adjusting the pH value of the system to 3.1-3.6 by using sodium dihydrogen phosphate solution, and fixing the volume to 1L by using ultrapure water;

2.5: finally, the solution was filtered through a 0.22 μm nylon filter to obtain a homogeneous solution, which was stored in a brown plastic bottle and sealed and protected from light at 2-8 ℃.

Furthermore, each component is prepared in the TMB color developing solution preparation process in a mother solution preparation mode, and except xylose, the components are mixed in sequence.

Further, the beaker, the glass rod and the ultrapure water used in the preparation process of the TMB color developing solution are subjected to high-temperature high-pressure sterilization treatment.

Furthermore, the former component prepared by the TMB system is mixed evenly and then the latter component is added.

Further, the nylon filter membrane is organic solvent resistant.

The invention has the beneficial effects that:

1. in the invention, the single-component TMB color developing solution has the characteristic of low detection background, and the detection rate of low-concentration samples is effectively improved; the 1788 low-viscosity polyvinyl alcohol is adopted, the polymerization degree is 1700, the alcoholysis degree is 88 percent, the low-viscosity polyvinyl alcohol is non-toxic to human bodies, has good biocompatibility, can be used as a stabilizer, a dispersant and the like, has good solubility and lower cost, and can better meet the preparation requirement by using the 1788 low-viscosity polyvinyl alcohol;

2. in the invention, the TMB color developing liquid is single-component liquid, and the traditional liquid A and liquid B do not need to be mixed, thereby providing convenience for the operation of a user and leading the invention to have outstanding progress on the whole.

Drawings

FIG. 1 is a graph plotting the color-developing activity as a function of storage time according to the data in Table 2.

Detailed Description

Example 1:

a high-sensitivity single-component TMB color developing solution comprises the following components: polyvinyl alcohol 0.2g/L, xylose 2g/L, citric acid 10mM, ethylene diamine tetraacetic acid 2mM, 30% aqueous hydrogen peroxide 4mM, TMB 2.5mM, DMSO20ml/L, Proclin 3000.04%, disodium hydrogen phosphate 2.5mM, and ultrapure water with constant volume of 1L.

Further, citric acid and disodium hydrogen phosphate buffer solution are adopted as the single-component TMB color developing solution, and the pH value of the buffer solution is 3.3.

Further, the polyvinyl alcohol is 1788 low viscosity type polyvinyl alcohol, and the polymerization degree and alcoholysis degree of the polyvinyl alcohol are 1700 and 88 respectively.

A preparation method of high-sensitivity single-component TMB color developing solution comprises the following steps:

the method comprises the following steps: preparing a reagent mother solution:

1.1: 50g/L of polyvinyl alcohol: accurately weighing 5g of 1788 low-viscosity polyvinyl alcohol in a beaker, adding a proper amount of ultrapure water, placing the beaker in a constant-temperature water bath kettle at 65 ℃, continuously stirring the beaker by a glass rod to uniformly disperse particles, continuously preserving heat for 2 hours after dissolution, and fixing the volume of the ultrapure water to 100ml and filtering the mixture by using a 0.45 mu m filter membrane;

1.2: preparing TMB mother liquor: accurately weighing TMB powder in a centrifuge tube, adding a proper amount of DMSO, performing vortex oscillation to fully dissolve the TMB powder, and keeping out of the sun for later use;

1.3: other reagents are prepared according to the concentration requirements of the formula respectively;

step two: preparing a TMB system:

2.1: firstly, adding a proper amount of ultrapure water into a beaker, adding 1788 low-viscosity polyvinyl alcohol mother liquor, then uniformly stirring on a magnetic stirrer, sequentially adding xylose, citric acid and disodium ethylene diamine tetraacetate, and uniformly stirring;

2.2: then, adding 30% aqueous hydrogen peroxide and stirring uniformly;

2.3: then, wrapping the beaker by using tin foil paper, adding TMB mother solution and uniformly stirring;

2.4: then adding Proclin300, stirring and mixing uniformly, adjusting the pH value of the system to 3.3 by using sodium dihydrogen phosphate solution, and fixing the volume of ultrapure water to 1L;

2.5: finally, the solution was filtered through a 0.22 μm nylon filter to obtain a homogeneous solution, which was stored in a brown plastic bottle and sealed and protected from light at 2-8 ℃.

Furthermore, each component is prepared in a mother liquor preparation mode in the TMB color developing solution preparation process, except xylose, and then the components are mixed in sequence.

Further, beakers, glass rods and ultrapure water used in the preparation process of the TMB color developing solution are subjected to high-temperature high-pressure sterilization treatment.

Furthermore, the former component prepared by the TMB system is mixed evenly and then the latter component is added.

Further, the nylon filter membrane is organic solvent resistant.

Example 2:

comparing the single-component TMB color developing solution prepared in the example 1 with a commercially available single-component TMB, and respectively detecting and verifying human serum samples and rat serum samples (without using the TMB color developing solution in the kit) by using a human gamma interferon ELISA kit (catalog number: 70-EK180-96) and a rat vascular endothelial growth factor ELISA kit (catalog number: 70-EK383-96) of Hangzhou Union Biotechnology corporation; the procedure was followed exactly as described in the product, and 100. mu.l/well of commercially available TMB and TMB prepared in example 1 were added during color development; after the color development is finished, adding the stop solution according to 100 mul/hole; the two-wavelength detection is carried out by using a microplate reader, and the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 570nm or 630nm is determined. The OD value after calibration is obtained by subtracting the measured value at 570nm or 630nm from the measured value at 450 nm; the results of human interferon gamma samples are shown in Table 1 below, and the results of rat vascular endothelial growth factor samples are shown in Table 2 below:

table 1: human IFN-. gamma.was assayed and compared using TMB prepared in example 1 with a single component TMB from a known manufacturer.

Table 2: the Rat VEGF was examined and compared in samples using TMB prepared in example 1 with a single component TMB from a known manufacturer.

As can be seen from tables 1 and 2, the detection rate of the Human IFN-gamma sample is improved from 37.5% to 50% in the single-component TMB developing solution prepared in example 1 compared with the commercial single-component TMB; the detection rate of the Rat VEGF sample is improved to 75% from 56.3%, which shows that the TMB of the formula can detect more low-concentration samples and has higher detection sensitivity.

Example 3:

the single-component TMB chromogenic substrate described in example 1 was subjected to a color development activity and background real-time test at 4 ℃ for 0 month, 3 months, 6 months, 12 months, 18 months and 24 months, respectively; the specific implementation method comprises the following steps:

diluting with streptavidin-labeled horse radish peroxidase (SA-HRP) (5 μ g/ml) 800 times as the highest concentration R1, and diluting with ultrapure water in a manner of four-fold dilution to obtain two subsequent concentration gradients, R2 and R3; adding 50 mul/well into 96-well enzyme label plate, using ultrapure water as negative control, adding 50 mul TMB color development solution into each well, incubating for 5-10 min under oscillation, and adding 100 mul 0.2M sulfuric acid for termination; performing dual-wavelength detection with microplate reader, measuring OD values at 450nm maximum absorption wavelength and 570nm or 630nm reference wavelength, and subtracting 570nm or 630nm measured value from 450nm measured value after calibration.

The chromogenic OD values at the respective time nodes were divided by the OD values at the corresponding concentrations of "0 month" to obtain the percent chromogenic activity, and the activity curves were plotted as the percent activities of R1, R2, and R3 (see FIG. 1).

Table 3: the color development of the SA-HRP at a fixed concentration was determined at a specific time point using TMB formulated in example 1.

	0 month	3 months old	6 months old	9 months old	12 months old	18 months old	24 months
								R1	1.610	1.591	1.596	1.597	1.603	1.575	1.550
R2	0.195	0.190	0.197	0.191	0.186	0.185	0.182
								R3	0.028	0.027	0.028	0.028	0.027	0.029	0.027
Blank	0.006	0.005	0.007	0.007	0.006	0.008	0.007

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.