CN107190050B - HRP activity determination and H2O2Concentration detection kit and application thereof - Google Patents
HRP activity determination and H2O2Concentration detection kit and application thereof Download PDFInfo
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Abstract
The invention relates to a biochemical detection kit, which comprises a reagent A: SDS/[ C2mim][BF4]Compounding reaction liquid; and (3) reagent B: solid powder of chromogenic substrate 3,3', 5' -Tetramethylbenzidine (TMB); and (3) reagent C: h2O2And (3) solution. And may further include D: HRP enzyme solution; and (3) reagent E: buffer solution PBS, and reagent F: substrate solvent dimethyl sulfoxide (DMSO-d)699.9%). The kit is based on SDS/[ C2mim][BF4]The compound solution can obviously improve HRP-H2O2Principle of color development efficiency of TMB reaction, measurement of HRP enzyme Activity and H Using the synergistic Effect2O2And (5) detecting the concentration. The kit has the advantages of rapid color development, sensitivity and stability, the reagent for detection is biologically nontoxic, the operation is convenient and rapid, the cost is low, and the kit has the advantages of large-scale HRP activity and H2O2The commercial potential of concentration detection.
Description
Technical Field
The invention relates to a biochemical detection method, in particular to a method for detecting HRP activity and H2O2A kit for detecting concentration.
Background
Horseradish peroxidase (HRP) is one of the most widely used enzyme preparations available at an early stage. Not only closely related to the metabolic processes of life, but also in the fields of clinical medicine, organic synthesis, biosensors, waste water treatment, environmental chemistry and food industryThe method has wide application. Since HRP can catalyze H2O2The method has the advantages of simple operation, low cost and little pollution, and can be distinguished by naked eyes. In addition, in recent years, the detection limit is continuously researched and refreshed, so that the sensitivity of the method is greatly improved, and the method is widely applied to HRP activity determination, H2O2Concentration determination, enzyme-linked immunosorbent assay (ELISA), biosensor and other fields.
The conventional HRP activity determination method comprises 3 methods: the o-methoxyphenol method, the Wooxinton (Worthington) method, the ABTS method. However, these methods have the disadvantages of high toxicity, slow color development rate, biological teratogenesis, environmental pollution and the like.
H2O2Is an intermediate product of glucose and cholesterol metabolism in the organism, and therefore, H in body fluid is measured2O2The concentration of (A) is of great significance for biochemical detection and clinical diagnosis. HRP catalyzes H2O2Oxidizing multiple chromogen substrates, according to which the enzyme catalysis method can be used to determine H2O2The method has the advantages of sensitivity, high efficiency, good selectivity, mild reaction conditions and the like. The results of comparison of the color sensitivity of 8 different substrates of HRP by Weringpu et al show that: h2O2The highest sensitivity of the TMB system. In addition, 3,3',5,5' -Tetramethylbenzidine (TMB) has a smaller K as a substrate with the most hydrophobic HRP than other substratesMThe chromogenic substrate has the best affinity with HRP, low detection limit, no toxicity and no biological teratogenicity, and is an ideal chromogenic substrate for HRP. However, the application of the compound is limited by the defects of poor water solubility, unstable and easily decomposed product and poor color development durability.
The surfactant can self-assemble in aqueous solution and organic solvent to form nano-sized aggregates such as micelles and reverse micelles by the characteristics of hydrophilic one end and hydrophobic one end, is used as a medium for enzyme catalytic reaction, has the synergistic effects of phase transfer, solubilization, stability augmentation and sensitivity enhancement, and can maintain or improve the activity and stability of enzyme, and even enable the enzyme to have super activity in some cases. The ionic liquid is a kind of new green chemicals, and is salt which is composed of anion and cation and is in liquid state near normal temperature. The method has the advantages of environmental protection, biocompatibility, molecular structure adjustability and the like, and the unique physicochemical property of the method enables the method to be widely applied to various fields, and particularly has wide application prospect as a green reaction medium in the biotransformation industry.
The surfactant and the ionic liquid are compounded, so that the enzyme catalysis efficiency can be further improved on the basis of an independent system, the micelle in the compounded system has a large specific surface area, and the compounded solution has the advantages of flexible composition, thermodynamic stability, better simulation of cell environment and the like, so that the surfactant/ionic liquid compounded system is used for the research of enzyme catalysis reaction, and the regulation and control means of people on the enzyme catalysis reaction are greatly enriched.
Kireyko et al reported work on HRP catalyzed oxidation of TMB in solutions of Sodium Dodecyl Sulfate (SDS) at various concentrations and found that only when the SDS concentration was 1.0X 10-5mol/L-1.0×10-3When the mol/L time is longer, a stable blue intermediate product can be obtained; the blue product is rapidly decomposed due to too low concentration, which is the same as that of the product without SDS; HRP inactivation is caused when the concentration of SDS is too high.
The research work of ionic liquid/enzyme homogeneous phase system is first developed by the professor issue group of Huangxinrong university in Shandong, the ionic liquid is introduced into an organic phase, and the formed microemulsion is beneficial to the expression of enzyme activity.
As mentioned above, the surfactant with appropriate concentration and the special ionic liquid reverse micelle system can improve the enzyme catalysis efficiency, but the work of using the compound solution of the surfactant and the ionic liquid for improving the enzyme catalysis reaction efficiency is only reported at present.
The inventor takes SDS aqueous solution with proper concentration as a reaction medium and introduces ionic liquid [ C2mim][BF4]The HRP-H can be further improved on the basis of the original compound solution2O2The color development efficiency of the-TMB reaction, wherein the SDS micelle can play a role in solubilizing a substrate, enhancing catalysis and stabilizing a product, and overcomes the defects of poor water solubility, unstable product and poor color development durability in a buffer solution, thereby completing the invention.
Disclosure of Invention
The invention aims to develop a method for measuring HRP activity and H2O2A kit for detecting concentration.
The purpose of the invention is realized as follows:
provided is a biochemical detection kit comprising a reagent A: SDS/[ C2mim][BF4]Compounding reaction liquid; and (3) reagent B: solid powder of chromogenic substrate 3,3', 5' -tetramethylbenzidine TMB; and (3) reagent C: h2O2And (3) solution.
The kit of the invention may further comprise a reagent D: HRP enzyme solution.
The kit may further comprise a reagent E: buffer solution PBS, and/or reagent F: substrate solvent dimethyl sulfoxide (DMSO-d)6，99.9％)。
The use principle of the kit is as follows: SDS/[ C2mim][BF4]The compound solution can obviously improve HRP-H2O2TMB color reaction efficiency and long-term maintenance of color development effect. Measuring the absorbance of the blue product at 652nm by using a UV-Vis spectrophotometer, and calculating to obtain HRP activity and H2O2And (4) concentration.
The reagent A contained in the kit comprises the following components:
SDS：2.5mmol/L
[C2mim][BF4]：2.5mmol/L
PBS(pH＝7.4)：1mmol/L
the reagent C contained in the kit can be as follows:
30wt％H2O2：8.8mol/L
the reagent D contained in the kit comprises the following components:
HRP：0.01g/L
PBS(pH＝7.4)：1mmol/L
the composition of the PBS described above was:
NaH2PO4：0.19mmol/L
Na2HPO4：0.81mmol/L
ultrapure water: 18.2 M.OMEGA.cm.
A preferred embodiment of the present invention provides a method for HRP activity determination and H2O2The kit for detecting the concentration comprises the following components in percentage by weight:
A.5mmol/LSDS/[C2mim][BF4]reaction solution:
SDS：2.5mmol/L
[C2mim][BF4]：2.5mmol/L
PBS(pH＝7.4)：1mmol/L
b.98wt% 3,3 '5, 5' -Tetramethylbenzidine (TMB) solid powder
C.30wt％H2O2：8.8mol/L
D.HRP：0.01g/L
PBS(pH＝7.4)：1mmol/L
E.1mmol/L Phosphate Buffer Solution (PBS):
NaH2PO4：0.19mmol/L
Na2HPO4：0.81mmol/L
ultrapure water: 18.2 M.OMEGA.cm
F.99％DMSO-d6。
Description of the drawings: due to the poor water solubility of TMB, the kit of this embodiment provides its good solvent DMSO exclusively. Since dissolved TMB is easily oxidized and decomposed, TMB needs to be prepared as it is when the test is carried out using a kit. For HRP activity detection, 5 reagents A, B, C, E and F are used; for H2O2The concentration detection needs to use A, B, C, D, E and F6 reagents.
The method for biochemical detection by using the kit of the invention comprises the following steps:
determination of HRP activity: preparing an enzyme solution to be detected by using E, dissolving B by using F, sequentially adding B, C after dissolving into the reaction solution A, finally adding the enzyme solution to be detected, quickly mixing, recording the change of the absorbance of a product at 652nm along with time by using a UV-Vis spectrophotometer (Shimadzu, UV-2250, Japan), and keeping the reaction and detection temperature at 25 +/-2 ℃. The enzyme activity was calculated from the change in absorbance of the product within 1min from the start of the reaction.
Measurement of H2O2The concentration of (a):
measurement of H2O2Standard curve of (2): dissolving B with F, diluting C with E to different concentrations, sequentially adding dissolved B and diluted C into reaction solution A, adding D, mixing, shaking, and measuring absorbance A of product at 652nm with UV-Vis spectrophotometer (Shimadzu, UV-2250, Japan) after 5min, wherein reaction and detection temperature is constant at 25 + -2 deg.C. Absorbance A and H2O2Concentration mapping to obtain H2O2Fitting a standard curve equation.
Determination of unknown concentration of H2O2: following the standard curve measurement described above, only diluted C was replaced by H of unknown concentration2O2The solution is prepared by substituting the absorbance A measured by the experiment into a standard curve equation, and calculating to obtain the H to be measured2O2The concentration of the solution.
HRP Activity and H were determined using the kit of the present invention2O2The concentration achieves good effect.
Specifically, the invention has the advantages that:
(1) the kit has the advantages of high color development speed, obvious color and luster and high sensitivity;
(2) the color development is lasting after the reaction, the product is stable, and the detection time is widened;
(3) when used for HRP enzyme activity determination, the response to low-concentration HRP is high;
(4) for H2O2The detection limit is low during concentration detection, the linearity is good, and the detection range is wide;
(5) the reaction system is biologically non-toxic and non-teratogenic and can be used for biochemical detection;
(7) the compound system is simple to prepare, the raw materials are cheap and easy to obtain, and the compound system is green and environment-friendly and biologically non-toxic;
(8) the application method is convenient and safe, has low cost, and can be used for large-scale HRP activity and H2O2The commercial potential of concentration detection.
Drawings
FIG. 1 is H in example 32O2Standard curve of concentrationA wire.
Detailed Description
The following examples are used to describe the invention in more detail. These examples are merely illustrative of embodiments of the present invention and do not limit the scope of the invention in any way.
[ example 1 ]
The enzyme activity determination method comprises the following steps:
(1) dissolving a substrate: 0.0127g B was dissolved in 0.5mL of F to prepare a 0.1mol/L TMB solution.
(2) Preparing an enzyme solution: the selected sample can be derived from natural peroxidase of animal and plant tissue cells and commercially purified peroxidase. If the detected object is animal and plant tissue cells, F is added firstly to grind and crush the cells, the cells are centrifuged, and the supernatant is separated to obtain a crude enzyme extracting solution; if the detected object is commercial peroxidase, dissolving the solid enzyme protein by using F to obtain enzyme solution to be detected. And (3) storing the prepared enzyme solution to be detected in a refrigerator at 4 ℃, and preheating in a water bath at 25 ℃ for 15min at constant temperature before detection.
(3) And (3) activity determination: adding 32 mu L of TMB (0.1mol/L) and 1 mu L C (8.8mol/L) into A in sequence to obtain 4mL of mixed reaction solution; finally, adding a small amount of enzyme solution to be detected, quickly mixing, recording the change of absorbance at 652nm along with time by using a UV-Vis spectrophotometer (Shimadzu, UV-2250, Japan) once the reaction starts, acquiring data by using UVProbe 2.33 software, selecting a kinetic module, and setting instrument parameters: scanning cycle 1 s/time, frequency 61, scanning time 60s, activity (change in absorbance per minute) range: 0-60 s. The reaction and detection temperature is constant at 25 +/-2 ℃, parallel experiments are carried out for 3 times, and the activity is averaged.
(4) And (3) activity calculation: the enzyme activity was calculated according to formula (1):
at 25 ℃, pH 7.4, the product produced 1mmol/L of product per minute as 1 enzyme activity unit (U).
(1) In the formula:
ΔA652nmis the increased value of the absorbance at 652nm within 1min of the initial reaction;
39000 molar extinction coefficient at wavelength 652nmλ652nmUnit L mol-1cm-1；
VHRPTaking the volume of enzyme solution in unit of mu L for determination;
[ example 2 ]
The activity of an unknown HRP sample was determined:
in this example, horseradish peroxidase produced by a certain reagent company was selected as a detection target. Dissolving the mixture by using F to prepare a solution to be detected with the concentration of 0.1 g/L. Activity measurement the volume of the enzyme solution added was 2. mu.L. In the same manner as in example 1, 3 sets of Δ A were measured652nmThe enzyme activity was calculated by substituting the formula (1), and the results are shown in Table 1.
TABLE 1 results of enzyme Activity measurement
[ example 3 ]
H2O2Method for measuring concentration of
Measurement of H2O2Standard curve of (2): a was used to formulate C into a panel of different concentrations of H2O2Standard, concentration range: 1 mu mol/L-100 mu mol/L; the TMB solution was prepared as described in example 1. Sequentially adding 32 mu L of TMB (0.1mol/L) and 2 mu L D into 4mL of standard sample, uniformly mixing by oscillation, measuring the absorbance of the product at 652nm after 5min, carrying out parallel measurement for 3 times in each group, and taking an average value. As absorbance (A) -H2O2Concentration (C)H2O2/mmol L-1) As shown in fig. 1, the equation of the standard curve is obtained as follows:
due to the high concentration of H2O2Has inhibitory effect on HRP activity, and the measured object is trace concentration of H2O2The sample can be derived from biological tissue cellsThe metabolism process of medium glucose and cholesterol, or diluted H2O2And (3) sampling.
[ example 4 ]
Determination of an unknown H2O2Concentration of the solution
This example selects a medical disinfectant (H)2O2Less than or equal to 2%) as a detection object. Firstly, diluting a sample to be tested by using A105Doubling; the TMB solutions were prepared as described in examples 1 and 3. Sequentially adding 32 mu L of TMB (0.1mol/L) and 2 mu L D into 4mL of standard sample, uniformly mixing by oscillation, measuring the absorbance of the product at 652nm after 5min, carrying out parallel measurement for 3 times in each group, and taking an average value. The results are shown in Table 2. Substituting A into the standard curve, and calculating to obtain H in the sample to be measured2O2Concentration C0. Calculating unknown H according to equation (3)2O2The concentration of the solution.
CH2O2＝10-3×C0×D(mol/L) (3)
(3) In the formula:
CH2O2to be measured for H2O2In mol L of-1；
C0Determined from the formula (2) in mmol L-1；
D is H2O2Dilution times;
TABLE 2 medical disinfectants H2O2Results of concentration measurement
[ example 5 ]
Determination of an unknown H2O2Concentration of the solution
This example selects laboratory Long-standing H2O2(AR，H2O2Less than or equal to 30 percent) as a detection object. Firstly, diluting a sample to be tested by using A105The fold was calculated and worked up as in example 4 and the results are shown in Table 3.
TABLE 3 laboratory Long-standing H2O2Results of concentration measurement
Claims (10)
1. A biochemical detection kit, characterized by comprising a reagent A: SDS/[ C2mim][BF4]Compounding reaction liquid; and (3) reagent B: solid powder of chromogenic substrate 3,3', 5' -tetramethylbenzidine TMB; and (3) reagent C: h2O2And (3) solution.
2. The kit of claim 1, further comprising a reagent D: HRP enzyme solution.
3. The kit of claim 1, further comprising a reagent E: buffer solution PBS and/or reagent F: substrate solvent 99.9% dimethyl sulfoxide DMSO-d6。
4. The kit of claim 1, wherein the composition of reagent a is:
SDS：2.5mmol/L
[C2mim][BF4]：2.5mmol/L
PBS pH＝7.4：1mmol/L。
5. the kit of claim 1, wherein the amount of reagent C is: 30 wt% H2O2，8.8mol/L。
6. The kit of claim 2, wherein the composition of reagent D is:
HRP：0.01g/L
PBS pH＝7.4：1mmol/L。
7. the kit of claim 3, 4 or 6, wherein the PBS has a composition of:
NaH2PO4：0.19mmol/L
Na2HPO4：0.81mmol/L
ultrapure water: 18.2 M.OMEGA.cm.
8. Use of a kit according to claim 1 for biochemical detection.
9. The use of claim 8, wherein the biochemical detection aspect is an HRP activity assay.
10. The use of claim 8, wherein the biochemical detection aspect is H2O2And (5) detecting the concentration.
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