Horseradish peroxidase inclusion compound as well as preparation method and application thereof
By forming supramolecular inclusions with horseradish peroxidase and cucurbiturium, the problems of enzyme inactivation and low sensitivity in enzyme-linked immunochromatography were solved, the enzyme activity and stability were improved, and the modification process was simplified. , and demonstrated high sensitivity and linear detection effects in tumor marker detection.
Patent Information
- Application Number
- CN202411285490.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-13
- Publication Date
- 2024-12-13
AI Technical Summary
In the prior art, horseradish peroxidase is prone to inactivate in enzyme-linked immunochromatography and has low sensitivity, and the existing improved methods are complex, time-consuming and low efficiency.
By mixing horseradish peroxidase with cucurbita, a supramolecular inclusion is formed, the activity and stability of the enzyme is improved, and its reversibility is made, and the enzyme is recycled.
It significantly improves the enzyme activity and stability of horseradish peroxidase, simplifies the modification process, reduces costs, and achieves high sensitivity and linear detection effects in tumor marker detection.
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Figure CN119120407A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of medical inspection, and specifically relates to a horseradish peroxidase inclusion compound and a preparation method and application thereof. Background Art
[0002] Immunochromatography technology has developed rapidly due to its simplicity, low cost, low sample volume, no need for other expensive analytical instruments or trained operators, and the ability to achieve rapid bedside testing. The labeling material used in immunochromatography plays a decisive role in the performance of the immunosensor.
[0003] Horseradish peroxidase (HRP) is the most widely used marker enzyme in enzyme-linked immunosorbent assay (ELISA), but the natural enzyme HRP is easily inactivated and has low sensitivity. Currently, complex technologies and methods such as genetic engineering or double-layer organic skeleton fixation of HRP are mainly used to improve or fix it in order to enhance its activity, but there are disadvantages such as complex procedures, long time consumption, and low efficiency. Summary of the invention
[0004] Based on the above technical background, the main purpose of the present invention is to provide a horseradish peroxidase inclusion compound and a preparation method and application thereof, so as to overcome the deficiencies in the prior art.
[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:
[0006] The first aspect of the present invention is to provide a horseradish peroxidase inclusion compound, which is prepared from horseradish peroxidase and cucurbituril, and the molar ratio of the horseradish peroxidase to the cucurbituril is 1:(1-5).
[0007] Preferably, the molar ratio of the horseradish peroxidase to the cucurbituril is 1:1.
[0008] The second aspect of the present invention is to provide a method for preparing the horseradish peroxidase inclusion complex described in the first aspect of the present invention, the method comprising the following steps:
[0009] The horseradish peroxidase and cucurbituril are mixed, shaken, and then centrifuged, and then a solvent is added to obtain a horseradish peroxidase inclusion complex solution.
[0010] Preferably, at room temperature, a sodium acetate solution containing horseradish peroxidase (HRP) is added to a cucurbituril (CB) solution, shaken at room temperature for 20 to 45 minutes, then centrifuged at 8000 to 12000 r / min for 5 to 20 minutes, and then sodium acetate solution is added to dissolve to obtain a horseradish peroxidase inclusion complex (HRP-CB) solution.
[0011] More preferably, at room temperature, a sodium acetate solution containing horseradish peroxidase is added to a cucurbituril solution, shaken at room temperature for 30 minutes, then centrifuged at 10,000 r / min for 10 minutes, and then sodium acetate solution is added to dissolve to obtain a horseradish peroxidase inclusion complex solution.
[0012] More preferably, the volume ratio of the sodium acetate solution containing 80-120 μM horseradish peroxidase to the 3-8 μM cucurbituril solution is 2-3:200.
[0013] More preferably, the volume ratio of the sodium acetate solution to the cucurbituril solution is (0.5-2):1.
[0014] The third aspect of the present invention is to provide an application of the horseradish peroxidase inclusion complex described in the first aspect of the present invention in the field of tumor marker detection technology.
[0015] The beneficial effects of the present invention are:
[0016] (1) The present invention adds cucurbituril to horseradish peroxidase, so that the horseradish peroxidase and cucurbituril react with each other to obtain a horseradish peroxidase inclusion complex, which can effectively improve the enzyme activity and stability of the horseradish peroxidase. At the same time, the horseradish peroxidase inclusion complex is reversible, so that the horseradish peroxidase can be recycled, and has the advantages of low cost, high practicality, and green environmental protection.
[0017] (2) The preparation method of the horseradish peroxidase inclusion complex of the present invention is simple, that is, the method for modifying horseradish peroxidase is simple, efficient, short in time, low in cost, and highly practical.
[0018] (3) The experiment found that in the presence of TMB (3,3',5,5'-tetramethylbenzidine) substrate and H2O2, the ultraviolet absorption spectrum signal of the horseradish peroxidase inclusion complex of the present invention at 370nm is negatively correlated with the antigen (tumor marker, carcinoembryonic antigen) concentration and has a linear relationship, which can be used for the detection of tumor markers. The detection method has the advantages of simplicity, convenience and sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 The figure shows the ultraviolet absorption signal intensity test graph of HRP and the HRP-CB inclusion complex prepared in Example 1;
[0020] Figure 2 The test results of the inclusion constant, enthalpy change and entropy change of the HRP-CB inclusion complex prepared in Example 1 are shown;
[0021] Figure 3 A schematic diagram showing the modification process of the detection antibody modified with adamantane;
[0022] Figure 4A schematic diagram showing that the small molecule ADA on the detection antibody Ab2 releases HRP from the CB;
[0023] Figure 5 The graph shows the UV absorption signal intensity test of CEA at different concentrations. DETAILED DESCRIPTION
[0024] The present invention will be described in detail below, and the characteristics and advantages of the present invention will become clearer and more distinct with the description.
[0025] The first aspect of the present invention is to provide a horseradish peroxidase inclusion compound, which is prepared from horseradish peroxidase and cucurbituril, and the molar ratio of the horseradish peroxidase to the cucurbituril is 1:(1-5).
[0026] Preferably, the molar ratio of the horseradish peroxidase to the cucurbituril is 1:1.
[0027] The invention adds cucurbituril (CB) to horseradish peroxidase (HRP) to make the two have supramolecular action, so that the activity and stability of horseradish peroxidase can be significantly improved. In addition, the inclusion compound prepared by horseradish peroxidase and cucurbituril is reversible, so that the horseradish peroxidase can be reused.
[0028] The second aspect of the present invention is to provide a method for preparing the horseradish peroxidase inclusion complex described in the first aspect of the present invention, the method comprising the following steps:
[0029] The horseradish peroxidase and cucurbituril are mixed, shaken, and then centrifuged, and then a solvent is added to obtain a horseradish peroxidase inclusion complex solution.
[0030] Preferably, under room temperature conditions, a sodium acetate solution containing horseradish peroxidase is added to a cucurbituril solution, and the solution is shaken at room temperature for 20 to 45 minutes to allow sufficient mixing and supramolecular action (intermolecular force, non-covalent bond action) to occur, followed by centrifugation at 8000 to 12000 r / min for 5 to 20 minutes using a high-speed centrifuge to remove excess cucurbituril, and then sodium acetate solution is added to dissolve the solution to obtain a horseradish peroxidase (HRP-CB) inclusion complex solution.
[0031] More preferably, at room temperature, a sodium acetate solution containing horseradish peroxidase is added to a cucurbituril solution, shaken at room temperature for 30 min, then centrifuged at 10000 r / min for 10 min using a high-speed centrifuge, and then sodium acetate solution is added to dissolve to obtain a horseradish peroxidase (HRP-CB) inclusion complex solution.
[0032] The volume ratio of the sodium acetate solution containing 80-120 μM horseradish peroxidase to the 3-8 μM cucurbituril solution is 2-3:200.
[0033] Preferably, the volume ratio of the sodium acetate solution containing 100 μM horseradish peroxidase to the 5 μM cucurbituril solution is 2.5:200.
[0034] The volume ratio of the sodium acetate solution to the cucurbituril solution is (0.5-2):1.
[0035] Preferably, the volume ratio of the sodium acetate solution to the cucurbituril solution is 1:1.
[0036] The third aspect of the present invention is to provide an application of the horseradish peroxidase inclusion complex described in the first aspect of the present invention in the field of tumor marker detection technology.
[0037] Example
[0038] The present invention is further described below by specific examples, which are limited to illustrating the present invention but not to limiting the scope of the present invention. The raw materials used in the examples of the present invention were all purchased.
[0039] Example 1
[0040] At room temperature, 2.5 μL of 100 μM HRP (horseradish peroxidase) sodium acetate solution was measured, and then 200 μL of 5 μM CB (cucurbituril) solution was added, and the mixture was shaken at room temperature for 30 min to allow it to be fully mixed and to undergo supramolecular action (intermolecular force, non-covalent bond action). The mixture was then transferred to a 10 kd ultrafiltration tube and centrifuged at 10,000 r / min for 10 min using a high-speed centrifuge to remove excess CB, and 200 μL of sodium acetate solution was added to dissolve the mixture to obtain an HRP-CB inclusion complex solution.
[0041] The HRP-CB inclusion complex solution was diluted with acetic acid-sodium acetate buffer solution at pH 4.76 to obtain a series of HRP-CB solutions with different concentrations. 3,3ˋ,5,5ˋ-tetramethylbenzidine (TMB) was used as the substrate. In the presence of H2O2, the ultraviolet absorption signal was detected by an enzyme marker. The concentration with the strongest HRP enzyme activity was screened out to be 2.5×10 -9 M.
[0042] Example 2
[0043] At room temperature, 2 μL of 80 μM HRP (horseradish peroxidase) sodium acetate solution was measured, and then 200 μL of 3 μM CB (cucurbituril) solution was added, and the mixture was shaken at room temperature for 20 min to allow it to be fully mixed and to undergo supramolecular action (intermolecular force, non-covalent bond action). The mixture was then transferred to a 10 kd ultrafiltration tube and centrifuged at 8000 r / min for 20 min using a high-speed centrifuge to remove excess CB, and 100 μL of sodium acetate solution was added to dissolve the mixture to obtain an HRP-CB inclusion complex solution.
[0044] Example 3
[0045] At room temperature, 3 μL of 120 μM HRP (horseradish peroxidase) sodium acetate solution was measured, and then 200 μL of 8 μM CB (cucurbituril) solution was added, and oscillated at room temperature for 45 min to allow sufficient mixing and supramolecular interaction (intermolecular force, non-covalent bond interaction) to occur. The mixture was then transferred to a 10 kd ultrafiltration tube and centrifuged at 12000 r / min for 5 min using a high-speed centrifuge to remove excess CB, and 400 μL of sodium acetate solution was added to dissolve the mixture to obtain an HRP-CB inclusion complex solution.
[0046] Experimental example
[0047] Experimental Example 1 Enzyme Activity Test
[0048] The enzyme activity of HRP and the HRP-CB inclusion complex prepared in Example 1 was tested respectively. The test process was as follows: 3,3 ˋ,5,5 ˋ-tetramethylbenzidine (TMB) was used as a substrate. In the presence of H2O2, HRP and the HRP-CB inclusion complex prepared in Example 1 were added respectively. The intensity of the ultraviolet absorption signal was detected by an ELISA instrument. The test results are shown in FIG. Figure 1 shown.
[0049] from Figure 1 It can be seen that after adding the HRP-CB inclusion complex, the absorbance value at 285nm decreased significantly (the absorption peak at 285nm is the absorption peak of the TMB substrate), while the absorbance values at 370nm and 650nm increased significantly, indicating that the enzyme activity of the HRP-CB inclusion complex formed by the supramolecular action of HRP and CB is higher than the enzyme activity of HRP, indicating that the addition of CB can significantly enhance the activity of the HRP enzyme.
[0050] Experimental Example 2 Determination of inclusion constant, enthalpy change and entropy change
[0051] At 298.15K, the inclusion constant, enthalpy change and entropy change of the HRP-CB inclusion complex prepared in Example 1 were measured by isothermal calorimetric titration (ITC). The test results are as follows: Figure 2 shown.
[0052] from Figure 2 It can be seen that the binding constant of HRP and CB is (7.52±0.984)×10 5 M -1 The N value shows that HRP and CB form a 1:1 inclusion complex, and △H is about -1.547×10 4 ±555.6cal / mol, △S is -25.0cal / mol / deg, and according to △G=△HT△S, it is calculated that △G<0. From the perspective of thermodynamics and kinetics, it can be seen that the inclusion compound of the present invention can be formed spontaneously and is reversible.
[0053] Experimental Example 3 Establishment of tumor marker detection technology based on CB supramolecular competition system and demonstration of the reversibility of HRP-CB inclusion complex
[0054] (1) Small molecules that strongly interact with CB (cucurbituril) are selected to modify antibodies.
[0055] Preparation of detection antibody (ADA-Ab2) modified with adamantane amine: Since adamantane amine (ADA) and CB have a strong interaction (supramolecular interaction) and the inclusion constant is much larger than HRP-CB (horseradish peroxidase inclusion complex), carboxylated adamantane amine is selected and modified onto detection antibody Ab2 in the presence of EDC / NHS (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysulfosuccinimide) to obtain detection antibody (ADA-Ab2) modified with adamantane amine. The specific steps are as follows: 200 μL of 1 mg / mL detection antibody (Ab2) solution is measured and added to a PBS solution containing a mixture of 10 mg EDC and 10 mg NHS, shaken at 10°C for 4 hours, transferred to a 10Kd ultrafiltration tube, centrifuged at 8000-10000 r / min for 10 minutes, and then dissolved in sodium acetate solution. Add 1 mL of 4.63 mM adamantane solution to the above solution and mix well. Place in a refrigerator and incubate overnight at 4°C. Take out the solution from the refrigerator the next day, centrifuge at 8000-10000 r / min for 10 min, and dissolve in PBS with a pH of 7.4 for later use. Figure 3 As shown in the figure, with the increase of ADA-Ab2 concentration, the absorbance value at 285nm increases, and the absorbance values at 370nm and 650nm decrease. This is because the inclusion complex of ADA and CB is stronger than the inclusion complex of HRP and CB, thus releasing HRP. This experiment verifies that the HRP-CB inclusion complex is reversible.
[0056] (2) The operating steps for coating the capture antibody (Ab1) on the microplate are as follows: the capture antibody Ab1 (coating antibody) is diluted to 1 μg / mL with a sodium carbonate buffer solution at pH 9.6, 100 mL of the solution containing the capture antibody is added to each well of the microplate, and the wells are sealed with a sealing film. The wells are incubated at 4°C overnight (24 h), and then the plate is washed twice with PBST (phosphate buffered saline), 400 μL of washing solution is added to each well, the washing solution is discarded, and the plate is patted dry with absorbent paper. Subsequently, 200 μL of 1%-2% BSA (bovine serum albumin) is added to each well for blocking, and then 0.5%-1% NaN3 preservative is added. The wells are placed in a refrigerator (4°C, 12-16 h), the liquid is removed, and the wells are dried at room temperature.
[0057] (3) The establishment of tumor marker detection technology based on the CB supramolecular competition system, using the antigen-antibody sandwich immune response model system, the antigen such as CEA (tumor marker, carcinoembryonic antigen) and the detection antibody ADA-Ab2 modified by adamantane amine are reacted, and a sandwich structure is formed in the micropores through the specific immune reaction of the antigen and antibody. Then, an appropriate amount of HRP-CB inclusion complex is added. Through the competitive inclusion and action of the small molecule ADA modified on the detection antibody Ab2 and HRP on CB, HRP is released from CB, such as Figure 4 shown.
[0058] The specific experimental steps of the colorimetric method for detecting tumor markers are as follows: a. Formation of sandwich immune structures of CEA (tumor marker) at different concentrations: At room temperature, add a series of PBS solutions of CEA at different concentrations to the microwells of a 96-well microplate coated with a capture antibody (Ab1) (the operation steps for coating the capture antibody are shown in (2)), place in a 37°C incubator for 2.0 hours, take out the 96-well microplate, wash the plate twice with PBST, discard the washing solution, pat dry with absorbent paper, then add 100 μL of 10 mg / mL Ab2-ADA (the preparation process of Ab2-ADA is shown in (1)) to each well, place in a 37°C incubator for 1.0 hour, take out the 96-well microplate, discard the excess liquid, continue to wash the plate twice with PBST, pat dry with absorbent paper, and sandwich immune structures of CEA with different concentrations binding to specific antibodies are formed. b. Determine the UV absorption signal of CEA with different concentrations in the presence of TMB substrate and enzyme. At room temperature, add 100 μL of 2.5×10 -9 Then add 200 μL of TMB and H2O2, place in a 37°C incubator for 15 min, take out and add 100 μL of 0.5 M H2SO4 to terminate the reaction, measure the UV absorption signal, and the test results are as follows: Figure 5 shown.
[0059] When HRP is released from CB, the activity of HRP enzyme decreases. In the presence of TMB (3,3',5,5'-tetramethylbenzidine) substrate and H2O2, the UV absorption spectrum at 370nm decreases. Figure 5 It can be seen that the UV absorption spectrum signal at 370nm is negatively correlated with the antigen concentration (each antigen binds to one ADA-Ab2, so the concentration of ADA-Ab2 is equal to the concentration of antigen CEA) and has a linear relationship. Based on this, a simple, convenient and sensitive method for detecting tumor markers can be established. At the same time, it shows that the HRP-CB inclusion complex is reversible.
[0060] Experimental Example 4 Stability Test of Inclusion Compound
[0061] In order to examine the stability of CB-HRP, HRP and HRP-CB inclusion complex were placed in a -18°C refrigerator for 7, 14, and 30 days, respectively. In the presence of substrates TMB and H2O2, the changes in their UV-vis signals were measured to examine their stability.
[0062] The results showed that after 30 days of storage, the UV absorption signals of the HRP-CB inclusion complex at 370nm and 650nm remained almost unchanged, while the absorbance value of HRP at 258nm increased slightly from 14 to 30 days, approaching the absorbance curve of the TMB substrate itself, indicating that the inclusion complex HRP-CB formed by HRP and CB has high stability, which is higher than the stability of HRP.
[0063] The present invention has been described in detail above in conjunction with specific implementations and exemplary examples, but these descriptions cannot be understood as limiting the present invention. Those skilled in the art understand that, without departing from the spirit and scope of the present invention, a variety of equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its implementation methods, all of which fall within the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims.
Claims
1. A horseradish peroxidase inclusion complex, characterized in that: The horseradish peroxidase inclusion compound is prepared from horseradish peroxidase and cucurbituril, and the molar ratio of the horseradish peroxidase to the cucurbituril is 1:(1-5).
2. The horseradish peroxidase inclusion complex according to claim 1, characterized in that The molar ratio of the horseradish peroxidase to the cucurbituril is 1:
1.
3. A method for preparing the horseradish peroxidase inclusion complex according to claim 1 or 2, characterized in that: The method comprises the following steps: The horseradish peroxidase and cucurbituril are mixed, shaken, and then centrifuged, and then a solvent is added to obtain a horseradish peroxidase inclusion complex solution.
4. The method according to claim 3, characterized in that Under room temperature, a sodium acetate solution containing horseradish peroxidase is added to a cucurbituril solution, shaken at room temperature for 20 to 45 minutes, then centrifuged at 8000 to 12000 r / min for 5 to 20 minutes, and then sodium acetate solution is added to dissolve to obtain a horseradish peroxidase inclusion complex solution.
5. The method according to claim 4, characterized in that At room temperature, a sodium acetate solution containing horseradish peroxidase was added to the cucurbituril solution, shaken at room temperature for 30 minutes, then centrifuged at 10000 r / min for 10 minutes, and then sodium acetate solution was added to dissolve to obtain a horseradish peroxidase inclusion complex solution.
6. The method according to claim 4, characterized in that The volume ratio of the sodium acetate solution containing 80-120 μM horseradish peroxidase to the 3-8 μM cucurbituril solution is 2-3:
200.
7. The method according to claim 4, characterized in that The volume ratio of the sodium acetate solution to the cucurbituril solution is (0.5-2):
1.
8. Use of the horseradish peroxidase inclusion complex according to claim 1 or 2 in the field of tumor marker detection technology.