CN115032389A - Paper-based sensing method for detecting thrombin and thrombin inhibitor - Google Patents
Paper-based sensing method for detecting thrombin and thrombin inhibitor Download PDFInfo
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- CN115032389A CN115032389A CN202210231955.2A CN202210231955A CN115032389A CN 115032389 A CN115032389 A CN 115032389A CN 202210231955 A CN202210231955 A CN 202210231955A CN 115032389 A CN115032389 A CN 115032389A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention belongs to the field of analysis and detection, and relates to a paper-based sensing method for detecting thrombin and a thrombin inhibitor. The method is based on the coagulation process of thrombin and fibrinogen in plasma, and converts the difference of water content released in the gelling process into the difference of flow distance or area on the paper base. The invention also provides a simple method for screening thrombin inhibition drugs. The inhibition drug obstructs the fibrinogen crosslinking process by inhibiting thrombin activity, reduces absorbed moisture, obtains different flowing distances on the paper base, and constructs a sensing device which is simple, portable, free of equipment, high in sensitivity and remarkable in specificity and can be used for instant detection.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to a visual paper-based bedside rapid detection method for thrombin and a drug inhibitor thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Bedside rapid test is a diagnostic method that is easy to operate, can respond quickly, and is low cost. By simplifying the diagnostic procedure, this method can be carried out at the bedside, in the ward or elsewhere than in the central laboratory. Paper-based lateral flow detection is the most widely studied biosensing method of the various point-of-care detection methods, and has been used for the detection of many biomolecules, including DNA, proteins, and viruses. The specific detection is generally realized by colloidal gold labeling or signal change of enzyme-catalyzed colorimetric reaction. The actual blood sample contains red blood cells, jaundice, hemolysis and the like, which can generate color interference on the experimental result, and generally needs to separate plasma as the sample for testing. However, the separation of plasma is time consuming and requires specialized equipment and procedures that do not meet the needs of real-time testing. The paper base based on viscosity change is a potential solution, and meets the requirements of portability, rapidness, simplicity and no professional operation.
Thrombin is a serine protease involved in the coagulation reaction and can catalyze the conversion of soluble fibrinogen into insoluble fibrin, activating platelets. Abnormal thrombin levels reflect coagulation dysfunction, leading to a variety of diseases such as alzheimer's disease, atherosclerosis, and the like. The research of thrombin inhibitors is urgently needed for the discovery of thrombolytic drugs and the clinical treatment of blood coagulation diseases. Therefore, the quantitative determination of thrombin and the screening of inhibitors are of great significance for disease diagnosis and drug evaluation.
Heretofore, as a method for detecting thrombin, there have been an electrochemical method, a fluorescence method, a colorimetric method and the like. These methods have good sensitivity and stability compared to conventional immunoassay methods. However, most of these methods are time-consuming and laborious, and have limitations in practical applications in combination with labeling of molecules, doping of aptamers, use of complicated instruments, and complicated procedures. The prothrombin time assay is commonly used for the diagnosis of congenital coagulation diseases of the extrinsic coagulation pathway, which requires more stringent experimental conditions and personnel training for determining thrombin activity by clotting time. The LC-MS technology has higher specificity and accuracy, is always used for screening novel thrombin inhibitors, but needs large-scale instruments and specialized operation. Therefore, it is important to develop label-free, simple, portable thrombin detection and inhibitor screening methods.
Disclosure of Invention
Aiming at the defects of complicated pretreatment steps, high requirement on the professional performance of personnel, long treatment time, low sensitivity of a detected object and the like in the thrombin detection process in the prior art. The invention provides a paper-based sensing method for detecting thrombin and an inhibitor thereof.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, a paper-based sensing method for detecting thrombin is provided, which comprises the following steps:
respectively forming a mixed solution by a plurality of groups of thrombin with known activity and plasma, and incubating to obtain a solution to be detected;
contacting the test strip with a solution to be tested, and standing until the aqueous solution is not diffused any more;
establishing a relation between the imprinting distance or area of the aqueous solution and the activity of the thrombin to be detected;
repeating the steps, testing the thrombin to be tested, and determining the activity of the thrombin to be tested.
The inventor finds that: the research application of paper-based sensing based on the viscosity change principle for detecting thrombin is not developed yet. Through the combination relationship between the fibrinogen and the thrombin, the invention establishes a method which is simple, portable, does not need equipment, has higher sensitivity and obvious specificity and can be used for detecting the thrombin and the inhibition drugs thereof in real time.
In a second aspect of the present invention, there is provided a paper-based sensing method for detecting thrombin inhibition drug, comprising:
dissolving an object to be detected containing an inhibitory drug by using an organic solvent, and diluting the object to be detected to a corresponding concentration by using a PBS (phosphate buffer solution) to obtain an inhibitory drug solution;
mixing the plasma solution with the thrombin solution, adding the inhibition drug solution, and incubating to obtain a solution to be tested;
contacting the test strip with a solution to be tested, and standing until the aqueous solution is not diffused any more;
establishing a relation between the moving area or distance of the aqueous solution in the whole test strip and the concentration of the inhibiting drug;
repeating the steps, testing the concentration of the inhibiting drug in the object to be tested, and determining the concentration of the inhibiting drug.
The principle of the invention lies in the blood coagulation insoluble fibrin formed by the fibrinogen in the blood plasma under the action of thrombin during the blood coagulation process. The cross-linking process can capture water molecules, which are fixed in the fibrin gel and can not flow with the test paper. When thrombin is inactivated by thrombin inhibition drugs, water is released and flows along the test paper to form a blot, and the thrombin inhibition drugs are quantified through the distance or area of the blot.
The invention has the beneficial effects that:
(1) the invention relates to a simple detection principle, and thrombin enables fibrinogen to be gelatinized and release an aqueous solution. The paper base has low cost, simple and convenient operation, quick process and easy preservation.
(2) The detection device is simple and easy, and detection can be completed only by a smart phone or a measurement scale which captures data. Compared with the traditional method, the method avoids the complexity of a large instrument and the professional training of detection personnel, can realize the function of instant detection, and has potential value of application and popularization.
(3) The invention provides a high-throughput detection method for detecting thrombin and inhibiting drugs, which has the advantages of capability of avoiding non-specificity, less interference of background signal factors and the like.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a schematic diagram of a paper-based sensor for detecting thrombin and its inhibitors;
FIG. 2(A) is a photograph of a paper substrate showing the data of detection of thrombin and (B) a linear relationship (C) indicating the optimized incubation time and the data (D) visually indicates 60min as the optimized time;
FIG. 3 is a standard curve for thrombin activity;
fig. 4(a) and (B) are argatroban assay data.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In a first aspect of the present invention, there is provided a method for detecting thrombin, comprising the steps of: the test substance is mixed with blood plasma to form a mixed solution. And after incubation under a proper condition, putting the test strip into the reaction hole, and waiting for the aqueous solution to diffuse to form different diffusion areas on the test strip. Different amounts of thrombin cause different degrees of fibrin gelling, and thus different amounts of released water, resulting in different migration effects on the pH strip. The content of the thrombin is measured by combining portable equipment such as a smart phone.
In a second aspect of the present invention, there is provided a method for detecting a thrombin inhibitor, comprising mixing a sample with thrombin and fibrinogen to form a solution. After incubation under appropriate conditions, the strip is placed in the reaction well and the aqueous solution is allowed to diffuse. The target molecule inhibits the enzymatic activity of thrombin, reducing fibrin gel formation to varying degrees. The mixed solution also generates different moving effects on the pH test strip, and can be combined with portable equipment such as a smart phone to measure the content of the thrombin.
The principle of the invention is that the fibrin cross-linking process formed by fibrinogen under the action of thrombin in the coagulation process can capture water molecules. Because the water molecules are immobilized in the fibrin gel and cannot flow with the test paper. When thrombin is inactivated by thrombin inhibition drugs, water is released and flows along the test paper to form a blot, and the thrombin and the inhibition drugs thereof are quantified through the distance or area of the blot.
FIG. 1 is a schematic diagram of a paper-based sensor for detecting thrombin and thrombin inhibitors
In a first aspect of the present invention, a method for detecting thrombin is provided, which comprises the following steps:
a. and (3) test strip treatment, namely, the indicating test strip used in the experiment is a pH test strip, and the test strip is cut into 5.75mm multiplied by 60 mm.
b. Detection of thrombin:
s1: lyophilized rabbit plasma was dissolved in PBS buffer.
S2: 10. mu.L of fibrinogen solution of S1 and 20. mu.L of analyte solution were mixed to obtain a detection solution with a total volume of 30. mu.L.
S3: and (3) incubating the solution at 37 ℃, putting the test strip in the a. into the reaction hole, and standing for 3min until the water is completely diffused.
c. Data processing:
s1, placing the test strip on a hydrophobic substrate for observation.
S2: and observing the moving distance of the test strip added with the enzyme to be detected, and recording by using photographing tools such as a smart phone, so that the target object is detected.
S2: tools such as Photoshop and Origin can be used for counting the proportion of the moving area or distance in the whole test strip for quantitative calculation.
It should be noted that, before the object to be tested is tested, a standard working curve needs to be made first, and then the signal of the actual sample is brought into the working curve.
In the present invention, the thrombin substrate is selected from fibrinogen-containing plasma solutions, including but not limited to lyophilized rabbit plasma, human whole blood, and isolated plasma.
According to the invention, the pH range of the PBS buffer solution is preferably 7.2-7.4.
Preferably, according to the invention, the incubation time for the S3 reaction is 60 min.
In the invention, the hydrophobic substrate material comprises a polycarbonate plate, a polystyrene plate, a polymethyl methacrylate plate, a PVC plate and the like.
Preferably, according to the present invention, the hydrophobic substrate is a PVC plate.
In the invention, in the experimental process, the test paper is placed on a hydrophobic substrate for reaction or is placed in a pore plate for reaction, and then is placed on the hydrophobic substrate for measurement.
In the invention, the quantitative basis of the data analysis process can select the measurement area, the distance, the pixel points and the like.
According to the optimization of the invention, the quantitative basis of the method is to measure the difference value between the front and the back of the pixel point, namely: the total pixel value of the water flow marks was measured.
FIG. 2(A) is a photograph of a paper substrate showing the data of detection of thrombin and (B) a linear relationship (C) indicating the optimized incubation time and the data (D) visually indicates 60min as the optimized time.
The second aspect of the invention provides a detection method for detecting thrombin inhibition drugs, and the technical scheme is as follows:
(1) and (3) test strip treatment, namely, the indicating test strip used in the experiment is a pH test strip, and the test strip is cut into 5.75mm multiplied by 60 mm.
(2) Detection of inhibitory drugs:
s1: lyophilized rabbit plasma was dissolved in PBS buffer.
S2: the test substance containing the inhibiting drug is dissolved by a small amount of organic solvent and then diluted to the corresponding concentration by PBS solution.
S3: s1 mu.L of the above-mentioned 10. mu.L plasma solution was mixed with 20. mu.L thrombin solution to obtain a total volume of 30. mu.L of a test solution. The inhibitory drug solution in S2 was added.
S4: the solution was incubated at 37 ℃ and the strips were placed in the wells and allowed to stand for 3min until the water was completely in solution.
(3) Data processing:
s1, placing the test strip on a hydrophobic substrate for observation.
S2: the moving distance of the added test strip is observed, and the moving distance is recorded by using photographing tools such as a smart phone, so that the target object is detected.
S3: and (4) counting the proportion of the moving area or distance in the whole test strip by using tools such as Photoshop and Origin to perform quantitative calculation.
In the present invention, the thrombin substrate is selected from fibrinogen-containing plasma solutions, including but not limited to lyophilized rabbit plasma, human whole blood, and isolated plasma.
According to the invention, the pH range of the PBS buffer solution is preferably 7.2-7.4.
In the present invention, an organic solution is used in S2, and is selected depending on the drug to be inhibited, for example, a solvent such as dimethyl sulfoxide.
Preferably, according to the invention, the incubation time of the S3 reaction is 60 min.
In the present invention, the hydrophobic substrate material includes a polycarbonate plate, a polystyrene plate, a polymethyl methacrylate plate, a PVC plate, and the like.
Preferably, according to the present invention, the hydrophobic substrate is a PVC plate.
In the invention, in the experimental process, the test paper is placed on a hydrophobic substrate for reaction or is placed on the hydrophobic substrate for measurement after reaction in a pore plate.
In the invention, the quantitative basis of the data analysis process can select the measurement area, the distance, the pixel points and the like.
According to the optimization of the invention, the quantitative basis of the method is to measure the difference value between the front and the back of the pixel point.
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.
In the following examples, all the products used are common commercial products unless otherwise specified.
Examples the invention is further illustrated by the use of argatroban as an example.
In the following examples, the thrombin ELISA kit was supplied by Nanjing Biotechnology, Inc., and OD values were obtained from Tecan Spark Multimode Microplate Reader. Phosphate buffered saline (PBS 10mM, pH 7.4) was purchased from bio biotechnology. Ultrapure water was used for all experiments and the resistivity was 18.25 M.OMEGA./cm.
Determination of thrombin Activity Using ELISA kits
The thrombin activity is accurately determined by adopting a thrombin-linked immunosorbent assay kit. Calibration curves were obtained by varying thrombin concentrations (0, 25, 50, 100, 200 mU/mL). The absorbance intensity of the final TMB developed solution was measured by a microplate reader at a wavelength of 450 nm. The resulting standard curve is shown in FIG. 3. Thrombin activity was measured to be 321U/mg according to the standard curve.
Example 1: the embodiment provides a method for screening an inhibitor argatroban, which realizes high-throughput screening of argatroban and has the molecular formula:
A. and (3) test strip treatment, wherein the indicating test strip used in the experiment is a pH test strip, and the test strip is cut into 5.75mm multiplied by 60 mm.
B. Detection of inhibitory drugs:
s1: lyophilized rabbit plasma was dissolved in PBS buffer.
S2: dissolving the substance to be detected containing argatroban in a dimethyl sulfoxide solvent, and diluting the substance to be detected to a corresponding concentration by using a PBS solution.
S3: s1 mu.L of the above plasma solution was mixed with 20 mu.L of thrombin solution to obtain a total volume of 30uL of test solution, and the argatroban solution obtained in S2 was added.
S4: the solution was also incubated at 37 ℃ and the strips were placed in the wells and allowed to stand for 3min until the water was completely diffused.
(3) Data processing:
s1: and observing the moving distance of the test strip, and recording by using photographing tools such as a smart phone, so that the target object is detected.
S2: and (4) counting the proportion of the moving area or distance in the whole test strip by using tools such as Photoshop and Origin to perform quantitative calculation.
FIG. 4(A) (B) shows the data of argatroban assay.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A paper-based sensing detection method for detecting thrombin is characterized by comprising the following steps:
respectively forming a mixed solution of multiple groups of thrombin with known activity and plasma, and incubating to obtain a solution to be detected;
contacting the test strip with a solution to be tested, and standing until the aqueous solution is not diffused any more;
establishing a relation between the imprinting distance or area of the aqueous solution and the activity of the thrombin to be detected;
repeating the steps, testing the thrombin to be tested, and determining the activity of the thrombin to be tested.
2. The paper-based sensing detection method for detecting thrombin according to claim 1, wherein the plasma is a plasma solution containing fibrinogen, preferably the plasma is freeze-dried rabbit plasma, human whole blood and separated plasma.
3. The paper-based sensing detection method for detecting thrombin according to claim 1, wherein the plasma is dissolved in PBS buffer, and preferably the pH range of the PBS buffer is 7.2-7.4.
4. The paper-based sensing detection method for detecting thrombin according to claim 1, wherein incubation is performed at 37-37.5 ℃ for 60-80 min.
5. The paper-based sensing detection method for thrombin according to claim 1, wherein the test strip is placed on a hydrophobic substrate for observation, preferably the hydrophobic substrate is a polycarbonate plate, a polystyrene plate, a polymethyl methacrylate plate or a PVC plate.
6. A paper-based sensing detection method for detecting thrombin inhibition drugs is characterized by comprising the following steps:
dissolving an object to be detected containing an inhibitory drug by using an organic solvent, and diluting the object to be detected to a corresponding concentration by using a PBS (phosphate buffer solution) to obtain an inhibitory drug solution;
mixing the plasma solution with the thrombin solution, adding an inhibition drug solution, and incubating to obtain a solution to be detected;
contacting the test strip with a solution to be tested, and standing until the aqueous solution is not diffused any more;
establishing a relation between the moving area or distance of the aqueous solution in the whole test strip and the concentration of the inhibiting drug;
repeating the steps, testing the concentration of the inhibiting medicament in the object to be tested, and determining the concentration of the inhibiting medicament.
7. The paper-based sensory test method for testing thrombin inhibitory drugs according to claim 6, wherein the organic solvent is dimethyl sulfoxide.
8. The paper-based sensing assay for detecting thrombin inhibitors as defined in claim 6 wherein said assay is performed by placing a strip on a hydrophobic substrate or reacting in a well plate and then measuring on a hydrophobic substrate.
9. The paper-based sensory test method for testing thrombin inhibitory drugs according to claim 6, wherein the quantitative basis of the data analysis process is area, distance or pixel point.
10. The paper-based sensing assay for the detection of thrombin inhibitory drugs according to claim 9, wherein the quantitative basis is the difference between the front and back of the measurement pixel.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115792231A (en) * | 2022-11-04 | 2023-03-14 | 山东大学 | DNase I biosensor based on enzyme cascade reaction regulated by thrombin aptamer |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115792231A (en) * | 2022-11-04 | 2023-03-14 | 山东大学 | DNase I biosensor based on enzyme cascade reaction regulated by thrombin aptamer |
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