A fluorescent antibody sealing solution, preparation method and use

Abstract

The present invention discloses a fluorescent antibody sealing solution, a preparation method and uses thereof. The fluorescent antibody sealing solution is mainly composed of 5% - 20% trehalose, 0.1% - 1.0% BSA, 0.1% - 1.0% polyvinylpyrrolidone, 0.1% - 5.0% sorbitol, and the rest is HEPES buffer solution by mass percentage. This sealing solution is suitable for sealing fluorescent microsphere antibodies; when used for sealing fluorescent dye antibodies, it also includes 0.05% - 0.1% sodium hyaluronate. The preparation method includes first preparing 20 mmol / L HEPES buffer solution, adjusting the pH to 7.4 - 7.6 with 1 mol / L sodium hydroxide, and then successively adding the substances in the sealing solution formula and dissolving them fully. The beneficial effects of the present invention are that the sealing solution has a simple formula and is convenient to prepare. After the fluorescent antibody sealed by it undergoes a labeling reaction, it can break free from the bondage and flow with the sample, and can maintain the antibody activity for a long time.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence immunoassay, and particularly relates to a fluorescent antibody sealing solution, a preparation method and uses thereof. Background Art

[0002] The three myocardial markers are three very commonly used indicators in clinical cardiovascular specialties, namely troponin, myoglobin and creatine kinase isoenzyme. These three types of indicators have different specificities and sensitivities for myocardial injury, and are manifested in different stages after myocardial injury. Therefore, it is of great significance for judging the severity and time of myocardial damage, which is conducive to early detection of myocardial injury and timely treatment intervention, and has good guiding significance for the judgment of later treatment and recovery. Myoglobin is the earliest indicator to change after myocardial injury, which helps to detect myocardial damage at an early stage. Troponin has great clinical guiding significance for the diagnosis and treatment of myocardial infarction. Creatine kinase isoenzyme is the most widely used in clinical practice and can be used as a routine examination for heart diseases.

[0003] At present, there are two products using colloidal gold and fluorescence chromatography. Both use the double antibody sandwich method to detect the substance to be tested in the blood sample, and then use the instrument to detect and quantitatively analyze the test object. Existing products basically use NC membrane as a carrier. The carrier is greatly affected by environmental factors. If the temperature and humidity do not meet the requirements, the NC membrane will have a very significant impact on the test results of the sample. Therefore, there are higher environmental control requirements during production and storage. At the same time, the NC membrane has large differences between batches and the amount of fixed antibodies is uneven. Secondly, the myocardial triple detection reagent prepared with NC membrane will cause the fluorescent antibody to be released with the flow of the sample due to the three factors of NC membrane, sample pad, and binding pad, which will also affect the test results, so it will cause the product repeatability CV to be larger. In addition, since the myocardial triple test combines the three myocardial test items of CKMB, CTNI and Myo on the same test reagent card, there are problems such as mutual interference between items, low detection sensitivity, and poor repeatability CV. To this end, the applicant invented a self-driving microfluidic chip and submitted a patent application to the State Intellectual Property Office on January 17, 2020. The application was published on April 28, 2020, and its publication number is CN111068801A. The self-driving microfluidic chip pre-stores the antibodies corresponding to the substance to be tested on the self-driving microfluidic chip, and the sample can form a double antibody sandwich through recognition reaction and immune reaction respectively. With the advantage of the microchannel formed by a single pure material, it can ensure the reliability and controllability of the sample test results through precise microfluidic control, and is expected to replace the immunochromatography (LIFCS) technology of multiple membrane materials commonly used in clinical practice, thereby providing new technical ideas for solving the challenges faced by current POCT detection technology. Afterwards, on May 22, 2020, the applicant submitted a patent application for a rapid immunofluorescence detection method based on a self-driven microfluidic chip to the State Intellectual Property Office. The application was published on August 21, 2020, and its publication number is CN111562380A; the method discloses the whole process of rapid immunofluorescence detection using the self-driven microfluidic chip.

[0004] During the manufacturing process of this self-driven microfluidic chip, an antibody labeled with a fluorescent substance needs to be sealed in the mixing zone, and another antibody is fixed on the micro-columns in the detection zone of the chip. When the sample to be detected flows through the chip, after the recognition reaction with the fluorescent antibody in the mixing zone, the antibody carrying the fluorescent marker will then undergo an immune reaction with the antibody on the micro-columns in the detection zone, thus forming a double-antibody sandwich marker retained on the micro-columns. The fluorescence detector obtains the detection result by detecting the fluorescence label of the double-antibody sandwich marker. Therefore, sealing the antibody labeled with the fluorescent substance in the mixing zone, so that after the recognition reaction, the fluorescent antibody can completely break free from the restraint of the mixing zone and the antibody can maintain its activity within the set time limit is the basis for obtaining accurate detection results. Currently, in the existing technology, there is no literature disclosing relevant technologies that can achieve the foregoing purposes. Summary of the Invention

[0005] The first object of the present invention is to provide a fluorescent antibody storage solution in view of the lack of antibody sealing technology in the preparation process of the existing self-driven microfluidic chip, so as to seal the fluorescent antibody in the mixing zone of the self-driven microfluidic chip. When the sample to be detected flows through the chip, after the recognition reaction with the fluorescent antibody in the mixing zone, the antibody carrying the fluorescent marker completely breaks free from the mixing zone and then undergoes an immune reaction with the antibody on the micro-columns, forming a double-antibody sandwich marker, so that the fluorescence detector obtains the detection result by detecting the fluorescence label of the double-antibody sandwich marker. The second object of the present invention is to provide a preparation method based on the foregoing storage solution to obtain a fluorescent antibody storage solution that meets the foregoing requirements. The third object of the present invention is to provide the use of the foregoing antibody storage solution for a self-driven microfluidic chip.

[0006] To achieve the first object, the present invention adopts the following technical solutions.

[0007] A fluorescent antibody storage solution is mainly prepared from trehalose, BSA, polyvinylpyrrolidone and sorbitol through HEPES buffer solution; wherein, by mass percentage, trehalose: 5% - 20%, BSA: 0.1% - 1.0%, polyvinylpyrrolidone: 0.1% - 1.0%, sorbitol: 0.1% - 5.0%, and the rest is HEPES buffer solution.

[0008] The present invention adopting the foregoing technical solution utilizes the colloidal component in the foregoing composition to encapsulate the fluorescent microsphere antibody in the mixing zone of the self-driven microfluidic chip, and after air-drying, uses the nutrient components to keep the antibody active within a set period. During the flow of the test sample in the chip, after the recognition reaction with the fluorescent antibody in the mixing zone, the antibody carrying the fluorescent microsphere can break free from the restraint of the mixing zone and then automatically flow into the detection zone to react immunologically with the antibody on the microcolumn, forming a double-antibody sandwich marker. Thus, the detection result is obtained by the fluorescence detector through the fluorescence labeling detection of the double-antibody sandwich marker. Among them, the air-drying after the encapsulation of the fluorescent microsphere antibody is natural air-drying under the environmental requirements of a 100,000-class clean area; the environmental temperature is 25 °C, and the humidity is kept constant at 50-60%.

[0009] Preferably, it further includes sodium hyaluronate. By mass percentage, sodium hyaluronate: 0.05% - 0.1%. The encapsulating solution with sodium hyaluronate is used to encapsulate the antibody labeled with a fluorescent dye through a chemical reaction in the mixing zone of the self-driven microfluidic chip. And after air-drying, uses the nutrient components to keep the antibody active within a set period. During the flow of the test sample in the chip, after the recognition reaction with the fluorescent antibody in the mixing zone, the antibody carrying the fluorescent dye can break free from the restraint of the mixing zone and then automatically flow into the detection zone to react immunologically with the antibody on the microcolumn, forming a double-antibody sandwich marker. Thus, the detection result is obtained by the fluorescence detector through the fluorescence labeling detection of the double-antibody sandwich marker. Among them, the air-drying after the encapsulation of the fluorescent dye antibody is natural air-drying under the environmental requirements of a 100,000-class clean area; the environmental temperature is 25 °C, and the humidity is kept constant at 50-60%.

[0010] To obtain a better encapsulation effect, for the encapsulating solution used for the encapsulation of the fluorescent dye antibody, by mass percentage, trehalose: 5% - 10%, BSA: 0.5% - 1.0%, polyvinylpyrrolidone: 0.5% - 1.0%, sorbitol: 0.5% - 1.0%, sodium hyaluronate: 0.05% - 0.1%, and the rest is HEPES buffer solution.

[0011] To obtain a better encapsulation effect, for the encapsulating solution used for the encapsulation of the fluorescent microsphere antibody, by mass percentage, trehalose: 5% - 10%, BSA: 0.5% - 1.0%, polyvinylpyrrolidone: 0.5% - 1.0%, sorbitol: 0.5% - 1.0%, and the rest is HEPES buffer solution.

[0012] To achieve the second object, the present invention adopts the following technical solution.

[0013] A preparation method of a fluorescent antibody encapsulating solution includes the following steps:

[0014] Step 1: Prepare a 20 mmol / L HEPES buffer solution;

[0015] Step 2: Adjust the pH to 7.4 - 7.6 with 1 mol / L sodium hydroxide;

[0016] Step 3: Sequentially add the remaining components of the fluorescent antibody sealing solution for achieving the first objective, and it can be used after being fully dissolved.

[0017] The preparation method of the fluorescent antibody sealing solution adopting the foregoing technical solution obtains a sealing solution for sealing fluorescent microsphere antibodies by sequentially adding trehalose, BSA, polyvinylpyrrolidone, and sorbitol according to the corresponding mass percentages and fully dissolving them; and obtains a sealing solution for sealing fluorescent dye antibodies by sequentially adding trehalose, BSA, polyvinylpyrrolidone, sorbitol, and sodium hyaluronate according to the corresponding mass percentages and fully dissolving them. Its operation method is simple. The two sealing solutions obtained by this method can be used for sealing antibodies with different fluorescent labeling methods, and the sealing effect is good.

[0018] To achieve the third objective, the present invention adopts the following technical solution.

[0019] Solution 1: A use of a fluorescent antibody sealing solution. The fluorescent antibody sealing solution prepared by HEPES buffer with trehalose, BSA, polyvinylpyrrolidone, and sorbitol obtained by the preparation method for achieving the second objective is used to dilute the activated fluorescent microsphere antibody and seal it in the mixing area of the self-driven microfluidic chip for myocardial detection. And after drying, use nutrients to keep the antibody active within a set time limit, so that during the flow of the test sample in the chip, after the recognition reaction with the fluorescent antibody in the mixing area, the antibody carrying the fluorescent microsphere can break free from the restraint of the mixing area and then automatically flow into the detection area to react with the antibody on the microcolumn to form a double-antibody sandwich marker, and thus the detection result is obtained by the fluorescence detector through the fluorescence labeling detection of the double-antibody sandwich marker. Among them, the drying after sealing the fluorescent microsphere antibody is naturally dried under the environmental requirements of a 100,000-class clean area; the environmental temperature is 25°C, and the humidity is kept constant at 50 - 60%.

[0020] To obtain a better sealing effect, the following steps are further included,

[0021] Step 1: Activate the fluorescent microspheres with the cross-linking agent EDC / NHS at 37°C for 20 min - 40 min, and the mass ratio of the fluorescent microspheres to EDC / NHS is 100:1 - 1:1;

[0022] Step 2: Add the activated fluorescent microspheres to the antibody solution and react at 37°C for 2 h - 3 h, and the mass ratio of the fluorescent microspheres to the antibody is 20:1 - 5:1;

[0023] Step 3: Centrifuge the solution after the antibody-fluorescent microsphere reaction using a refrigerated centrifuge. After removing the unlabeled antibody, add HEPES buffer to resuspend the antibody microsphere conjugate;

[0024] Step 4: Repeat Step 3 at least once;

[0025] Step 5: Centrifuge the solution after the antibody microsphere reaction again using a refrigerated centrifuge. After removing the unlabeled antibody microspheres, add the aforementioned fluorescent antibody sealing solution to resuspend the antibody microsphere conjugate.

[0026] To further obtain a better sealing effect, during the resuspension process of adding the HEPES buffer, calculate by the mass percentage after resuspension, and add the HEPES buffer at a ratio of 0.5% of the solid content of the fluorescent antibody microsphere conjugate in the mixed solution; during the resuspension process of adding the fluorescent antibody sealing solution, calculate by the mass percentage after resuspension, and add the fluorescent antibody sealing solution at a ratio of 0.2% of the solid content of the fluorescent antibody microsphere conjugate in the mixed solution. The resuspension is carried out by means of ultrasonic energy, that is, insert the ultrasonic cleaning head into the mixed liquid to make the antibody microspheres form a re-suspended state.

[0027] Solution 2, a use of a fluorescent antibody sealing solution. The fluorescent antibody sealing solution prepared by trehalose, BSA, polyvinylpyrrolidone, sorbitol and sodium hyaluronate through HEPES buffer obtained by the preparation method for achieving the second object is used to seal the activated fluorescent dye antibody in the mixing area of the self-driven microfluidic chip for myocardial detection. And after drying, use nutrients to keep the antibody active within a set period. When the test sample flows in the chip and undergoes an identification reaction with the fluorescent antibody in the mixing area, the antibody carrying the fluorescent dye can break free from the restraint of the mixing area and then automatically flow into the detection area to undergo an immune reaction with the antibody on the microcolumn, forming a double-antibody sandwich marker, so that the fluorescence detector can obtain the detection result by detecting the fluorescence label of the double-antibody sandwich marker. Among them, the drying after sealing the fluorescent dye antibody is natural drying under the environmental requirements of a 100,000-class clean area; the environmental temperature is 25 °C, and the humidity is a constant temperature and humidity condition of 50-60%.

[0028] To obtain a better sealing effect, it also includes,

[0029] Step 1: Mix the fluorescent dye and the antibody at a mass ratio of 1:1 to 1:10, and react at 37 °C for 20 min to 40 min;

[0030] Step 2: Remove the excess fluorescent dye by dialysis or purification;

[0031] Step 3: Dilute to an antibody concentration of 0.2 mg / ml with the aforementioned fluorescent antibody sealing solution.

[0032] The beneficial effects of the present invention are that the two fluorescent antibody sealing solutions can respectively seal the fluorescent microsphere antibody and the fluorescent dye antibody on the chip, and enable the antibody to have long-term activity. After the labeled reaction occurs, the sealed fluorescent antibody can break free from the restraint and flow with the sample; the preparation method of the sealing solution is simple. Through detection and verification, it has a strong ability to break free after the labeled reaction, the antibody activity can be maintained for a long time, and the corresponding sealing effect is good. When used in the self-driven microfluidic chip for routine examination of heart diseases, the detection efficiency is high and the detection accuracy is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic structural diagram of a self-driven microfluidic chip applying the fluorescent antibody sealing solution of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention will be further described below in conjunction with the accompanying drawings, but the present invention is not limited to the scope of the embodiments described herein.

[0035] Example 1, see Figure 1 , a fluorescent antibody sealing solution, used in a self-driven microfluidic chip for routine examination of heart diseases, seals the fluorescent microsphere antibody in the mixing zone 3, and is prepared from trehalose, BSA, polyvinylpyrrolidone and sorbitol through HEPES buffer; wherein, by mass percentage, trehalose: 5% - 20%, BSA: 0.1% - 1.0%, polyvinylpyrrolidone: 0.1% - 1.0%, sorbitol: 0.1% - 5.0%, and the rest is HEPES buffer.

[0036] As a preferred scheme, by mass percentage, the foregoing components are: trehalose: 5% - 10%, BSA: 0.5% - 1.0%, polyvinylpyrrolidone: 0.5% - 1.0%, sorbitol: 0.5% - 1.0%, and the rest is HEPES buffer.

[0037] Example 2, see Figure 1 , a fluorescent antibody sealing solution, used in a self-driven microfluidic chip for routine examination of heart diseases, seals the fluorescent dye antibody in the mixing zone 3, and is prepared from trehalose, BSA, polyvinylpyrrolidone, sorbitol and sodium hyaluronate through HEPES buffer; wherein, by mass percentage, trehalose: 5% - 20%, BSA: 0.1% - 1.0%, polyvinylpyrrolidone: 0.1% - 1.0%, sorbitol: 0.1% - 5.0%, sodium hyaluronate: 0.05% - 0.1%, and the rest is HEPES buffer.

[0038] As a preferred solution, in terms of mass percentage, trehalose: 5% - 10%, BSA: 0.5% - 1.0%, polyvinylpyrrolidone: 0.5% - 1.0%, sorbitol: 0.5% - 1.0%, sodium hyaluronate: 0.05% - 0.1%, and the rest is HEPES buffer solution.

[0039] Example 3, A method for preparing a fluorescent antibody sealing solution, comprising the following steps:

[0040] The first step is to prepare a 20 mmol / L HEPES buffer solution;

[0041] The second step is to adjust the pH to 7.4 - 7.6 with 1 mol / L sodium hydroxide;

[0042] The third step is to sequentially add the remaining components constituting the fluorescent antibody sealing solution of Example 1 or Example 2, and it can be used after being fully dissolved.

[0043] Example 4, The use of a fluorescent antibody sealing solution, the fluorescent antibody sealing solution as in Example 1 prepared by trehalose, BSA, polyvinylpyrrolidone and sorbitol through HEPES buffer solution obtained by the preparation method of Example 3 is used to dilute the activated fluorescent microsphere antibody and seal it in the mixing zone 3 of the self-driven microfluidic chip for myocardial detection. And after air-drying, the antibody is maintained active within a set period by using nutrients, so that during the flow of the test sample in the chip, after the recognition reaction with the fluorescent antibody in the mixing zone 3, the antibody carrying the fluorescent microsphere can break free from the restraint of the mixing zone and then automatically flow into the detection zone 5 to have an immune reaction with the antibody on the microcolumn, forming a double-antibody sandwich marker, and thus the detection result is obtained by the fluorescence detector through the fluorescence labeling detection of the double-antibody sandwich marker. Among them, the air-drying after the fluorescent microsphere antibody is sealed is natural air-drying under the environmental requirements of a 100,000-class clean area; the environmental temperature is 25°C, and the humidity is 50 - 60% under constant temperature and humidity conditions.

[0044] To obtain better results, it also includes,

[0045] The first step is to activate the fluorescent microspheres with the cross-linking agent EDC / NHS at 37°C for 20 min - 40 min, and the mass ratio of the fluorescent microspheres to EDC / NHS is 100:1 - 1:1;

[0046] The second step is to add the activated fluorescent microspheres to the antibody solution and react at 37°C for 2 h - 3 h, and the mass ratio of the fluorescent microspheres to the antibody is 20:1 - 5:1;

[0047] Step 3: Centrifuge the solution after the antibody-fluorescent microsphere reaction using a refrigerated centrifuge. After removing the unlabeled antibody, add HEPES buffer to resuspend the antibody microsphere conjugate;

[0048] Step 4: Repeat Step 3 at least once;

[0049] Step 5: Centrifuge the solution after the antibody microsphere reaction again using a refrigerated centrifuge. After removing the unlabeled antibody microspheres, add the aforementioned fluorescent antibody sealing solution to resuspend the antibody microsphere conjugate.

[0050] Among them, during the resuspension process of adding the HEPES buffer, based on the mass percentage after resuspension, add the HEPES buffer at a ratio of 0.5% of the solid content of the fluorescent antibody microsphere conjugate in the mixture; during the resuspension process of adding the fluorescent antibody sealing solution, based on the mass percentage after resuspension, add the fluorescent antibody sealing solution at a ratio of 0.2% of the solid content of the fluorescent antibody microsphere conjugate in the mixture. Resuspension is carried out by means of ultrasonic energy, that is, insert the ultrasonic cleaning head into the mixed liquid to make the antibody microspheres form a resuspended state again.

[0051] Example 5, Use of a fluorescent antibody sealing solution. The fluorescent antibody sealing solution prepared by trehalose, BSA, polyvinylpyrrolidone, sorbitol, and sodium hyaluronate through HEPES buffer obtained by the preparation method of Example 3 is used to seal the activated fluorescent dye antibody in the mixing zone 3 of the self-driven microfluidic chip for myocardial detection. After drying, use nutrients to keep the antibody active within a set time limit. When the test sample flows through the chip and undergoes an identification reaction with the fluorescent antibody in the mixing zone 3, the antibody carrying the fluorescent dye can break free from the restraint of the mixing zone 3 and then automatically flow into the detection zone 5 to undergo an immune reaction with the antibody on the microcolumn, forming a double-antibody sandwich marker. Thus, the detection result is obtained by the fluorescence detector detecting the fluorescence label of the double-antibody sandwich marker. Among them, the drying after sealing the fluorescent dye antibody is natural drying under the environmental requirements of a 100,000-class clean area; the environmental temperature is 25°C, and the humidity is a constant temperature and humidity condition of 50-60%.

[0052] To obtain a better sealing effect, it also includes,

[0053] Step 1: Mix the fluorescent dye and the antibody at a mass ratio of 1:1 to 1:10 and react at 37°C for 20 min to 40 min;

[0054] Step 2: Remove the excess fluorescent dye by dialysis or purification;

[0055] Step 3: Dilute with the aforementioned fluorescent antibody sealing solution to an antibody concentration of 0.2 mg / ml.

[0056] According to the following requirements for the sealing of fluorescent antibodies, the sealed fluorescent antibodies should flow away completely with the test samples without residue, and at the same time, the activity of the antibodies should be ensured to be unaffected. The applicant verified the two sealing solutions through the following experiments, and obtained experimental data after testing with a fluorescence analyzer. Table 1 is the experimental data record of the first sealing solution for sealing fluorescent dye antibodies under different ratios. In the table, the residual test of fluorescent antibodies is the ability of fluorescent antibodies to break free after the recognition reaction with the sample. The ability to break free is inversely correlated with the residual value, that is, the stronger the ability to break free, the less residual, and vice versa; among them, "+" indicates residual, "++" indicates obvious residual, "+++++" indicates severe residual, and "-" indicates no residual; the antibody activity is characterized by the fluorescence signal intensity test results after 30 days in a 37°C environment, and the signal attenuation index, among which "+" indicates attenuation, "++" indicates obvious attenuation, "+++" indicates severe attenuation, and "-" indicates no attenuation. Table 2 is the test data record of the second sealing solution used for sealing fluorescent microsphere antibodies under different ratio conditions. In the table, the fluorescent antibody residue test is the ability of the fluorescent antibody to break free after the recognition reaction with the sample. The ability to break free is inversely correlated with the residual value, that is, the stronger the ability to break free, the less the residue, and vice versa; among them, "+" indicates residue, "++" indicates obvious residue, "++++" indicates severe residue, and "-" indicates no residue; the antibody activity is the signal attenuation degree after accelerated aging at 37°C for 1 month, among which "+" indicates attenuation, "++" indicates obvious attenuation, and "+++" indicates severe attenuation. Among them, the unit of fluorescence signal intensity is CPS.

[0057] Table 1, experimental data record of the first sealing solution under different ratios of solid content

[0058]

[0059] The percentages listed in Table 1 are all mass percentages of solid content, and the rest are HEPES buffer.

[0060] Table 2, experimental data record of the second sealing liquid under different ratio solid content conditions

[0061]

[0062] The percentages listed in Table 2 are all mass percentages of solid content, and the rest are HEPES buffer.

[0063] In the process of evaluating the antibody activity of the first sealing solution and the second sealing solution, the relevant fluorescence signal intensity was tested at regular intervals during the 30 days; the specific test data are shown in Tables 3 and 4, respectively. Table 3 shows the intermittent time test results of the group corresponding to the first sealing solution; Table 4 shows the intermittent time test results of the group corresponding to the second sealing solution.

[0064] Table 3. Record of the test results of the interruption time for the group corresponding to the first sealing liquid

[0065]

[0066] Table 4. Record of the test results of the interruption time for the group corresponding to the second sealing liquid

[0067]

[0068] In Table 3 and Table 4, the change rate of the signal intensity is the percentage of the difference between the signal detection value at 30-day intervals and the detection value on the day of sealing relative to the signal detection value on the same day. A negative number indicates attenuation, and a positive number indicates enhancement. In theory, enhancement should not occur, but considering detection errors, a slight enhancement of the signal value is acceptable. The evaluation criteria are as follows: when the absolute value of the signal change rate is less than or equal to 10%, it is defined as no attenuation, denoted as "-"; when it is greater than 10% and less than or equal to 20%, it is defined as having attenuation, denoted as "+"; when it is greater than 20% and less than or equal to 50%, it is defined as obvious attenuation, denoted as "++"; when it is greater than 50%, it is defined as severe attenuation, denoted as "+++".

[0069] Example 6. A self-driven microfluidic chip includes a mixing zone 3 and a detection zone 5. The mixing zone 3 is sealed with a fluorescent antibody, and the detection zone 5 is fixed with a capture antibody through micro-columns on the microchannels of the chip bottom plate. Among them, the fluorescent antibody is sealed with the fluorescent antibody sealing liquid of Example 1 or 2 prepared by the method described in Example 3. When the antibody sealed in the mixing zone 3 is a fluorescent microsphere antibody, the fluorescent antibody sealing liquid of Example 1 is used; when the antibody sealed in the mixing zone 3 is a fluorescent dye antibody, the fluorescent antibody sealing liquid of Example 2 is used.

[0070] When the chip is used for the triple detection of myocardial markers, it includes a chip bottom plate as shown in Figure 1 . The chip bottom plate is sequentially provided with a sample loading zone 1, a blood filtration zone 2, a mixing zone 3, a speed-limiting zone 4, and a detection zone 5. The speed-limiting zone 4 is provided with a time-controlled valve to slow down the flow rate of the sample to be tested in the speed-limiting zone 4, extend the residence time of the sample to be tested in the mixing zone 3, and increase the reaction time between the sample to be tested and the reagents in the mixing zone 3. Three fluorescent antibodies are sealed in the mixing zone 3, which are a CKMB antibody, a CTNI antibody, and a Myo antibody respectively. Three antibodies are sequentially fixed through the micro-columns on the microchannels of the chip bottom plate in the detection zone 5, and the three fixed antibodies are a CKMB antibody, a CTNI antibody, and a Myo antibody respectively.

[0071] During use, after the blood sample of the whole blood, serum or plasma to be tested is added dropwise in the sample addition area 1 through a pipette, the sample automatically enters the blood filtration area 2 for filtration according to the capillary principle in the direction indicated by arrow A. After that, it enters the mixing area 3 to carry out an identification reaction with the fluorescent antibody. After the fluorescent antibody binds to the blood sample, it breaks free from the restraint of the mixing area 3 and flows with the blood sample. Subsequently, it enters the detection area 5, and a double-antibody sandwich detection marker is formed after binding with the immobilized antibody. Finally, the detection result is obtained by detecting the signal intensity of the marker with a fluorescence analyzer. Among them, a zero value area and a quality control area are provided in the detection area. The quality control serves as the basis for judging the invalidation of the test card and the detection time, and the zero value point serves as the background deduction.

[0072] The above has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. Use of a fluorescent antibody sealing solution, characterized in that, It is used to seal the activated fluorescent dye antibody after dilution in the mixing area of the self-driven microfluidic chip for myocardial detection; it includes the following steps: First step, mix the fluorescent dye and the antibody at a mass ratio of 1:1 to 1:10, and react at 37 °C for 20 min to 40 min; Second step, remove the excess fluorescent dye by dialysis or purification; Third step, dilute it to an antibody concentration of 0.2 mg / ml with the fluorescent antibody sealing solution; The fluorescent antibody sealing solution is prepared from trehalose, BSA, polyvinylpyrrolidone, sorbitol and sodium hyaluronate through HEPES buffer; among them, by mass percentage, trehalose: 5% - 10%, BSA: 0.5% - 1.0%, polyvinylpyrrolidone: 0.5% - 1.0%, sorbitol: 0.5% - 1.0%, sodium hyaluronate: 0.05% - 0.1%, and the rest is HEPES buffer; The fluorescent antibody sealing solution is prepared by the following method: First step, prepare 20 mmol / L HEPES buffer; Second step, adjust the pH to 7.4 - 7.6 with 1 mol / L sodium hydroxide; Third step, sequentially add the remaining components of the fluorescent antibody sealing solution, and it can be obtained after fully dissolving, to obtain the fluorescent antibody sealing solution.

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