Fabrication method of a self-driven microfluidic chip and a self-driven microfluidic chip

Abstract

The present invention discloses a manufacturing method of a self-driven microfluidic chip and the self-driven microfluidic chip. The manufacturing method includes steps of antibody encapsulation and antibody immobilization; wherein, antibody encapsulation includes a fluorescent dye encapsulation method or a fluorescent microsphere encapsulation method; antibody immobilization includes an EPI immobilization method or a latex microsphere immobilization method, and both encapsulation and immobilization are quantitatively carried out with the aid of an instrument. The self-driven microfluidic chip encapsulates and immobilizes antibodies by the foregoing method. The beneficial effects of the present invention are that the antibodies encapsulated and immobilized by the manufacturing method have a long active time. After the labeled reaction occurs for the encapsulated fluorescent antibodies, they can break free from the restraint and flow with the sample; the antibodies immobilized in the detection area remain in place after the immune reaction to retain the double antibody sandwich label on the micro-columns in the detection area of the chip. When the self-driven microfluidic chip is used for routine examination of heart diseases, the detection result can be obtained by detecting the fluorescence labeling signal intensity of the double antibody sandwich retained in the detection area with a fluorescence detector.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence immunoassay, and particularly relates to a manufacturing method of a self-driven microfluidic chip and a self-driven microfluidic chip. Background Art

[0002] The three myocardial markers are three very commonly used indicators in the clinical practice of 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 at 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, 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] Currently, there are products using two methods, colloidal gold and fluorescence chromatography, both of which use the double antibody sandwich method to detect the substance to be detected in the blood sample, and then detect it by an instrument for quantitative analysis of the analyte. Existing products basically use the NC membrane as the carrier. This carrier is greatly affected by the environment. If the temperature and humidity do not meet the requirements, it will cause a very significant impact on the detection results of the NC membrane for the sample. Therefore, there are high environmental control requirements during production and storage. At the same time, there are large differences between batches of the NC membrane, and the amount of immobilized antibody is not uniform. Secondly, the myocardial triple detection reagent prepared with the NC membrane will cause the problem of the release of fluorescent antibody with the sample flow due to these three factors: the NC membrane, the sample pad, and the conjugate pad, which will also affect the detection results, so the repeatability CV of the product is relatively large. In addition, since the myocardial triple detection combines three detection items, CKMB, CTNI, and Myo of myocardial three items, on the same detection reagent card, there are problems such as mutual interference between items, low detection sensitivity, and poor repeatability CV. For this reason, the applicant invented a self-driven microfluidic chip and submitted a patent application to the State Intellectual Property Office on January 17, 2020. This application was published on April 28, 2020, and its publication number is CN111068801A. The self-driven microfluidic chip pre-stores the antibody corresponding to the substance to be detected on the self-driven 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 and through precise microfluidic control, it can ensure the reliability and controllability of the sample detection results, and is expected to replace the current clinically commonly used immunochromatography (LIFCS) technology with various membrane materials, thus providing a new technical idea for solving the challenges faced by the current POCT detection technology. Subsequently, the applicant submitted a patent application for a rapid immunofluorescence detection method based on the self-driven microfluidic chip to the State Intellectual Property Office on May 22, 2020. This application was published on August 21, 2020, and its publication number is CN111562380A; this method discloses the whole process of using this self-driven microfluidic chip for rapid immunofluorescence detection.

[0004] During the fabrication process of the self-driven microfluidic chip, a strain of antibody labeled with a fluorescent substance needs to be sealed in the mixing zone, and another strain of antibody is fixed on the micro-columns in the detection zone of the chip. During the flow of the sample to be detected through the chip, after the recognition reaction with the fluorescent antibody in the mixing zone, the antibody carrying the fluorescent marker then undergoes an immune reaction with the antibody on the micro-columns, thereby 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 confinement of the mixing zone, and the antibody fixed on the micro-columns can firmly adhere to the micro-columns after the immune reaction, so as to obtain an accurate detection result when detected by the fluorescence detector, is the key to the fabrication technology of this self-driven microfluidic chip, and the activity of the antibody after sealing and fixation is also an important part of this technology. Currently, in the existing technology, there is no literature disclosing related technologies that can achieve the aforementioned purpose. Summary of the Invention

[0005] The first object of the present invention is to address the deficiency of the lack of antibody pre-storage technology in the preparation process of the existing self-driven microfluidic chip, and to provide a method for fabricating a self-driven microfluidic chip. This method seals the fluorescent antibody in the mixing zone of the self-driven microfluidic chip by the fluorescent dye encapsulation method or the fluorescent microsphere encapsulation method, and fixes the antibody in the reaction zone of the self-driven microfluidic chip by the EPI fixation method or the latex microsphere fixation method. During the flow of the sample to be detected through the chip, after the recognition reaction with the fluorescent antibody in the mixing zone, the antibody carrying the fluorescent marker then undergoes an immune reaction with the antibody on the micro-columns, thereby forming a double-antibody sandwich marker, and then 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 also provides a self-driven microfluidic chip based on the aforementioned method for antibody encapsulation and antibody fixation.

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

[0007] A method for fabricating a self-driven microfluidic chip, including the steps of antibody encapsulation and antibody fixation; wherein,

[0008] The antibody encapsulation includes the fluorescent dye encapsulation method or the fluorescent microsphere encapsulation method;

[0009] The fluorescent dye encapsulation method includes labeling the fluorescent dye on the antibody through a chemical reaction and removing the excess fluorescent dye; then, diluting the fluorescent antibody to the required concentration with a transparent first antibody encapsulation solution; finally, quantitatively sealing the fluorescent antibody onto the mixing zone of the self-driven microfluidic chip by an instrument and drying it.

[0010] The fluorescent microsphere encapsulation method includes reacting an antibody with fluorescent microspheres through a crosslinking agent EDC / NHS; then, diluting the antibody-fluorescent microspheres to the required concentration with a transparent second antibody encapsulation solution; finally, quantitatively depositing the fluorescent antibody onto the mixing zone of the self-driven microfluidic chip by an instrument and air-drying it;

[0011] The first antibody encapsulation solution is prepared from trehalose, BSA, polyvinylpyrrolidone, sorbitol, and sodium hyaluronate through HEPES buffer;

[0012] The second antibody encapsulation solution is prepared from trehalose, BSA, polyvinylpyrrolidone, and sorbitol through HEPES buffer;

[0013] The antibody immobilization includes the EPI immobilization method or the latex microsphere immobilization method;

[0014] The EPI immobilization method includes first reacting an antibody with polyethyleneimine with a molecular weight of 10,000 - 1,000,000 through a crosslinking agent BS3 or DSS; then, quantitatively depositing the reacted mixed solution onto the microcolumns in the detection zone of the self-driven microfluidic chip by an instrument and air-drying it;

[0015] The latex microsphere immobilization method includes first reacting an antibody with latex microspheres through a crosslinking agent EDC / NHS to form an antibody - microsphere conjugate; mixing the reacted antibody - microsphere conjugate with a fixing solution of hyaluronic acid; finally, quantitatively depositing the mixed solution onto the microcolumns in the detection zone of the self-driven microfluidic chip by an instrument and air-drying it;

[0016] The fixing solution is prepared from any one of hydroxyethyl cellulose, sodium hyaluronate, sodium alginate, and polyvinyl alcohol through HEPES buffer; or a composition formed by two or more of them prepared separately.

[0017] The present invention adopting the foregoing technical solution seals the fluorescent antibody in the mixing area of the self-driven microfluidic chip by the fluorescent dye encapsulation method or the fluorescent microsphere encapsulation method and dries it, and fixes the antibody in the reaction area of the self-driven microfluidic chip by the EPI fixation method or the latex microsphere fixation method and dries it. During the flow of the sample to be detected in the chip, after the recognition reaction with the fluorescent antibody in the mixing area, the antibody carrying the fluorescent marker then undergoes an immune reaction with the antibody on the microcolumn to form a double-antibody sandwich marker, so that 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 antibody fixation and encapsulation is natural 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%. The fixing solutions are prepared by HEPES buffer with hydroxyethyl cellulose, sodium hyaluronate, sodium alginate and polyvinyl alcohol respectively to form a variety of different fixing solutions; these different fixing solutions can be used alone or in any combination of two or more.

[0018] Preferably, in the EPI fixation method, the process of obtaining the mixed solution after reaction includes,

[0019] S11, mixing polyethyleneimine and antibody at a mass ratio of 1:3 to 1:6;

[0020] S12, adding BS3 to the mixture of polyethyleneimine and antibody and mixing evenly, where the mass ratio of BS3 to polyethyleneimine is 1:10 to 1:2;

[0021] S13, reacting at 37 °C for 2 h to 3 h.

[0022] Through reasonable ratio and reaction conditions, the antibody can be fixed on the microcolumn in the reaction area of the chip based on the structural characteristics of the microcolumn carrier on the chip. In addition to maintaining the antibody activity, the antibody can also be firmly fixed on the microcolumn.

[0023] Preferably, in the latex microsphere fixation method, the process of obtaining the solution after mixing with the fixing solution includes,

[0024] S21, activating the latex microspheres with the crosslinking agent EDC / NHS at 37 °C for 20 min to 40 min, and the mass ratio of the latex microspheres to EDC / NHS is 100:1 to 1:1;

[0025] S22, adding the activated latex microspheres to the antibody solution and reacting at 37 °C for 2 h - 3 h, and the mass ratio of the latex microspheres to the antibody is 20:1 to 5:1;

[0026] S23, centrifuging the solution after the antibody microsphere reaction with a refrigerated centrifuge, and after removing the unlabeled antibody, adding HEPES buffer to resuspend the antibody microsphere conjugate;

[0027] S24. Repeat S23 at least once;

[0028] S25. Then, centrifuge the solution after the antibody microsphere reaction using a refrigerated centrifuge. After removing the unlabeled antibody, add the fixing solution to resuspend the antibody microsphere conjugate.

[0029] To obtain an antibody with a long active retention time through reasonable ratios and reaction conditions, and based on the structural characteristics of the microcolumn carrier on the chip, with the aid of the characteristics of the fixing solution and latex microspheres, fix the antibody on the microcolumns in the reaction area of the chip. Among them, 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.

[0030] Further preferably, during the resuspension process of adding the HEPES buffer solution, calculated by the mass percentage after resuspension, add the HEPES buffer solution at a ratio of 0.5% of the solid content of the antibody microsphere conjugate in the mixed liquid; during the resuspension process of adding the fixing solution, calculated by the mass percentage after resuspension, add the fixing solution at a ratio of 0.1% of the solid content of the antibody microsphere conjugate in the mixed liquid. To finally form a fixing mixture that meets the requirements of microsphere antibody fixation and maintains antibody activity through reasonable ratios.

[0031] Preferably, in the fixing solution prepared by HEPES buffer solution from hydroxyethyl cellulose, sodium hyaluronate, sodium alginate, and polyvinyl alcohol respectively, by mass percentage, hydroxyethyl cellulose accounts for 0.01% - 1.0%, sodium hyaluronate accounts for 0.01% - 0.1%, sodium alginate accounts for 0.01% - 0.1%, polyvinyl alcohol accounts for 0.01% - 1.0%, and the rest is HEPES buffer solution. To finally form a variety of fixing solutions that meet the functional characteristics of microsphere antibody fixation and maintain antibody activity through reasonable ratios.

[0032] Preferably, in the fluorescence dye encapsulation method, the process of obtaining the diluted fluorescent antibody mixed solution includes

[0033] S31. Mix the fluorescent dye and the antibody at a mass ratio of 1:1 - 1:10, and react at 37°C for 20 min - 40 min;

[0034] S32. Remove the excess fluorescent dye by dialysis or purification;

[0035] S33. Dilute to an antibody concentration of 0.2 mg / ml with a transparent first antibody sealing solution.

[0036] Through reasonable proportioning and reaction conditions, the antibody can be evenly dyed with fluorescent dye. By removing the excess fluorescent dye, the accuracy of the test result marked by fluorescence can be ensured. At the same time, the activity of the antibody is maintained with the help of the sealing solution. Utilizing the weak binding ability of the fluorescent dye to the sealing solution, it is ensured that after the recognition reaction occurs in the mixing area of the test sample, it can break free from the restraint of the carrier and flow with the sample in the microchannel of the chip.

[0037] Preferably, in the fluorescent microsphere sealing method, the process of obtaining the diluted fluorescent microsphere mixture includes

[0038] S41, Activate the fluorescent microspheres with the cross-linking agent EDC / NHS at 37°C for 20 min to 40 min, and the mass ratio of the fluorescent microspheres to EDC / NHS is 100∶1 to 1∶1;

[0039] S42, Add the activated fluorescent microspheres to the antibody solution and react at 37°C for 2 h to 3 h, and the mass ratio of the fluorescent microspheres to the antibody is 20∶1 to 5∶1;

[0040] S43, Centrifuge the solution after the reaction of the antibody and fluorescent microspheres with a refrigerated centrifuge. After removing the unlabeled antibody, add HEPES buffer to resuspend the antibody microsphere conjugate;

[0041] S44, Repeat S43 at least once;

[0042] S45, Then centrifuge the solution after the reaction of the antibody and microspheres with a refrigerated centrifuge. After removing the unlabeled antibody microspheres, add a transparent second antibody sealing solution to resuspend the antibody microsphere conjugate.

[0043] Through reasonable proportioning and reaction conditions, the antibody can be fully combined with the fluorescent microspheres. By removing the excess fluorescent microspheres, the accuracy of the detection result can be ensured. At the same time, the activity of the antibody is maintained with the help of the sealing solution. Utilizing the weak binding ability of the fluorescent antibody microspheres to the sealing solution, it is ensured that after the recognition reaction occurs in the mixing area of the test sample, the fluorescent antibody microspheres can break free from the restraint of the carrier and flow with the sample in the microchannel of the chip. Among them, resuspension is carried out by means of ultrasonic energy supply, that is, the ultrasonic cleaning head is inserted into the mixed liquid to make the fluorescent antibody microspheres form a resuspended state.

[0044] Further preferably, during the resuspension process of adding the HEPES buffer, calculated by 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 mixed liquid; during the resuspension process of adding the second antibody sealing solution, calculated by the mass percentage after resuspension, add the second antibody sealing solution at a ratio of 0.2% of the solid content of the fluorescent antibody microsphere conjugate in the mixed liquid. Through reasonable proportioning, a sealing mixture that meets the requirements of fluorescent antibody sealing and maintaining antibody activity is finally formed.

[0045] Preferably, for the first antibody storage solution, 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 balance is HEPES buffer solution; for the second antibody storage solution, by mass percentage, trehalose: 5% - 20%, BSA: 0.1% - 1.0%, polyvinylpyrrolidone: 0.1% - 1.0%, sorbitol: 0.1% - 5.0%, and the balance is HEPES buffer solution. Through reasonable ratios, the functional characteristics of the storage solution that can meet the requirements of fluorescent antibody storage and maintain antibody activity are finally formed.

[0046] Through reasonable ratios and reaction conditions, based on the structural characteristics of the flow channel carrier in the chip mixing area and the characteristics of the storage solution, with the help of the storage solution, the antibody can be sealed in the chip mixing area. In addition to maintaining antibody activity, after the test sample undergoes an identification reaction with the antibody labeled with a fluorescent substance, it can break free from the restraint of the carrier and flow with the sample in the microfluidic channel, so as to smoothly enter the reaction area and undergo an immune reaction with the antibody on the microcolumn.

[0047] To achieve the second object, the present invention adopts the following technical solutions.

[0048] A self-driven microfluidic chip performs antibody storage and antibody immobilization by the method for achieving the first object.

[0049] For the self-driven microfluidic chip that performs antibody storage and antibody immobilization by the aforementioned technical solutions, during the routine examination of heart diseases, when the test sample flows in the chip and undergoes an identification reaction with the fluorescent antibody in the mixing area, it breaks free from the restraint and flows with the sample. After the antibody carrying the fluorescent marker undergoes an immune reaction with the antibody on the microcolumn in the detection area, a double-antibody sandwich marker is formed, and thus the detection result is obtained by the fluorescence detector detecting the fluorescence label of the double-antibody sandwich marker.

[0050] The beneficial effects of the present invention are that the antibody stored and immobilized on the chip by the manufacturing method has long-term activity. After the stored fluorescent antibody undergoes a labeling reaction, it can break free from the restraint and flow with the sample; the antibody immobilized in the detection area remains unchanged after the immune reaction and can retain the double-antibody sandwich marker on the microcolumn in the detection area of the chip. When the self-driven microfluidic chip is used for the routine examination of heart diseases, the detection result can be obtained by the fluorescence detector detecting the fluorescence label signal intensity of the double-antibody sandwich retained in the detection area.

[0051] Description of the drawings

[0052] Figure 1It is a schematic structural diagram of a self-driven microfluidic chip applying the method of the present invention. Detailed implementation manners

[0053] 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 described embodiments.

[0054] Example 1, a manufacturing method of a self-driven microfluidic chip, including steps of antibody encapsulation and antibody immobilization; wherein, the antibody encapsulation is carried out according to the fluorescent dye encapsulation method;

[0055] The fluorescent dye encapsulation method includes labeling the fluorescent dye on the antibody through a chemical reaction and removing the excess fluorescent dye; then, diluting the fluorescent antibody to the required concentration with a transparent first antibody encapsulation solution; finally, quantitatively encapsulating the fluorescent antibody into the mixing zone 3 of the self-driven microfluidic chip through an instrument and naturally drying it under the environmental requirement conditions of a 100,000-class clean area; the first antibody encapsulation solution is prepared from trehalose, BSA, polyvinylpyrrolidone, sorbitol and sodium hyaluronate through HEPES buffer solution;

[0056] The antibody immobilization is carried out according to the EPI immobilization method;

[0057] The EPI immobilization method includes first reacting the antibody with polyethyleneimine having a molecular weight of 10,000 - 1,000,000 through a cross-linking agent BS3 or DSS; then, quantitatively immobilizing the antibody on the micro-columns in the detection zone 5 of the self-driven microfluidic chip through an instrument for the reacted mixed solution and naturally drying it under the environmental requirement conditions of a 100,000-class clean area;

[0058] Wherein, during the EPI immobilization process, the process of obtaining the reacted mixed solution includes,

[0059] S11, mixing polyethyleneimine and the antibody at a mass ratio of 1:3 - 1:6;

[0060] S12, adding BS3 to the mixed solution of polyethyleneimine and the antibody and mixing evenly, wherein the mass ratio of BS3 to polyethyleneimine is 1:10 - 1:2;

[0061] S13, reacting at 37 °C for 2 h - 3 h.

[0062] Wherein, during the fluorescent dye encapsulation process, the process of obtaining the diluted fluorescent antibody mixed solution includes,

[0063] S31, mixing the fluorescent dye and the antibody at a mass ratio of 1:1 - 1:10 and reacting at 37 °C for 20 min - 40 min;

[0064] S32, removing the excess fluorescent dye by dialysis or purification;

[0065] S33. Dilute the antibody to an antibody concentration of 0.2 mg / ml with a transparent primary antibody blocking solution.

[0066] In the primary antibody blocking solution by mass percentage, trehalose accounts for 5% - 20%, BSA accounts for 0.1% - 1.0%, polyvinylpyrrolidone accounts for 0.1% - 1.0%, sorbitol accounts for 0.1% - 5.0%, sodium hyaluronate accounts for 0.05% - 0.1%, and the rest is HEPES buffer.

[0067] To obtain a better blocking effect, in the primary antibody blocking solution by mass percentage, trehalose: 5% - 10%, BSA: 0.5% - 1%, polyvinylpyrrolidone: 0.5% - 1.0%, sorbitol: 0.5% - 1.0%, sodium hyaluronate: 0.05% - 0.1%, and the rest is HEPES buffer.

[0068] Example 2. A method for fabricating a self - driven microfluidic chip, including steps of antibody blocking and antibody immobilization; among them, the antibody immobilization is carried out according to the latex microsphere immobilization method.

[0069] The latex microsphere immobilization method includes first reacting the antibody with latex microspheres through a cross - linker EDC / NHS to form an antibody - microsphere conjugate; mixing the reacted antibody - microsphere conjugate with a hyaluronic acid fixing solution; finally, quantitatively fixing the antibody to the micro - columns in the detection area 5 of the self - driven microfluidic chip through an instrument and air - drying. Among them, the fixing solution is prepared from any one of hydroxyethyl cellulose, sodium hyaluronate, sodium alginate, and polyvinyl alcohol through HEPES buffer.

[0070] During the latex microsphere immobilization process, the process of obtaining the solution after mixing with the fixing solution includes

[0071] S21. Activate the latex microspheres with the cross - linker EDC / NHS at 37 °C for 20 min - 40 min, and the mass ratio of latex microspheres to EDC / NHS is 100∶1 - 1∶1.

[0072] S22. Add the activated latex microspheres to the antibody solution and react at 37 °C for 2 h - 3 h; and the mass ratio of latex microspheres to antibody is 20∶1 - 5∶1.

[0073] S23. Centrifuge the solution after the antibody - microsphere reaction with a refrigerated centrifuge, and after removing the unlabeled antibody, add HEPES buffer to resuspend the antibody - microsphere conjugate.

[0074] S24. Repeat S23 at least once.

[0075] S25. Then, centrifuge the solution after the antibody microsphere reaction using a refrigerated centrifuge. After removing the unlabeled antibody, add the fixing solution to resuspend the antibody microsphere conjugate.

[0076] During the resuspension process of adding the HEPES buffer solution, calculate by the mass percentage after resuspension, and add the HEPES buffer solution at a ratio of 0.5% of the solid content of the antibody microsphere conjugate in the mixed solution; during the resuspension process of adding the fixing solution, calculate by the mass percentage after resuspension, and add the fixing solution at a ratio of 0.1% of the solid content of the antibody microsphere conjugate in the mixed solution.

[0077] Among them, the fixing solution can be prepared by mixing hydroxyethyl cellulose, sodium hyaluronate, sodium alginate, and polyvinyl alcohol with the HEPES buffer solution respectively. When preparing separately, calculate by mass percentage, hydroxyethyl cellulose accounts for 0.01% - 1%, sodium hyaluronate accounts for 0.01% - 0.1%, sodium alginate accounts for 0.01% - 0.1%, polyvinyl alcohol accounts for 0.01% - 1%, and the rest is the HEPES buffer solution.

[0078] The remaining steps of this example are the same as those of Example 1 and will not be elaborated here.

[0079] In this example, the fixing solution can also be formed by mixing any two or more of the liquids prepared by mixing hydroxyethyl cellulose, sodium hyaluronate, sodium alginate, and polyvinyl alcohol with the HEPES buffer solution respectively.

[0080] To obtain a better fixing effect, during the process of preparing the antibody fixing solution separately, calculate by mass percentage, hydroxyethyl cellulose accounts for 0.01% - 0.05%, sodium hyaluronate accounts for 0.01% - 0.05%, sodium alginate accounts for 0.01% - 0.05%, polyvinyl alcohol accounts for 0.01% - 0.05%, and the rest is the HEPES buffer solution.

[0081] The fixing solution in this example can also be composed of any two or more combinations of hydroxyethyl cellulose, sodium hyaluronate, sodium alginate, and polyvinyl alcohol, and can be prepared together with the HEPES buffer solution constituting the remaining content according to the aforementioned solid content ratio.

[0082] Example 3. A method for fabricating a self-driven microfluidic chip includes steps of antibody encapsulation and antibody fixation; among them, the antibody encapsulation is carried out according to the fluorescence microsphere encapsulation method.

[0083] The fluorescent microsphere encapsulation method includes reacting an antibody with fluorescent microspheres through a cross-linking agent EDC / NHS; then, diluting the antibody-fluorescent microspheres to the required concentration with a transparent secondary antibody encapsulation solution; finally, quantitatively encapsulating the fluorescent antibody into the mixing zone 3 of the self-driven microfluidic chip by an instrument and naturally drying it under the environmental requirements of a Class 100,000 clean area; the secondary antibody encapsulation solution is prepared from trehalose, BSA, polyvinylpyrrolidone, and sorbitol through a HEPES buffer solution;

[0084] During the fluorescent microsphere encapsulation process, the process of obtaining the diluted fluorescent microsphere mixture includes,

[0085] S41, activating the fluorescent microspheres with the cross-linking agent EDC / NHS at 37 °C for 20 min to 40 min, and the mass ratio of the fluorescent microspheres to EDC / NHS is 100:1 to 1:1;

[0086] S42, adding the activated fluorescent microspheres to the antibody solution and reacting at 37 °C for 2 h to 3 h, and the mass ratio of the fluorescent microspheres to the antibody is 20:1 to 5:1;

[0087] S43, centrifuging and separating the solution after the antibody-fluorescent microsphere reaction with a refrigerated centrifuge, and after removing the unlabeled antibody, adding a HEPES buffer solution to resuspend the antibody microsphere conjugate;

[0088] S44, repeating S43 at least once;

[0089] S45, centrifuging and separating the solution after the antibody microsphere reaction with a refrigerated centrifuge again, and after removing the unlabeled antibody microspheres, adding the secondary antibody encapsulation solution to resuspend the antibody microsphere conjugate.

[0090] During the resuspension process of adding the HEPES buffer solution, calculated by the mass percentage after resuspension, the HEPES buffer solution is added 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 secondary antibody encapsulation solution, calculated by the mass percentage after resuspension, the transparent antibody encapsulation solution is added at a ratio of 0.2% of the solid content of the fluorescent antibody microsphere conjugate in the mixture.

[0091] Calculated by mass percentage, for the secondary antibody encapsulation solution, trehalose: 5% - 20%, BSA: 0.1% - 1%, polyvinylpyrrolidone: 0.1% - 1.0%, sorbitol: 0.1% - 5.0%, and the rest is the HEPES buffer solution.

[0092] In order to obtain a better sealing effect, the second antibody sealing solution is calculated by mass percentage as follows: trehalose: 5%-10%, BSA: 0.5%-1.0%, polyvinyl pyrrolidone: 0.5%-1.0%, sorbitol: 0.5%-1.0%, and the rest is HEPES buffer.

[0093] The remaining steps of this embodiment are the same as those of Embodiment 1 or Embodiment 2 and will not be described again.

[0094] In the above-mentioned embodiments, the preparation method of the first and second sealing solutions is to first prepare 20mmol / L HEPES buffer, adjust the pH to 7.4-7.6 with 1mol / L sodium hydroxide, and then add the substances in the sealing solution formula in sequence until they are fully dissolved.

[0095] The preparation process of the fixative solution is the same as that of the sealing solution.

[0096] According to the following requirements for antibody sealing and fixation, the sealed fluorescent antibody flows away completely with the test sample without residue, and at the same time, the activity of the antibody must be ensured not to be affected; the fixed antibody is fixed stably, that is, the higher the detection signal value, the better, and the product repeatability CV is within the minimum requirement of 15%. The applicant verified the two sealing solutions and fixatives 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 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 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 at 37°C, 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 that there is residue, "++" indicates that there is obvious residue, "++++" indicates that there is serious residue, and "-" indicates that there is no residue; the antibody activity is measured by the signal attenuation degree after accelerated aging at 37°C for 1 month, among which "+" indicates attenuation, "++" indicates obvious attenuation, and "+++" indicates serious attenuation; Table 3 is the test data record of different fixatives, among which the antibody fluorescence signal detection value after fixation and the repeatability CV evaluation index are recorded, among which CV is less than 15%, and the smaller the better. Among them, the unit of fluorescence signal intensity is CPS.

[0097] Table 1. Test data records of the first sealing liquid under different solid content ratios

[0098]

[0099] The percentages listed in Table 1 are all mass percentages of the solid content, and the rest is the HEPES buffer solution.

[0100] Table 2. Test data records of the second sealing liquid under different solid content ratios

[0101]

[0102] The percentages listed in Table 2 are all mass percentages of the solid content, and the rest is the HEPES buffer solution.

[0103] Table 3. Fluorescence signal detection values and repeatability CV values after the antibody is fixed by the fixing liquid

[0104]

[0105] The fixing liquid listed in Table 3 is prepared by sodium hyaluronate, hydroxyethyl cellulose, sodium alginate, polyvinyl alcohol and HEPES buffer solution respectively. The corresponding percentages are all mass percentages of the solid content, and the rest is the HEPES buffer solution.

[0106] During the evaluation of the antibody activity sealed by the first sealing liquid and the second sealing liquid, the fluorescence signal intensity was tested at regular intervals during the 30-day process, and the change rate was calculated. The specific data are shown in Table 4 and Table 5 respectively. Table 4 is the test results of the intermittent time of the corresponding group of the first sealing liquid; Table 5 is the test results of the intermittent time of the corresponding group of the second sealing liquid.

[0107] Table 4. Test results record of the intermittent time of the corresponding group of the first sealing liquid

[0108]

[0109] Table 5. Test results record of the intermittent time of the corresponding group of the second sealing liquid

[0110]

[0111] In Tables 4 and 5, 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 day of sealing. 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: an absolute value of the signal change rate less than or equal to 10% is defined as no attenuation, denoted as "-"; greater than 10% and less than or equal to 20% is defined as having attenuation, denoted as "+"; greater than 20% and less than or equal to 50% is defined as obvious attenuation, denoted as "++"; greater than 50% is defined as severe attenuation, denoted as "+++".

[0112] Example 4. A self-driven microfluidic chip includes a mixing zone 3 and a detection zone 5. A fluorescent antibody is sealed in the mixing zone 3, and a fixed antibody is fixed on the micro-columns on the micro-channel of the chip bottom plate in the detection zone 5. Among them, the sealing of the fluorescent antibody and the fixation of the fixed antibody are respectively carried out by the methods described in Example 1, Example 2, or Example 3.

[0113] When the chip is used for triple detection of myocardial markers, it includes a chip bottom plate as shown in Figure 1 A sampling area 1, a blood filtration area 2, a mixing area 3, a speed-limiting area 4, and a detection area 5 are sequentially arranged on the chip bottom plate. A time-controlled valve is provided in the speed-limiting area 4 to slow down the flow rate of the sample to be tested in the speed-limiting area 4, extend the residence time of the sample to be tested in the mixing area 3, and increase the reaction time between the sample to be tested and the reagent in the mixing area 3. Three fluorescent antibodies are sealed in the mixing area 3, and the three fluorescent antibodies are respectively one CKMB antibody, one CTNI antibody, and one Myo antibody. Three antibodies are sequentially fixed on the micro-columns on the micro-channel of the chip bottom plate in the detection area, and the three fixed antibodies are respectively one CKMB antibody, one CTNI antibody, and one Myo antibody.

[0114] During use, after a blood sample of the whole blood, serum, or plasma to be tested is added to the sampling 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, and then 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, and then enters the detection area 5. After binding to the fixed antibody, a double-antibody sandwich detection marker is formed. Finally, the signal intensity of the marker is detected by a fluorescence analyzer to obtain the detection result.

[0115] The above shows and describes 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 of the present invention is defined by the appended claims and their equivalents.

Claims

1. A manufacturing method of a self-driven microfluidic chip, characterized in that It includes the steps of antibody sequestration and antibody immobilization; among them, the antibody sequestration is the fluorescent dye sequestration method; the fluorescent dye sequestration method includes labeling the fluorescent dye on the antibody through a chemical reaction and removing the excess fluorescent dye; then, diluting the fluorescent antibody to the required concentration with a transparent first antibody sequestration solution; finally, quantitatively sequestering the fluorescent antibody onto the mixing area of the self-driven microfluidic chip by an instrument and air-drying it; the first antibody sequestration solution is prepared from trehalose, BSA, polyvinylpyrrolidone, sorbitol and sodium hyaluronate through HEPES buffer; for the first antibody sequestration solution, by mass percentage, trehalose: 5% - 20%, BSA: 0.1% - 1.0%, polyvinylpyrrolidone: 0.1% - 1.0%, sorbitol: 0.1% - 5%, sodium hyaluronate: 0.05% - 0.1%, and the rest is HEPES buffer; the antibody immobilization is the latex microsphere immobilization method; the latex microsphere immobilization method includes first reacting the antibody with the latex microspheres through the cross-linking agent EDC / NHS to form an antibody-microsphere conjugate; mixing the reacted antibody-microsphere conjugate with the immobilization solution of hyaluronic acid; finally, quantitatively immobilizing the antibody onto the micro-columns in the detection area of the self-driven microfluidic chip by an instrument and air-drying it; the immobilization solution is formed by preparing any one of hydroxyethyl cellulose, sodium alginate and polyvinyl alcohol through HEPES buffer. When preparing separately, by mass percentage, hydroxyethyl cellulose accounts for 0.01% - 0.1%, sodium alginate accounts for 0.01% - 0.1%, polyvinyl alcohol accounts for 0.01% - 0.1%, and the rest is HEPES buffer.

2. The manufacturing method of the self-driven microfluidic chip according to claim 1, wherein In the latex microsphere immobilization method, the process of obtaining the solution after mixing with the immobilization solution includes S21, activating the latex microspheres with the cross-linking agent EDC / NHS at 37°C for 20 min - 40 min, and the mass ratio of the latex microspheres to EDC / NHS is 100:1 - 1:1; S22, adding the activated latex microspheres to the antibody solution and reacting at 37°C for 2 h - 3 h, and the mass ratio of the latex microspheres to the antibody is 20:1 - 5:1; S23, centrifuging and separating the solution after the antibody-microsphere reaction with a refrigerated centrifuge, and after removing the unlabeled antibody, adding HEPES buffer to resuspend the antibody-microsphere conjugate; S24, repeating S23 at least once; S25, centrifuging and separating the solution after the antibody-microsphere reaction with a refrigerated centrifuge again, and after removing the unlabeled antibody, adding the immobilization solution to resuspend the antibody-microsphere conjugate.

3. The manufacturing method of the self-driven microfluidic chip according to claim 2, characterized in that, During the resuspension process of adding the HEPES buffer, by mass percentage of the resuspended solution, the HEPES buffer is added at a ratio of 0.5% of the solid content of the antibody-microsphere conjugate in the mixed solution; during the resuspension process of adding the immobilization solution, by mass percentage of the resuspended solution, the immobilization solution is added at a ratio of 0.1% of the solid content of the antibody-microsphere conjugate in the mixed solution.

4. The manufacturing method of the self-driven microfluidic chip according to any one of claims 1 to 3, characterized in that, In the fluorescent dye sequestration method, the process of obtaining the diluted fluorescent antibody mixed solution includes S31. 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; S32. Remove the excess fluorescent dye by dialysis or purification; S33. Dilute with the first antibody sealing solution to an antibody concentration of 0.2 mg / ml.

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