CN114088936A - Fluorescent antibody sealing liquid, preparation method and application - Google Patents
Fluorescent antibody sealing liquid, preparation method and application Download PDFInfo
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- CN114088936A CN114088936A CN202111323989.6A CN202111323989A CN114088936A CN 114088936 A CN114088936 A CN 114088936A CN 202111323989 A CN202111323989 A CN 202111323989A CN 114088936 A CN114088936 A CN 114088936A
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- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 19
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- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 2
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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Abstract
The invention discloses a fluorescent antibody sealing liquid, a preparation method and application, wherein the fluorescent antibody sealing liquid is mainly prepared from 5-20% of trehalose, 0.1-1.0% of BSA, 0.1-1.0% of polyvinylpyrrolidone, 0.1-5.0% of sorbitol and the balance of HEPES buffer solution by mass percent, and is suitable for sealing fluorescent microsphere antibody; when used for sealing the fluorescent dye antibody, the fluorescent dye antibody also comprises 0.05 to 0.1 percent of sodium hyaluronate; the preparation method comprises the steps of preparing 20mmol/L HEPES buffer solution, adjusting the pH value to 7.4-7.6 by using 1mol/L sodium hydroxide, then sequentially adding substances in the formula of the sealing solution, and fully dissolving the substances. The invention has the advantages that the formula of the sealing liquid is simple, the preparation is convenient, the sealed fluorescent antibody can break loose and be bound to flow along with a sample after the marking reaction occurs, and the activity of the antibody can be kept for a long time.
Description
Technical Field
The invention belongs to the technical field of fluorescence immunoassay, and particularly relates to fluorescent antibody encapsulation liquid, a preparation method and application.
Background
The three cardiac muscle indexes are three indexes which are very commonly used in clinical practice of cardiovascular department, namely troponin, myoglobin and creatine kinase isozyme, and have different specificities and sensitivities for cardiac muscle injury and are shown in different stages after the cardiac muscle injury, so that the three indexes have important significance for judging the severity and time of the cardiac muscle injury, are beneficial to finding the cardiac muscle injury in early stage and carrying out treatment intervention in time, and have good guiding significance for treating and recovering judgment in later stage. Myoglobin is an indicator of the earliest change after myocardial injury, and helps to detect myocardial damage early. Troponin has great clinical guiding significance for diagnosis and treatment of myocardial infarction. The creatine kinase isoenzyme is most widely applied in clinic and can be used for routine examination of heart diseases.
At present, the products of two methods, namely colloidal gold and fluorescence chromatography, adopt a double antibody sandwich method to detect substances to be detected in blood samples, and then detect the substances through an instrument to quantitatively analyze the detected substances. The existing product basically adopts an NC membrane as a carrier, the carrier is greatly influenced by the environment, if the temperature and the humidity do not meet the requirements, the NC membrane can cause very obvious influence on the detection result of a sample, so the high environment control requirements are met in the production and storage processes, meanwhile, the quantity of fixed antibodies with large batch difference of the NC membrane is not uniform, and secondly, the problem that fluorescent antibodies are released along with the flow of the sample due to the 3 factors of the NC membrane, a sample pad and a combination pad can be caused by the myocardial triple detection reagent prepared by the NC membrane, so the detection result can be influenced, and the product repeatability CV can be greatly caused. In addition, the three myocardial joint tests combine 3 detection items of three myocardial CKMB, CTNI and Myo on the same detection reagent card, so that the problems of mutual interference among the items, low detection sensitivity, poor repeatability CV and the like exist. To this end, the applicant invented a self-driven microfluidic chip and filed a patent application to the national intellectual property office on 17.01.2020, which was published on 28.4.2020 with publication number CN 111068801A. The self-driven micro-fluidic chip prestores the antibody corresponding to the substance to be detected on the self-driven micro-fluidic chip, and the sample can form a double-antibody sandwich through recognition reaction and immunoreaction respectively. The micro-flow channel formed by a single pure material has the advantages of ensuring the reliability and controllability of a sample detection result through accurate micro-flow control, and is expected to replace the immunochromatography (LIFCS) technology of various membrane materials commonly used in clinic at present, so that a new technical idea is provided for solving the challenges faced by the POCT detection technology at present. Then, the applicant submits a patent application of a rapid immunofluorescence detection method based on a self-driven microfluidic chip to the national intellectual property office in 5/22/2020, wherein the application is published in 21/8/2020, and the publication number is CN 111562380A; the method discloses the whole process of using the self-driven micro-fluidic chip to carry out rapid immunofluorescence detection.
In the manufacturing process of the self-driven micro-fluidic chip, one antibody marked with fluorescent substances needs to be sealed in a mixing area, the other antibody is fixed on a micro-column of a chip detection area, so that in the process that a detected sample flows in the chip, after the detected sample and fluorescent antibodies in the mixing area perform recognition reaction, antibodies carrying fluorescent markers perform immunoreaction with the antibodies on the micro-column of the detection area, and therefore double-antibody sandwich markers retained on the micro-column are formed, and a detection result is obtained by the fluorescent detector through the fluorescent marker detection of the double-antibody sandwich markers. Therefore, the antibody labeled with the fluorescent substance is sealed in the mixed region, so that the fluorescent antibody can completely break loose from the bound mixed region after the recognition reaction occurs, and the antibody can keep activity for a set period of time is the basis for obtaining an accurate detection result. At present, no document in the prior art discloses a related art capable of achieving the aforementioned object.
Disclosure of Invention
The first purpose of the present invention is to provide a fluorescent antibody sealing liquid for sealing a fluorescent antibody in a mixed region of a self-driven microfluidic chip against the shortage of an antibody sealing technology in the preparation process of the existing self-driven microfluidic chip, and in the process of a sample to be detected flowing in the chip, after a recognition reaction with the fluorescent antibody in the mixed region occurs, an antibody carrying a fluorescent marker breaks away from the mixed region completely, and then an immunoreaction with an antibody on a microcolumn occurs, a double antibody sandwich marker is formed, so that a detection result is obtained by a fluorescence detector through the detection of the fluorescent marker of the double antibody sandwich marker. The second purpose of the invention is to provide a preparation method based on the sealing liquid, so as to obtain the fluorescent antibody sealing liquid meeting the requirements. The third purpose of the invention is to provide the application of the antibody encapsulating solution for the self-driven microfluidic chip.
In order to achieve the first object, the invention adopts the following technical scheme.
A fluorescent antibody sealing solution is mainly prepared from trehalose, BSA, polyvinylpyrrolidone and sorbitol through HEPES buffer solution; wherein, according to the mass percentage, the trehalose: 5% -20%, BSA: 0.1% -1.0%, polyvinylpyrrolidone: 0.1% -1.0%, sorbitol: 0.1-5.0 percent of buffer solution and the balance of HEPES buffer solution.
According to the invention adopting the technical scheme, the fluorescent microsphere antibody can be sealed in the mixing region of the self-driven microfluidic chip by using the colloid component in the composition, and the antibody can keep activity in a set period by using nutritional components after being dried, so that in the process that a detected sample flows in the chip, after the fluorescent microsphere antibody and the fluorescent antibody in the mixing region are subjected to recognition reaction, the antibody carrying the fluorescent microsphere can break loose the constraint of the mixing region, and then automatically flows into the detection region to be subjected to immunoreaction with the antibody on the microcolumn, a double-antibody sandwich marker is formed, and thus a detection result is obtained by a fluorescence detector through the fluorescence marker detection of the double-antibody sandwich marker. The drying after the fluorescent microsphere antibody is sealed is naturally performed under the condition that the environmental requirement of a 10 ten thousand grade clean area is met; the environment temperature is 25 ℃, and the humidity is constant humidity and constant temperature condition of 50-60%.
Preferably, the composition also comprises sodium hyaluronate, wherein the mass percentage of the sodium hyaluronate: 0.05 to 0.1 percent. And the sealing liquid with the sodium hyaluronate is used for sealing the antibody marked with the fluorescent dye through chemical reaction in a mixing area of the self-driven microfluidic chip. And the antibody keeps activity within a set period by utilizing nutrient components after being dried, so that in the process that a sample to be detected flows in a chip, after the sample and the fluorescent antibody in the mixed region have identification reaction, the antibody carrying fluorescent dye can break loose the constraint of the mixed region, and then automatically flows into a detection region to have immunoreaction with the antibody on the microcolumn, so that the double-antibody sandwich marker is formed, and a detection result is obtained by a fluorescence detector through the fluorescence labeling detection of the double-antibody sandwich marker. The air drying after the fluorescent dye antibody is sealed is carried out naturally under the condition that the environmental requirement of a 10 ten thousand grade clean area is met; the environment temperature is 25 ℃, and the humidity is constant humidity and constant temperature condition of 50-60%.
In order to obtain better sealing effect, the sealing liquid for sealing the fluorescent dye antibody comprises the following components in percentage by mass: 5% -10%, BSA: 0.5% -1.0%, polyvinylpyrrolidone: 0.5% -1.0%, sorbitol: 0.5% -1.0%, sodium hyaluronate: 0.05 to 0.1 percent of buffer solution and the balance of HEPES buffer solution.
In order to obtain a better sealing effect, the sealing liquid for sealing the fluorescent microsphere antibody comprises the following components in percentage by mass: 5% -10%, BSA: 0.5% -1.0%, polyvinylpyrrolidone: 0.5% -1.0%, sorbitol: 0.5-1.0 percent of buffer solution and the balance of HEPES buffer solution.
In order to achieve the second object, the invention adopts the following technical scheme.
A preparation method of fluorescent antibody sealing liquid comprises the following steps:
firstly, preparing 20mmol/L HEPES buffer solution;
secondly, adjusting the pH value to 7.4-7.6 by using 1mol/L sodium hydroxide;
and thirdly, sequentially adding the other components of the fluorescent antibody encapsulating solution for realizing the first purpose, and fully dissolving.
According to the preparation method of the fluorescent antibody sealing solution in the technical scheme, trehalose, BSA, polyvinylpyrrolidone and sorbitol are added in sequence according to corresponding mass percent, and the sealing solution for sealing the fluorescent microsphere antibody is obtained after the trehalose, the BSA, the polyvinylpyrrolidone and the sorbitol are fully dissolved; trehalose, BSA, polyvinylpyrrolidone, sorbitol and sodium hyaluronate are added in sequence according to the corresponding mass percentage, and the mixture is fully dissolved to obtain the sealing liquid for sealing the fluorescent dye antibody. The operation method is simple, the two sealing solutions obtained by the method can be used for sealing antibodies in different fluorescence labeling modes, and the sealing effect is good.
In order to achieve the third object, the invention adopts the following technical scheme.
The fluorescent antibody encapsulating solution prepared from trehalose, BSA, polyvinylpyrrolidone and sorbitol through HEPES buffer solution and obtained by the preparation method for realizing the second purpose is used for diluting the activated fluorescent microsphere antibody and then encapsulating the diluted fluorescent microsphere antibody in a mixing area of a self-driven microfluidic chip for myocardial detection. And the antibody keeps activity within a set period by utilizing nutrient components after being dried, so that in the process that a sample to be detected flows in the chip, after the sample and the fluorescent antibody in the mixed region have identification reaction, the antibody carrying the fluorescent microspheres can break loose the constraint of the mixed region, and then automatically flows into the detection region to have immunoreaction with the antibody on the microcolumn, so that the double-antibody sandwich marker is formed, and a detection result is obtained by the fluorescent detector through the fluorescent label detection of the double-antibody sandwich marker. The drying after the fluorescent microsphere antibody is sealed is naturally performed under the condition that the environmental requirement of a 10 ten thousand grade clean area is met; the environment temperature is 25 ℃, and the humidity is constant humidity and constant temperature condition of 50-60%.
In order to obtain better sealing effect, the method also comprises the following steps,
firstly, activating fluorescent microspheres by using a cross-linking agent EDC/NHS for 20-40 min at 37 ℃, wherein the mass ratio of the fluorescent microspheres to the EDC/NHS is 100: 1-1: 1;
secondly, adding the activated fluorescent microspheres into an antibody solution, and reacting for 2-3 h at 37 ℃, wherein the mass ratio of the fluorescent microspheres to the antibody is 20: 1-5: 1;
thirdly, centrifugally separating the solution after the antibody fluorescent microsphere reaction by using a refrigerated centrifuge, and adding HEPES buffer solution to resuspend the antibody microsphere conjugate after removing the unlabeled antibody;
fourthly, repeatedly executing the third step at least once;
and fifthly, centrifuging the solution after the antibody microsphere reaction by using a refrigerated centrifuge, and adding the fluorescent antibody sealing solution to resuspend the antibody microsphere conjugate after removing the unlabeled antibody microspheres.
In order to further obtain a better sealing effect, in the process of adding the HEPES buffer solution for resuspension, the HEPES buffer solution is added according to the proportion that the solid content of the fluorescent antibody microsphere conjugate in the mixed solution is 0.5 percent in terms of mass percentage after resuspension; in the process of adding the fluorescent antibody sealing solution for resuspension, the fluorescent antibody sealing solution is added according to the proportion that the solid content of the fluorescent antibody microsphere conjugate in the mixed solution is 0.2 percent in terms of mass percentage after resuspension. The resuspension is carried out by an ultrasonic energizing means, namely, an ultrasonic cleaning head is extended into the mixed liquid to ensure that the antibody microspheres are in a resuspension state.
And the fluorescent antibody sealing solution prepared from trehalose, BSA, polyvinylpyrrolidone, sorbitol and sodium hyaluronate through a HEPES buffer solution and obtained by the preparation method for realizing the second purpose is used for sealing the activated fluorescent dye antibody in a mixing area of the self-driven microfluidic chip for myocardial detection. And the antibody keeps activity within a set period by utilizing nutrient components after being dried, so that in the process that the sample to be detected flows in the chip, after the sample and the fluorescent antibody in the mixed region have identification reaction, the antibody carrying fluorescent dye can break loose the constraint of the mixed region, and then automatically flows into the detection region to have immunoreaction with the antibody on the microcolumn, so that the double-antibody sandwich marker is formed, and the fluorescence detector obtains a detection result through the fluorescence label detection of the double-antibody sandwich marker. The air drying after the fluorescent dye antibody is sealed is carried out naturally under the condition that the environmental requirement of a 10 ten thousand grade clean area is met; the environment temperature is 25 ℃, and the humidity is constant humidity and constant temperature condition of 50-60%.
In order to obtain better sealing effect, the method also comprises the following steps,
firstly, mixing a fluorescent dye and an antibody according to the mass ratio of 1: 1-1: 10, and reacting for 20-40 min at 37 ℃;
secondly, removing redundant fluorescent dye by dialysis or purification;
thirdly, the solution was diluted with the aforementioned fluorescent antibody-blocking solution to an antibody concentration of 0.2 mg/ml.
The invention has the advantages that the two fluorescent antibody sealing solutions can respectively seal the fluorescent microsphere antibody and the fluorochrome antibody on the chip, and the antibodies have long-term activity, and the sealed fluorescent antibodies can break loose and be bound to flow along with a sample after the labeled reaction; the preparation method of the sealing liquid is simple, and the sealing liquid has strong breaking-off capability after the identification reaction, long retention time of the activity of the antibody and good corresponding sealing effect through detection and verification. When the self-driven micro-fluidic chip is used for the routine examination of heart diseases, the detection efficiency and the detection precision are high.
Drawings
Fig. 1 is a schematic structural diagram of a self-driven microfluidic chip using the fluorescent antibody sealing solution of the present invention.
Detailed Description
The present invention is further described with reference to the accompanying drawings, but the invention is not limited thereby within the scope of the described embodiments.
Example 1, referring to fig. 1, a fluorescent antibody-encapsulating solution, which is used in a self-driven microfluidic chip for routine examination of heart diseases, is prepared by encapsulating a fluorescent microsphere antibody in a mixing region 3 and preparing trehalose, BSA, polyvinylpyrrolidone and sorbitol by a HEPES buffer solution; wherein, according to the mass percentage, the trehalose: 5% -20%, BSA: 0.1% -1.0%, polyvinylpyrrolidone: 0.1% -1.0%, sorbitol: 0.1-5.0 percent of buffer solution and the balance of HEPES buffer solution.
As a preferable scheme, the weight percentage of the components is as follows: 5% -10%, BSA: 0.5% -1.0%, polyvinylpyrrolidone: 0.5% -1.0%, sorbitol: 0.5-1.0 percent of buffer solution and the balance of HEPES buffer solution.
Example 2, referring to fig. 1, a fluorescent antibody-encapsulating solution, which is used in a self-driven microfluidic chip for routine examination of heart diseases, is prepared by encapsulating a fluorescent dye antibody in a mixing region 3 and preparing trehalose, BSA, polyvinylpyrrolidone, sorbitol, and sodium hyaluronate with a HEPES buffer; wherein, according to the mass percentage, the trehalose: 5% -20%, BSA: 0.1% -1.0%, polyvinylpyrrolidone: 0.1% -1.0%, sorbitol: 0.1% -5.0%, sodium hyaluronate: 0.05 to 0.1 percent of buffer solution and the balance of HEPES buffer solution.
As a preferable scheme, the components are calculated according to mass percent, and the ratio of trehalose: 5% -10%, BSA: 0.5% -1.0%, polyvinylpyrrolidone: 0.5% -1.0%, sorbitol: 0.5% -1.0%, sodium hyaluronate: 0.05 to 0.1 percent of buffer solution and the balance of HEPES buffer solution.
firstly, preparing 20mmol/L HEPES buffer solution;
secondly, adjusting the pH value to 7.4-7.6 by using 1mol/L sodium hydroxide;
and a third step of adding the remaining components constituting the fluorescent antibody-encapsulating solution of example 1 or example 2 in order and dissolving them sufficiently.
Example 4 use of a fluorescent antibody blocking solution, the fluorescent antibody blocking solution of example 1 prepared from trehalose, BSA, polyvinylpyrrolidone and sorbitol in HEPES buffer according to the preparation method of example 3, to dilute the activated fluorescent microsphere antibody and then block it in the mixing region 3 of the self-driven microfluidic chip for myocardial detection. And the antibody keeps activity within a set period by utilizing nutrient components after being dried, so that in the process that the sample to be detected flows in the chip, after the sample and the fluorescent antibody in the mixing region 3 are subjected to identification reaction, the antibody carrying the fluorescent microspheres can break loose the constraint of the mixing region, and then automatically flows into the detection region 5 to be subjected to immunoreaction with the antibody on the microcolumn, so that the double-antibody sandwich marker is formed, and a detection result is obtained by a fluorescence detector through the fluorescence label detection of the double-antibody sandwich marker. The drying after the fluorescent microsphere antibody is sealed is naturally performed under the condition that the environmental requirement of a 10 ten thousand grade clean area is met; the environment temperature is 25 ℃, and the humidity is constant humidity and constant temperature condition of 50-60%.
In order to obtain better results, it is also included,
firstly, activating fluorescent microspheres by using a cross-linking agent EDC/NHS for 20-40 min at 37 ℃, wherein the mass ratio of the fluorescent microspheres to the EDC/NHS is 100: 1-1: 1;
secondly, adding the activated fluorescent microspheres into an antibody solution, and reacting for 2-3 h at 37 ℃, wherein the mass ratio of the fluorescent microspheres to the antibody is 20: 1-5: 1;
thirdly, centrifugally separating the solution after the antibody fluorescent microsphere reaction by using a refrigerated centrifuge, and adding HEPES buffer solution to resuspend the antibody microsphere conjugate after removing the unlabeled antibody;
fourthly, repeatedly executing the third step at least once;
and fifthly, centrifuging the solution after the antibody microsphere reaction by using a refrigerated centrifuge, and adding the fluorescent antibody sealing solution to resuspend the antibody microsphere conjugate after removing the unlabeled antibody microspheres.
During the resuspension process of adding the HEPES buffer solution, adding the HEPES buffer solution according to the proportion that the solid content of the fluorescent antibody microsphere conjugate in the mixed solution is 0.5 percent in terms of mass percentage after resuspension; in the process of adding the fluorescent antibody sealing solution for resuspension, the fluorescent antibody sealing solution is added according to the proportion that the solid content of the fluorescent antibody microsphere conjugate in the mixed solution is 0.2 percent in terms of mass percentage after resuspension. The resuspension is carried out by an ultrasonic energizing means, namely, an ultrasonic cleaning head is extended into the mixed liquid to ensure that the antibody microspheres are in a resuspension state.
Example 5 use of a fluorescent antibody blocking solution prepared from trehalose, BSA, polyvinylpyrrolidone, sorbitol, and sodium hyaluronate in HEPES buffer, obtained according to the preparation method of example 3, to block activated fluorescent dye antibody in the mixing zone 3 of a self-driven microfluidic chip for myocardial detection. And the antibody keeps activity within a set period by utilizing nutrient components after being dried, so that in the process that the sample to be detected flows in the chip, after the sample and the fluorescent antibody in the mixed region 3 are subjected to identification reaction, the antibody carrying fluorescent dye can break loose the constraint of the mixed region 3, and then automatically flows into the detection region 5 to be subjected to immunoreaction with the antibody on the microcolumn, so that a double-antibody sandwich marker is formed, and a detection result is obtained by a fluorescence detector through the fluorescence label detection of the double-antibody sandwich marker. The air drying after the fluorescent dye antibody is sealed is carried out naturally under the condition that the environmental requirement of a 10 ten thousand grade clean area is met; the environment temperature is 25 ℃, and the humidity is constant humidity and constant temperature condition of 50-60%.
In order to obtain better sealing effect, the method also comprises the following steps,
firstly, mixing a fluorescent dye and an antibody according to the mass ratio of 1: 1-1: 10, and reacting for 20-40 min at 37 ℃;
secondly, removing redundant fluorescent dye by dialysis or purification;
thirdly, the solution was diluted with the aforementioned fluorescent antibody-blocking solution to an antibody concentration of 0.2 mg/ml.
According to the requirement of the fluorescent antibody blocking, the blocked fluorescent antibody completely flows away along with the detection sample without residue, and simultaneously, the activity of the antibody is not influenced. The applicant verifies the two sealing liquids through the following tests, and obtains experimental data after the tests are carried out by a fluorescence analyzer, wherein table 1 is a test data record of the first sealing liquid for sealing the fluorescent dye antibody under different proportioning conditions, and in the table, the residual test of the fluorescent antibody is the break-off capability after the fluorescent antibody and a sample have identification reaction, and the break-off capability is inversely related to a residual value, namely, the stronger the break-off capability is, the less the residue is, and vice versa; wherein "+" indicates residue, "+ + + + +" indicates significant residue, "+ + + + + +" indicates severe residue, "-" indicates no residue; antibody activity was measured by fluorescence signal intensity measurements at 37 ℃ for 30 days, and was characterized by signal decay indicators, where "+" indicates a decay, "+ + + +" indicates a significant decay, and "+ + + +" indicates a severe decay "-" indicates no decay. Table 2 is a test data record of the second encapsulation liquid for encapsulation of the fluorescent microsphere antibody under different mixture ratios, in the table, the fluorescent antibody residue is tested by the break-away ability of the fluorescent antibody after the fluorescent antibody and the sample have identification reaction, and the break-away ability is inversely related to the residue value, i.e. the stronger the break-away ability is, the less the residue is, and vice versa; wherein "+" indicates residue, "+ + + + +" indicates significant residue, "+ + + + + +" indicates severe residue, and "-" indicates no residue; antibody activity was measured by signal decay after 1 month of accelerated aging at 37℃, where "+" indicates decay, "+ + +" indicates significant decay, and "+ + + +" indicates severe decay. Wherein the fluorescence signal intensity unit is CPS.
TABLE 1 test data record for first confining liquid at different ratios of solids
The percentages listed in table 1 are mass percentages of solids, with the remainder being HEPES buffer.
TABLE 2 test data record for the second confining liquid at different ratios of solids
The percentages listed in table 2 are mass percentages of solids, with the remainder being HEPES buffer.
In the evaluation of the activity of the blocked antibodies of the first and second blocking solutions, the measurement of the intensity of the relevant fluorescence signal was carried out at intervals during the course of 30 days; the specific test data are respectively shown in tables 3 and 4, and the table 3 shows the test result of the interruption time of the corresponding group of the first sealing liquid; table 4 shows the results of the intermittent time tests of the corresponding group of the second sealing solution.
TABLE 3 recording of the results of the intermittent time test of the corresponding group of the first sealing solution
TABLE 4 recording of the results of the intermittent time test for the corresponding group of the second sealing solution
In tables 3 and 4, the rate of change of the signal intensity is the percentage of the difference between the signal detection value at the time of 30 days interval and the detection on the date of sequestration relative to the signal detection value on the day, and the negative number is attenuation and the positive number is enhancement, and theoretically, enhancement does not occur, but a slight enhancement of the signal value is acceptable in consideration of the detection error. The evaluation criterion is that no attenuation is defined as the absolute value of the signal change rate is less than or equal to 10 percent, and is marked as "-"; greater than 10% and less than or equal to 20% are attenuated, marked as "+"; greater than 20% and less than or equal to 50% is significant attenuation, denoted as "+"; greater than 50% is severely attenuated, which is marked as "+++".
Embodiment 6, a self-driven microfluidic chip, comprising a mixing region 3 and a detection region 5, wherein a fluorescent antibody is sealed in the mixing region 3, and the detection region 5 is fixed with an immobilized antibody through a microcolumn on a micro channel of a chip bottom plate; wherein, the fluorescent antibody is sealed by the fluorescent antibody sealing liquid prepared in the example 1 or 2 and prepared by the method described in the example 3. Wherein, when the antibody sealed in the mixed region 3 is a fluorescent microsphere antibody, the fluorescent antibody sealing solution of the embodiment 1 is adopted; when the antibody to be blocked in the mixed region 3 is a fluorochrome antibody, the fluorescent antibody blocking solution of example 2 is used.
The chip is used for the three-phase combined examination of the cardiac muscle, and comprises a chip bottom plate as shown in figure 1, wherein a sample adding area 1, a blood filtering area 2, a mixing area 3, a speed limiting area 4 and a detection area 5 are sequentially arranged on the chip bottom plate, the speed limiting area 4 is provided with a time control valve so as to slow down the slow flowing speed of a sample to be detected in the speed limiting area 4, prolong the detention time of the sample to be detected in the mixing area 3 and increase the reaction time of the sample to be detected and a reagent in the mixing area 3; three fluorescent antibodies are sealed in the mixing region 3, wherein the three fluorescent antibodies are respectively a CKMB antibody, a CTNI antibody and a Myo antibody, the three antibodies are sequentially fixed in the detection region 5 through micro-columns on a micro-channel of a chip base plate, and the three fixed antibodies are respectively a CKMB antibody, a CTNI antibody and a Myo antibody.
When the double-antibody sandwich detection marker is used, a blood sample of detected whole blood, serum or plasma is injected into the sample injection region 1 through a straw, the sample automatically enters the blood filtration region 2 for filtration according to the direction indicated by an arrow A by virtue of the capillary principle, then enters the mixing region 3 for carrying out recognition reaction with a fluorescent antibody, the fluorescent antibody is bound with the blood sample and then flows along with the blood sample, and then enters the detection region 5, and the double-antibody sandwich detection marker is formed after the fluorescent antibody is bound with the fixed antibody. And finally, detecting the signal intensity of the marker by a fluorometer to obtain a detection result. Wherein, be equipped with zero value district and matter accuse district in the detection zone, matter accuse is as the judgement basis of detection card inefficacy and detection time, and the zero value point is as background deduction.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A fluorescent antibody sealing solution is characterized by being mainly prepared from trehalose, BSA, polyvinylpyrrolidone and sorbitol through an HEPES buffer solution; wherein, according to the mass percentage, the trehalose: 5% -20%, BSA: 0.1% -1.0%, polyvinylpyrrolidone: 0.1% -1.0%, sorbitol: 0.1-5.0 percent of buffer solution and the balance of HEPES buffer solution.
2. The fluorescent antibody-containing sealant solution according to claim 1, further comprising sodium hyaluronate, wherein the ratio of sodium hyaluronate: 0.05 to 0.1 percent.
3. The fluorescent antibody-encapsulating solution according to claim 2, wherein the ratio of trehalose: 5% -10%, BSA: 0.5% -1.0%, polyvinylpyrrolidone: 0.5% -1.0%, sorbitol: 0.5% -1.0%, sodium hyaluronate: 0.05 to 0.1 percent of buffer solution and the balance of HEPES buffer solution.
4. The fluorescent antibody-encapsulating solution according to claim 1, wherein the ratio of trehalose: 5% -10%, BSA: 0.5% -1.0%, polyvinylpyrrolidone: 0.5% -1.0%, sorbitol: 0.5-1.0 percent of buffer solution and the balance of HEPES buffer solution.
5. A preparation method of fluorescent antibody sealing liquid is characterized by comprising the following steps:
firstly, preparing 20mmol/L HEPES buffer solution;
secondly, adjusting the pH value to 7.4-7.6 by using 1mol/L sodium hydroxide;
and a third step of adding the remaining components constituting the fluorescent antibody-encapsulating solution according to any one of claims 1 to 4 in order and dissolving them sufficiently.
6. The use of the fluorescent antibody-containing solution according to claim 1 or 4 obtained by the preparation method of claim 5 in a mixing region of a self-driven microfluidic chip for myocardial detection after dilution of the activated fluorescent microsphere antibody.
7. The use according to claim 6, further comprising the step of,
firstly, activating fluorescent microspheres by using a cross-linking agent EDC/NHS for 20-40 min at 37 ℃, wherein the mass ratio of the fluorescent microspheres to the EDC/NHS is 100: 1-1: 1;
secondly, adding the activated fluorescent microspheres into an antibody solution, and reacting for 2-3 h at 37 ℃, wherein the mass ratio of the fluorescent microspheres to the antibody is 20: 1-5: 1;
thirdly, centrifugally separating the solution after the antibody fluorescent microsphere reaction by using a refrigerated centrifuge, and adding HEPES buffer solution to resuspend the antibody microsphere conjugate after removing the unlabeled antibody;
fourthly, repeatedly executing the third step at least once;
and fifthly, centrifuging the solution after the antibody microsphere reaction by using a refrigerated centrifuge, and adding the fluorescent antibody sealing solution of claim 1 or 4 to resuspend the antibody microsphere conjugate after removing the unlabeled antibody microspheres.
8. The use according to claim 7, wherein during the resuspension process, the HEPES buffer solution is added in a proportion that the solid content of the fluorescent antibody microsphere conjugate in the mixed solution is 0.5% in terms of mass percentage after resuspension; in the process of adding the fluorescent antibody sealing solution for resuspension, the fluorescent antibody sealing solution is added according to the proportion that the solid content of the fluorescent antibody microsphere conjugate in the mixed solution is 0.2 percent in terms of mass percentage after resuspension.
9. Use of the fluorescent antibody-containing solution according to claim 2 or 3 obtained by the method according to claim 5 for diluting the activated fluorescent dye antibody and storing the diluted fluorescent dye antibody in a mixing region of a self-driven microfluidic chip for myocardial detection.
10. The use according to claim 9, further comprising,
firstly, mixing a fluorescent dye and an antibody according to the mass ratio of 1: 1-1: 10, and reacting for 20-40 min at 37 ℃;
secondly, removing redundant fluorescent dye by dialysis or purification;
in a third step, the antibody is diluted to a concentration of 0.2mg/ml with the blocking solution of claim 2 or 3.
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