CN115792246B - Direct antiglobulin microfluidic detection chip card and its application in the detection of red blood cells - Google Patents

Abstract

The invention provides a direct anti-human globulin microfluidic detection chip card and its application in the detection of red blood cells. The chip card includes a negative and a cover. The area is marked with fluorescent probes, the fluorescent probes are respectively labeled with anti-IgG antibody and C3d antibody, and the reaction area is coated with anti-IgG antibody and C3d antibody respectively. The invention saves multi-step dilution, is simple to operate, and has high sensitivity, and can directly obtain accurate quantified results for samples with different intensities, and has higher accuracy.

Description

Direct antiglobulin microfluidic detection chip card and its application in the detection of red blood cells

technical field

The invention belongs to the field of biological detection, in particular to a direct anti-human globulin microfluidic detection chip card and its application in the detection of red blood cells.

Background technique

Antihuman globulin test is a classic method for detecting incomplete red blood cell antibodies, and is an important basis for diagnosing hemolytic disease of the newborn, autoimmune hemolytic disease, and immune hemolytic transfusion reactions. Most of the incomplete antibodies are IgG antibodies, which can bind to the corresponding antigen. Since they can only bind to the determinants of one red blood cell antigen, but cannot connect to the determinant of both red blood cell antigens at the same time, there is no visible reaction under normal conditions. Anti-human globulin antibody acts as a bridge for the second antibody, connecting the specific antibody that binds to the red blood cell antigen to agglutinate red blood cells.

At present, the commonly used antiglobulin detection methods mainly include test tube method, microplate method, and microcolumn gel method. However, existing methods have their limitations. For example, the test tube method needs to judge the degree of agglutination by naked eyes, which is greatly affected by subjective factors, is prone to manual errors, and the results cannot be saved; moreover, red blood cells sensitized by IgG antibodies need to be added to the negative reaction test tube to confirm the negative reaction. Whether the reaction is accurate and reliable, the operation is cumbersome. The micro-column gel method is sensitive to the amount of incomplete antibody sensitized on the surface of red blood cells and the concentration of red blood cells. Therefore, when the amount of incomplete antibody sensitized or the concentration of red blood cells is low, false negatives often occur in the detection In addition, the reagent gel is easy to deform and generate bubbles during transportation, and the temperature will also affect the size of the gel molecular sieve, thereby affecting the stability and reliability of the final test result.

The microfluidic chip card has the advantages of easy operation, short reaction time, accurate diagnostic results and high sensitivity, but if it is applied to the anti-human globulin project, the reaction time will be prolonged, the sensitivity will be reduced and the results will be repeated due to the large red blood cells blocking the fluorescent signal Problems such as bad sex. In view of this, the present invention is committed to developing an anti-human globulin detection chip card with simple operation, high sensitivity, good specificity and stable quality.

Contents of the invention

In view of this, the present invention aims to propose a direct anti-human globulin microfluidic detection chip card to solve the problems of complex detection, insensitive operation, inaccuracy and reliability of test tube method, microplate method and microcolumn gel method.

In order to achieve the above object, technical solution of the present invention is achieved in that way:

Direct anti-globulin microfluidic detection chip card, including a negative film and a cover film. The surface of the negative film is equipped with a sample loading area, a labeling area, and a reaction area. The labeling area is marked with fluorescent probes, which are respectively labeled with anti-IgG antibodies and C3d Antibodies, the reaction area is coated with anti-IgG antibody and C3d antibody respectively.

Further, a drainage column is arranged downstream of the fluorescent probe on the labeling area.

Further, there are multiple rows of drainage columns, and the distance between adjacent rows of drainage columns gradually decreases from 60 μm to 8 μm along the sample flow direction;

Preferably, the distances between adjacent rows of drainage posts are respectively set to 45-55 μm, 25-35 μm, 15-25 μm and 8-12 μm along the sample flow direction.

Further, the fluorescent probe is a fluorescent microsphere or a latex microsphere, and its particle size ranges from 1 μm to 10 μm.

Further, the bottom sheet is also provided with a waste liquid area in the downstream direction of the reaction zone, and the cover sheet is provided with a sample injection hole facing the sample application area, and the bottom of the cover sheet is provided with a microchannel facing the reaction zone position, and the bottom of the cover sheet is directly facing the reaction zone. A waste liquid tank is provided at the position of the waste liquid area, and a ventilation hole is provided at the bottom of the waste liquid tank.

The present invention also provides an application of the direct anti-human globulin microfluidic detection chip card in detecting red blood cells.

Further, detecting red blood cells includes the following steps:

Take a certain amount of packed erythrocytes and erythrocyte chromogenic fluorescent dyes and add them to normal saline to make a suspension. Take the suspension and add it to the sample hole of the chip card. After standing at room temperature for 5-8 minutes, read on the fluorescence detection machine.

Further, the erythrocyte chromogenic fluorescent dyes are Di series dyes.

Further, the mass fraction of packed erythrocytes in the suspension is 2-4%, and the mass fraction of erythrocyte chromogenic fluorescent dye in the suspension is 0.05-0.2%.

Further, the sample volume is 30-40 μL.

Compared with the prior art, the present invention has the following beneficial effects:

1. The red blood cells in the sample are firstly stained with fluorescent dyes, and the red blood cells with fluorescent signals themselves are used as fluorescent signal probes. Compared with the traditional method, it solves the problem that the fluorescent signal is difficult to monitor due to the large volume of red blood cells blocking the fluorescent microspheres. question.

2. Compared with the traditional microfluidic chip technology, the present invention adopts fluorescent microspheres with larger diameters, which improves the ability to capture specific red blood cells and enriches specific red blood cells, which can play a role among multiple specific red blood cells. The bridging function solves the problems of signal occlusion, long reaction time and poor repeatability of results caused by too large red blood cell volume and too small volume of fluorescent microspheres.

3. Two kinds of fluorescent signals, fluorescent dye and fluorescent microsphere, can amplify the signal in two directions, further improving the sensitivity.

4. The structure of the drainage column above the labeling area can promote the full reaction of the red blood cells and the labeled antibody, and at the same time, the distance between adjacent drainage columns is gradually reduced to 10 μm, and finally only a single red blood cell-labeled antibody fluorescent complex is allowed to sequentially through its step-by-step shunting. Orderly flow to the channel reaction area, thereby avoiding aggregation of red blood cells and improving the repeatability and stability of the test results.

5. The fluorescent microsphere-labeled anti-human globulin reagent anti-IgG antibody and C3d antibody form a fluorescent microsphere-antibody conjugate. The anti-IgG antibody and C3d antibody can specifically capture red blood cells and flow in the microchannel, while a unit of Fluorescent microsphere molecules can be combined with multiple anti-IgG antibodies and C3d antibodies, so the sensitivity of specific red blood cell capture can be improved, and specific red blood cells can be specifically captured even when there are a small number of specific red blood cells.

6. The anti-human globulin reagents coated in the reaction area, anti-IgG antibody and C3d antibody can specifically recognize red blood cells and effectively enrich them again.

7. Finally, the coated antibody, specific red blood cells and labeled antibody form antibody-red blood cell-composite fluorescent substances in the reaction area. The high-efficiency capture of labeled antibodies, the specific enrichment of coated antibodies, and the combination of fluorescent probes can quickly develop color and significantly improve the sensitivity of the reagents, effectively avoiding missed detection when the number of specific red blood cells is small.

8. The capture antibody is marked with fluorescent signal microspheres. The red blood cells themselves are also stained with fluorescent dyes before pretreatment to have fluorescent signals. The fluorescent signals can be converted into digital signal values by using instruments. Linear positive correlation, allowing quantification of sample strength. After the two-way amplification of fluorescent microspheres and fluorescent dyes, the final complex formed in the reaction zone will have a stronger fluorescent signal, which is more sensitive than the naked eye, and the results can be objectively judged by reading the fluorescent signal through the instrument. Avoid subjective errors caused by naked eye judgment.

9. At present, the commonly used methods in the market have complicated operation steps. For samples of different strengths, it is necessary to perform multi-step dilution of samples under unknown conditions to obtain rough results, and it takes a long time. The method of the present invention can save multi-step dilution, sample pretreatment is simple, and the required time is short, and the result can be interpreted within 5 minutes after adding the sample. Compared with the traditional method, the operation steps are simple and convenient, and the samples of different intensities can be directly obtained after quantification Accurate results greatly shorten the detection time, avoid manual errors caused by multi-step dilution of samples, have higher accuracy, and truly achieve effective and rapid detection.

Description of drawings

The drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the schematic embodiments and descriptions of the present invention are used to explain the present invention, and do not constitute an improper limitation of the present invention. In the attached picture:

Fig. 1 is a schematic diagram of the film structure described in the embodiment of the present invention;

Fig. 2 is a schematic structural diagram of the cover sheet described in the embodiment of the present invention;

3 is a schematic cross-sectional view of the cover sheet described in the embodiment of the present invention;

Fig. 4 is a schematic diagram of the channel structure described in the embodiment of the present invention;

Fig. 5 is an enlarged effect diagram of the structure of the drainage column described in the embodiment of the present invention.

Explanation of reference signs:

1. Negative film; 11. Sample loading area; 12. Marking area; 121. Drainage column; 13. Reaction area; 14. Waste liquid area; Liquid tank; 24, ventilation hole.

Detailed ways

Unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods, unless otherwise specified, are conventional methods.

As shown in Figures 1 to 5, the direct antiglobulin microfluidic detection chip card of the present invention includes a negative film 1 and a cover film 2, and the surface of the negative film 1 is provided with a sample loading area 11, a marking area 12, and a reaction area 13, The marking area 12 is marked with a fluorescent probe, the fluorescent probe is coated with an anti-human globulin reagent, the reaction area 13 is coated with an anti-human globulin reagent channel, and the marking area 12 is provided with a drainage column 121 downstream of the fluorescent probe , there are multiple rows of drainage columns 121, and the distance between adjacent rows of drainage columns 121 gradually decreases from 60 μm to 8 μm along the sample flow direction. The fluorescent probes are fluorescent microspheres or latex microspheres, and the particle size ranges from 1 μm to 10 μm. The bottom sheet 1 is also provided with a waste liquid area 14 in the downstream direction of the reaction zone 13; The channel 22 and the bottom of the cover sheet 2 are provided with a waste liquid tank 23 at the position facing the waste liquid area 14 , and the bottom of the waste liquid tank 23 is provided with a ventilation hole 24 .

The implementation and beneficial effects of the present invention will be described in detail below through comparative examples and specific examples.

Embodiment 1: Preparation of detection reagent card

1. Antihuman globulin reagent pretreatment

React 1mL of broad-spectrum anti-human globulin reagent (anti-IgG and C3d antibody) with 2mg of EZ-LINK biotin, and dialyze with 0.01M PBS at 4°C overnight, then measure the protein concentration with a UV spectrophotometer . Biotinylated anti-IgG and biotinylated C3d antibodies were formed, respectively.

2. Preparation of reaction solution

Dilute biotinylated anti-IgG and biotinylated C3d antibody with 0.01M PBS to final concentrations of 0.01-10mg/mL and 0.01-10mg/mL respectively as required to form a reaction solution in the reaction zone. In this example, biotinylated The concentrations of the C3d antibody reaction solution and the biotinylated anti-IgG reaction solution were both 1 mg/mL.

3. Preparation of microsphere-erythrocyte monoclonal antibody conjugate

Take 1 mL of latex microspheres, add anti-IgG antibody with a final concentration of 0.1 mg/mL, react at room temperature for 20 min, add casein with a mass concentration of 1% to block for 10 min, centrifuge to get the supernatant, and finally use 0.1 mL of PPBS buffer, store for later use .

Take 1 mL of latex microspheres, add anti-C3d antibody with a final concentration of 0.1 mg/mL, react at room temperature for 20 min, add casein with a mass concentration of 1% to block for 10 min, centrifuge to get the supernatant, and finally use 0.1 mL of PBS buffer, store for later use .

4. Chip fabrication

Take an avidin solution with a final concentration of 5 mg/mL, apply it to the reaction area, incubate at room temperature for 1 h, and wash with 0.01 M PBS. There are two sites in the reaction area, which are detection site 1 and detection site 2 from bottom to top. Spot the biotinylated C3d antibody reaction liquid with a concentration of 1 mg/mL obtained in step 2 on the detection site 1 of the reaction zone, and react the biotinylated anti-IgG with a concentration of 1 mg/mL obtained in step 2 Spot the liquid on the detection site 2 of the reaction area, mix the microsphere-anti-IgG antibody conjugate obtained in step 3 and the microsphere-C3d antibody conjugate at a ratio of 1:1, and take 1 μL Spot the sample on the marked area, and after drying at room temperature, take the cover sheet and press it on the negative sheet.

5. Diluent preparation and packaging

A certain amount of NaCl was dissolved in pure water to prepare a 0.9% NaCl solution, and the diluted solution was dispensed into 1 mL centrifuge tubes, 500 μL per tube, and the Did red blood cell fluorescent dye was dispensed into 0.5 ml centrifuge tubes.

6. Packing and sealing

Put the one-person reagent card in an aluminum foil bag, put in a desiccant, seal it, and store it at 2-8°C.

Test example: comparative study of detection method of the present invention and other antihuman globulin detection methods

The comparison kits used were anti-human globulin (anti-IgG, C3d) detection kit (test tube method) purchased from Shanghai Blood Biomedical Co., Ltd., and anti-human globulin (anti-human globulin (anti-IgG) Anti-IgG, anti-C3d) detection card (micro-column gel method) and the traditional microfluidic chip detection card prepared by our company do not have a drainage column structure.

1. Test tube method: Add 3% erythrocyte suspension into a test tube, wash with normal saline three times, add 2 drops of antihuman globulin, place at room temperature for 5-10 minutes, centrifuge, and observe the results with the naked eye or light microscope.

2. Micro-column gel method: Take a certain amount of packed red blood cells and add them to normal saline to make a 3% suspension, take 50 μL and put them into a micro-column gel card, incubate in a 37°C incubator for 15 minutes, then centrifuge for 5 minutes, and observe the results.

3. Traditional microfluidic method: Take a certain amount of packed red blood cells and add them to normal saline to make a 3% suspension, take 35 μL and add them to the reagent card, and put them at room temperature for 5-8 minutes before reading on the computer. The structure of the microfluidic chip used is a traditional chip structure, and no drainage column structure is set.

4. The method used in the present invention: take a certain amount of packed erythrocytes and add them to normal saline to make a 3% suspension stained with fluorescent dyes, take 35 μL and add them to the reagent card, leave it at room temperature for 5-8 minutes, and then read it on the computer. The microfluidic chip used in the method of the present invention is an innovatively modified chip, and its structure is a step-by-step shunt structure of drainage columns. As shown in Figure 5, the distance between adjacent drainage columns is set to 50 μm, 30 μm, 20μm and 10μm.

5. Use four methods to test 10 positive and 5 negative samples respectively, and record the experimental results.

Table 1 Comparison of four detection methods

Table 2 Comparison of positive rates of four detection methods

Table 3 Fluorescence signal value results of microfluidic method

The result shows: Positive sample 1 is a strong positive sample. After diluting the sample 256 times by the test tube method, IgG can see 78 agglutinations, and C3d can see 78 agglutinations. The signal values read by the traditional microfluidic method are respectively 165948 (strong positive) and 195366 (strong positive), the signal values read by the method of the present invention are 156859 (strong positive) and 256311 (strong positive) respectively.

Positive sample 2 is also a strong positive sample. After diluting the sample 128 times by the test tube method, IgG can see 70 agglutinations, and C3d can see 56 agglutinations. The signal values read by the traditional microfluidic method are 102284 ( Strong positive) and 215129 (strong positive), the read signal values of the method of the present invention are 125541 (strong positive) and 195002 (strong positive) respectively.

Positive sample 3 is a strong positive sample. After diluting the sample 256 times by the test tube method, 76 agglutinations can be seen for IgG, 72 agglutinations can be seen for C3d, and the signal values read by the traditional microfluidic method are 96654 (strong positive) and 115688 (strong positive), the read signal values of the method of the present invention are 163594 (strong positive) and 231688 (strong positive) respectively.

Positive sample 4 is a strong positive sample. Through the test tube method, IgG can see 68 agglutinations when diluted 256 times, and C3d can see 78 agglutinations when diluted 128 times. The signal values read by traditional microfluidic methods are 126843 (strong Positive) and 65537 (moderately positive), the signal values read by the method of the present invention are 168436 (strongly positive) and 186349 (strongly positive) respectively.

Positive sample 5 is a moderately positive sample. After diluting the sample 64 times by the test tube method, IgG can see 56 agglutinations, C3d can see 64 agglutinations, and the signal values read by traditional microfluidic methods are 46337 (middle Positive) and 102678 (strongly positive), the signal values read by the method of the present invention are 88433 (medium positive) and 113699 (medium positive) respectively.

Positive sample 6 is a medium positive sample. After diluting the sample 64 times by the test tube method, 32 agglutinations can be seen for IgG, 64 agglutinations can be seen for C3d, and the signal values read by traditional microfluidic methods are 23684 (weak positive) and 63114 (moderately positive), the signal values read by the method of the present invention are 80322 (moderately positive) and 120059 (moderately positive) respectively.

Positive sample 7 is a moderately positive sample. After diluting the sample 32 times by the test tube method, 28 agglutinations can be seen for IgG, 16 agglutinations can be seen for C3d, and the signal values read by the traditional microfluidic method are 76332 (middle Positive) and 32697 (strongly positive), the signal values read by the method of the present invention are 65664 (medium positive) and 83678 (medium positive) respectively.

Positive sample 8 is a weakly positive sample. After diluting the sample 2 times by the test tube method, 6 agglutinations can be seen for IgG, 6 agglutinations can be seen for C3d, and the signal values read by the traditional microfluidic method are 1005 (negative ) and 2641 (negative), the signal values read by the method of the present invention are 6029 (weak positive) and 10026 (weak positive) respectively.

Positive sample 9 is a weak positive sample. After diluting the sample 4 times by the test tube method, 6 agglutinations can be seen for IgG, and 2 agglutinations can be seen for C3d after dilution 8 times. The signal values read by the traditional microfluidic method are respectively are 2001 (negative) and 1322 (negative), and the read signal values of the method of the present invention are 15266 (weak positive) and 21335 (weak positive) respectively.

Positive sample 10 is a weak positive sample. After diluting the sample 4 times by the test tube method, IgG can see 2 agglutinations, and C3d can see 4 agglutinations. The signal values read by the traditional microfluidic method are 2638 (negative ) and 998 (negative), the signal values read by the method of the present invention are 9988 (weak positive) and 16645 (weak positive) respectively. The detection results of 5 negative samples test tube method, traditional microfluidic method and the method of the present invention are all negative.

The method of the present invention has better consistency and compliance with the test tube method, but the traditional microfluidic method has problems such as positive missed detection, poor compliance, long reaction time and the like. When interpreting the results of the test tube method, first dilute it to a certain multiple, and then count the number of agglutinations. If the number of agglutinations is too large, continue to dilute and then count until it is diluted to a more appropriate multiple to count the number of agglutinations to record the results. The steps to read the results are complicated, and it is easy to cause human error due to subjective judgment. However, the method of the present invention directly outputs results in the form of fluorescence signal values, and the result reading method is simpler, the required reaction time is shorter, and there is no subjective factor of human eyes, which is more accurate.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (4)

1. The direct antiglobulin microfluidic detection chip card is characterized in that: it includes a negative film and a cover film. The surface of the negative film is provided with a sample loading area, a labeling area, and a reaction area. The labeling area is marked with a fluorescent probe, and the fluorescent probes are respectively Anti-IgG antibody and C3d antibody are labeled, and the reaction area is coated with anti-IgG antibody and C3d antibody respectively. The fluorescent probe is fluorescent microsphere or latex microsphere, and its particle size ranges from 1 μm to 10 μm. A drainage column is arranged downstream of the fluorescent probe on the labeling area, There are multiple rows of drainage columns, and the distance between adjacent rows of drainage columns gradually decreases from 60 μm to 8 μm along the direction of sample flow. The application of the direct antiglobulin microfluidic detection chip card to detect red blood cells includes the following steps: Take a certain amount of packed erythrocytes and erythrocyte chromogenic fluorescent dyes and add them into normal saline to make a suspension. Take the suspension and add it to the sample hole of the chip card. After standing at room temperature for 5-8 minutes, read on the fluorescence detection machine. Erythrocyte chromogenic fluorescent dyes are Di series dyes. 2. The direct antiglobulin microfluidic detection chip card according to claim 1, characterized in that: the bottom sheet is also provided with a waste liquid area in the downstream direction of the reaction area, and the cover sheet is provided with a In the sample injection hole, a microchannel is opened at the position facing the reaction zone at the bottom of the cover slip, and a waste liquid tank is opened at the position facing the waste liquid area at the bottom of the cover slip. 3. The direct antiglobulin microfluidic detection chip card according to claim 1, characterized in that: the mass fraction of packed erythrocytes in the suspension is 2-4%, and the mass fraction of erythrocytes in the suspension is chromogenic fluorescent dye The fraction is 0.05-0.2%. 4. The direct antiglobulin microfluidic detection chip card according to claim 1, characterized in that: the sample volume is 30-40 μL.

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