CN115184598A - A numbered capture microsphere and convenient visual multiplex detection kit and method - Google Patents

Abstract

The invention provides a numbered capture microsphere, a convenient visual multiple detection kit and a method. The invention applies the number capture microspheres which are independently researched and developed to the multiple detection kit, has the advantages of low production cost, simple structure and convenient operation, can complete the detection of the multi-component sample in one convenient experiment, and has wide application prospect.

Description

A numbered capture microsphere and convenient visual multiplex detection kit and method

technical field

The invention belongs to the technical field of immunological detection, and relates to a numbered capture microsphere and a convenient visual multiplex detection kit and method.

Background technique

xMAP技术等，是近20多年逐渐发展起来的多指标联合诊断技术。该技术以荧光编码微球为核心，集流式原理、激光分析、高速数字信号处理等多种技术于一体，多指标并行分析，最多可同时准确定量检测2-500种不同的生物分子，具有高通量、高灵敏度、并行检测等特点。该项技术是继生物芯片技术、化学发光技术之后的新一代高通量分子诊断技术平台，可用于免疫分析、核酸研究、酶学分析、受体、配体识别分析等多方面、多领域的研究，是临床诊断领域及生命科学研究中的一大热点。Flow fluorescence technology, also known as liquid chip, suspended array and

xMAP technology, etc., is a multi-index joint diagnosis technology developed gradually in the past 20 years. The technology is based on fluorescently encoded microspheres and integrates various technologies such as flow principle, laser analysis, high-speed digital signal processing, and multi-index parallel analysis. High throughput, high sensitivity, parallel detection and other characteristics. This technology is a new generation of high-throughput molecular diagnostic technology platform following biochip technology and chemiluminescence technology. Research is a hot spot in the field of clinical diagnosis and life science research.

100/200。但是其昂贵的机器成本，也使得一些实验室望而却步。我国也已有不少厂家将先进的流式荧光技术平台用于临床检验领域高端的体外诊断试剂开发和生产，如国内的透景生命、益善生物及协和洛克等，这些仪器的成本虽然有所降低，但设备结构和操作较为复杂，同时，荧光编码微球作为流式荧光技术的一大技术核心，虽然一些国内企业在流式细胞仪方面实现了突破，但具备自主研发荧光编码微球技术能力的企业却寥寥无几。并且，流式荧光技术操作复杂，需要专业设备及专业人员经过复杂的操作才能得出检测结果。Flow Fluorescence Technology is the first FDA-approved high-throughput diagnostic technology for clinical applications, and was awarded the "2005 Diagnostic Technology Innovation Award" by Frost & Sullivan, an authoritative organization in the global technology industry and industry. After the launch of the Luminex platform, the market has received good feedback, and more and more companies and laboratories are developing reagents and scientific research based on this technology. Its representative products are:

100/200. But its expensive machine cost has also discouraged some laboratories. There are also many manufacturers in my country that have used the advanced flow fluorescence technology platform for the development and production of high-end in vitro diagnostic reagents in the field of clinical testing, such as domestic perspective life, Yishan biological and Xiehe Locke, etc. Although the cost of these instruments is high. However, the structure and operation of the equipment are relatively complicated. At the same time, fluorescence-encoded microspheres are a major technical core of flow fluorescence technology. Although some domestic companies have achieved breakthroughs in flow cytometry, they have the ability to independently develop fluorescent-encoded microspheres. There are very few companies with technical capabilities. In addition, the flow fluorescence technology is complicated to operate, and requires professional equipment and professionals to obtain the test results through complex operations.

SUMMARY OF THE INVENTION

In view of the problems existing in the prior art, the purpose of the present invention is to provide a numbered capture microsphere and a convenient and visualized multiplex detection kit and method. The numbered capture microspheres independently developed and designed are applied to the multiplex detection kit. The production cost is low, the structure is simple, the operation is convenient, and the utility model has wide application prospects.

In order to realize the purpose of the present invention, the technical scheme adopted in the present invention is:

The present invention provides a numbered capture microsphere, comprising a capture hemisphere and a numbered hemisphere, wherein the capture hemisphere is provided with a cavity, and the numbered hemisphere is provided with a counterweight microsphere.

Preferably, the diameter of the numbered capture microspheres ranges from 1 to 5 mm.

Preferably, the density of the numbered hemispheres is greater than the density of the capture hemispheres, and the total density of the numbered capture microspheres is ≥1.

The present invention also provides a convenient visual multiplex detection kit, comprising the above-mentioned numbered capture microspheres.

Preferably, it also includes detection microspheres and a detection dish, the detection dish is made of transparent material and the width only allows the numbered capture microspheres to be arranged in a single layer.

More preferably, the width of the detection dish is 110%-120% of the diameter of the numbered capture microspheres.

Preferably, the diameter range of the detection microspheres is 20-200 nm.

Preferably, the upper end of the detection dish is provided with an upper screw cover, the lower end is provided with a lower screw cover, and a filter screen is arranged in the lower screw cover.

Preferably, the numbered hemisphere is composed of an inert material that does not adsorb proteins and the surface contains coded information, the capture hemisphere is composed of an active material and the surface is capable of adsorbing or coupling proteins; the detection microsphere is composed of an active material and the surface is capable of Adsorbed or coupled proteins.

The present invention also provides a convenient and visual multiple detection method, which is realized by the above-mentioned kit, including: adding a reaction solution, numbered capture microspheres, samples to be tested and detection microspheres into a detection dish, and mixing; reaction at room temperature; removal of the reaction liquid, rinse with clean water, add water and mix well, and then take pictures to read the data after standing.

The beneficial effects of the present invention are:

When there is an object to be detected in the sample, it is captured by the corresponding numbered capture microsphere, and then the detection microsphere identifies the object to be detected on the numbered capture microsphere, and accumulates color on its surface. The numbered capture microsphere is due to its own weight. And the size of the detection dish makes it form the numbered hemisphere on the bottom, the capture hemisphere on the top and the monolayer microsphere arrangement, and then carry out visual observation or photographed image analysis. The invention can complete the detection of multi-component samples in one convenient experiment, the production cost of the kit is low, the structure is simple, it can be operated without professional equipment and professional technicians, it is suitable for ordinary people to use, and has wide application prospects.

Description of drawings

Figure 1 is a schematic structural diagram of a numbered capture microsphere in the present invention.

FIG. 2 is a schematic diagram of the capture state of the microspheres in the embodiment of the present invention.

Figure 3 is a schematic diagram of the detection results of the present invention.

in:

1. Numbered capture microspheres, 11. Capture hemispheres, 12. Cavity, 13. Numbered hemispheres, 14. Weighted microspheres; 2. Blue latex microspheres; 3. Mouse anti-human IgE monoclonal antibody; 4. Samples middle IgE; 5. Allergen protein; 6. Test dish, 61, upper screw cap, 62, filter screen, 63, lower screw cap.

Detailed ways

In order to illustrate the present invention more clearly, the present invention will be further described in detail below with reference to the embodiments and the accompanying drawings. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not limit the protection scope of the present invention.

It can be understood that, unless otherwise specified, the materials and reagents used in the following examples are existing materials and reagents, and their corresponding reactions can also be realized by conventional technical means, which will not be repeated in this application. .

The multiple detection kit provided by the present invention is mainly composed of numbered capture microspheres, detection microspheres and detection dishes; the numbered capture microspheres are composed of numbered hemispheres (heavier) and capture hemispheres (lighter), and the numbered hemispheres have coding information, The capture hemisphere is coated with protein, which can specifically capture the object to be detected in the sample; the detection antibody is coupled to the detection microsphere, which can identify the object to be detected captured on the numbered capture microsphere; the detection dish is composed of transparent glass or plastic, and its The size only allows the numbered capture microspheres to be arranged in a single layer; when there is an object to be detected in the sample, it is captured by the corresponding numbered capture microsphere, and then the detection microsphere identifies the object to be detected on the numbered capture microsphere and accumulates on its surface For color development, the numbered capture microspheres form a numbered hemisphere at the bottom due to its own weight and the size of the detection dish, the capture hemisphere is at the top, and the monolayer microspheres are arranged in a single-layer microsphere, and then carry out visual observation or photographed image analysis.

Example

Preparation and use of allergen-specific IgE antibody detection kit (10 items in inhalation group)

1. Preparation of numbered capture microsphere 1: The structure of numbered capture microsphere 1 is shown in Figure 1, wherein the capture hemisphere 11 is composed of polystyrene material, a hemisphere with a diameter of 3 mm, and a hemispherical cavity 12 with a diameter of 1.5 mm in the center of the hemisphere ; The numbered hemisphere 13 is composed of polypropylene material, a hemisphere with a diameter of 3mm, and the bottom of the hemisphere is embedded with iron beads with a diameter of 1mm as a counterweight microsphere 14; the surface of the numbered hemisphere 13 has 3 uniform 1mm*1mm coding areas at equal distances, and the coding area has 3*3 squares, each square is black or white, forming 512 codes; the numbered capture microspheres can be suspended in the reaction solution, and the upper half is the capture hemisphere, and the lower half is the numbered hemisphere.

2. Numbered capture microspheres 1 coupled to allergen protein 5: 12 types of numbered capture microspheres with different numbers were used to coat dust mites, mugwort, common ragweed, cockroaches, cat epithelium, dog epithelium, house dust, cross-linking Spore mold, mucor, willow, BSA (blank microspheres), hemoglobin capture antibody (quality control microspheres); take the numbered microspheres 1 coated with dust mites as an example: take 1000 number 1 capture microspheres in a 500mL beaker, Wash three times with 200ml 10mM pH 7.4 PBS buffer, add 100ml 20mM pH 9.6 sodium bicarbonate buffer, add 1mg dust mite allergen under magnetic stirring, after 2 hours of magnetic stirring at room temperature, add 10ml 10% BSA to block, room temperature magnetic After stirring for 4 hours, it was washed three times with 200 ml of 10 mM PBS buffer, pH 7.4, to obtain D. dust mite-numbered capture microspheres, as shown in FIG. 2 .

3. Preparation of detection microspheres: take 100 μl of blue latex microspheres 2 to 1 ml of 10mM pH 7.4 PBS buffer and ultrasonically disperse them for 1 minute, add 10 μl of 50 mg/ml EDC and 10 μl of 50 mg/ml NHSS, mix well, and shake at room temperature React for 30 min; remove the supernatant by magnetic washing, add 1 ml of 10 mM PB buffer pH 6.0 for reconstitution, add 10 μl of 1 mg/ml mouse anti-human IgE monoclonal antibody 3, mix well, and shake at room temperature for 2 hours; add 100 μl of 10% The BSA solution was blocked, and the reaction was shaken at room temperature for 4 hours; the microspheres were magnetically washed 3 times and reconstituted with 1 ml of PBST for later use.

4. Testing dish: The testing dish 6 is made of polycarbonate (PC), with an internal size of 35mm*3.2mm*45mm, transparent color, and is provided with an upper screw cover 61 and a lower screw cover 63, and the lower screw cover 63 is also provided with There is a filter 62.

5. Kit assembly: Unscrew the upper screw cap 61 and add one each of the 12 types of capture microspheres in step 2, 1 mL of reaction solution, and 100 μl of the detection microspheres in step 3 into the detection dish 6 to assemble a one-serving kit.

6. Kit detection: press the finger with a disposable peripheral blood collection device, squeeze the finger and draw 10 μl of blood with a disposable self-priming quantitative micro blood collection pipette; invert the test dish for 1 minute and add all the blood in the pipette to the test dish; upside down After 1min, let the test dish stand on a horizontal table for 20min reaction at room temperature; unscrew the upper screw cap 61 and the lower screw cap 63 to remove the reaction solution, rinse with clean water for 1min, screw the lower screw cap 63, fill up with water and screw the upper screw cap 61. Mix it upside down for 1 min, put it on a horizontal table and stand for 1 min, open the APP to take pictures and analyze the results (regardless of whether there is an analyte in the sample, the blank microspheres will not develop color, and the quality control microspheres will develop color, otherwise the test results are invalid and need to be Analyze the cause and retest).

Obviously, the above-mentioned embodiments of the present invention are merely examples for illustrating the present invention more clearly, rather than limiting the embodiments of the present invention. For those of ordinary skill in the art, on the basis of the above descriptions, Changes or changes in other different forms are made, and all implementation methods cannot be exhaustively listed here, and all obvious changes or changes derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims (10)

1. A numbered capture microsphere, comprising a capture hemisphere and a numbered hemisphere, wherein a cavity is provided in the capture hemisphere, and a counterweight microsphere is provided in the numbered hemisphere. 2 . The numbered capture microsphere according to claim 1 , wherein the diameter range of the numbered capture microsphere is 1-5 mm. 3 . 3 . The numbered capture microspheres according to claim 1 , wherein the density of the numbered hemispheres is greater than the density of the capture hemispheres, and the total density of the numbered capture microspheres is greater than or equal to 1. 4 . 4. A convenient and visualized multiplex detection kit, comprising a numbered capture microsphere according to any one of claims 1-3. 5. A convenient and visualized multiplex detection kit according to claim 4, further comprising detection microspheres and a detection dish, the detection dish is made of a transparent material and the width only allows the numbered capture microspheres Arranged in a single layer. 6 . The convenient and visualized multiplex detection kit according to claim 5 , wherein the width of the detection dish is 110% to 120% of the diameter of the numbered capture microspheres. 7 . 7 . The convenient and visualized multiplex detection kit according to claim 5 , wherein the detection microspheres have a diameter range of 20-200 nm. 8 . 8. A convenient and visualized multiple detection kit according to claim 5, wherein the upper end of the detection dish is provided with an upper screw cap, the lower end is provided with a lower screw cap, and the lower screw cap is provided with a filter. network. 9. A convenient and visualized multiplex detection kit according to claim 5, wherein the numbered hemisphere is composed of an inert material that does not adsorb proteins and the surface contains coded information, and the capture hemisphere is composed of an active material and has a surface. Capable of adsorbing or coupling proteins; the detection microspheres are composed of active materials and have surfaces capable of adsorbing or coupling proteins. 10. A convenient visual multiple detection method, realized by a convenient visual multiple detection kit according to claim 5, comprising: adding a reaction solution, numbering capture microspheres, samples to be tested and detection microspheres to the detection dish, Mix well; react at room temperature; remove the reaction solution, rinse with water, add water, mix well, stand and take pictures to read the data.

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