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

Abstract

The present invention provides a numbered capture microsphere and a convenient visualized multiple detection kit and method. A numbered capture microsphere comprises a capture hemisphere and a numbered hemisphere, wherein a cavity is provided in the capture hemisphere, and a weighted microsphere is provided in the numbered hemisphere. The present invention applies the independently developed and designed numbered capture microsphere to a multiple detection kit. The kit has low production cost, simple structure, and is easy to operate. It can complete the detection of multi-component samples in a convenient experiment, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of immunological detection, and relates to a numbered capture microsphere and a convenient visualized multiple detection kit and method. Background Art

[0002] Flow fluorescence technology, also known as liquid chip, suspension array and xMAP technology is a multi-index combined diagnostic technology that has gradually developed in the past 20 years. This technology is based on fluorescent coded microspheres, integrating multiple technologies such as flow principle, laser analysis, and high-speed digital signal processing. It can analyze multiple indicators in parallel and accurately and quantitatively detect up to 2-500 different biological molecules at the same time. It has the characteristics of high throughput, high sensitivity, and parallel detection. This technology is a new generation of high-throughput molecular diagnostic technology platform after biochip technology and chemiluminescence technology. It can be used for immunoassay, nucleic acid research, enzymatic analysis, receptor, ligand recognition analysis and other aspects and fields of research. It is a hot spot in the field of clinical diagnosis and life science research.

[0003] Flow cytometry is the first high-throughput diagnostic technology for clinical applications that has passed FDA certification, and was awarded the "2005 Diagnostic Technology Innovation Award" by Frost & Sullivan, an authority in the global science and technology industry. After the Luminex platform was launched, the market feedback was good, and more and more companies and laboratories are developing reagents and conducting scientific research based on this technology. Its representative products are 100 / 200. However, its expensive machine cost also makes some laboratories discouraged. In my country, many manufacturers have used advanced flow fluorescence technology platforms for the development and production of high-end in vitro diagnostic reagents in the field of clinical testing, such as Transgene, Yishan Biotechnology and Concord. Although the cost of these instruments has been reduced, the equipment structure and operation are relatively complex. At the same time, fluorescently encoded microspheres are a major technical core of flow fluorescence technology. Although some domestic companies have achieved breakthroughs in flow cytometry, there are very few companies that have the ability to independently develop fluorescently encoded microsphere technology. In addition, flow fluorescence technology is complex to operate, and professional equipment and professionals are required to perform complex operations to obtain test results. Summary of the invention

[0004] 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 visualized multiple detection kit and method, and to apply the independently developed and designed numbered capture microspheres to the multiple detection kit. The kit has low production cost, simple structure, and is easy to operate, and has broad application prospects.

[0005] In order to achieve the purpose of the present invention, the technical solution adopted by the present invention is:

[0006] The invention provides a numbered capture microsphere, comprising a capture hemisphere and a numbered hemisphere, wherein a cavity is arranged in the capture hemisphere, and a weighted microsphere is arranged in the numbered hemisphere.

[0007] Preferably, the diameter of the numbered capture microspheres ranges from 1 to 5 mm.

[0008] 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.

[0009] The present invention also provides a convenient visual multiplex detection kit, comprising the above-mentioned numbered capture microspheres.

[0010] Preferably, it further comprises detection microspheres and a detection dish, wherein the detection dish is made of a transparent material and has a width that only allows the numbered capture microspheres to be arranged in a single layer.

[0011] More preferably, the width of the detection dish is 110% to 120% of the diameter of the numbered capture microspheres.

[0012] Preferably, the diameter of the detection microspheres ranges from 20 to 200 nm.

[0013] Preferably, an upper screw cover is provided at the upper end of the detection dish, and a lower screw cover is provided at the lower end, and a filter is provided in the lower screw cover.

[0014] Preferably, the numbering hemisphere is composed of an inert material that does not adsorb proteins and has coded information on its surface, the capturing hemisphere is composed of an active material and has a surface that can adsorb or couple proteins; the detection microsphere is composed of an active material and has a surface that can adsorb or couple proteins.

[0015] The present invention also provides a convenient visualized multiple detection method, which is implemented by the above-mentioned kit, including: adding reaction solution, numbered capture microspheres, samples to be tested and detection microspheres into the detection dish, and mixing; reacting at room temperature; removing the reaction solution, rinsing with clean water, then filling up with clean water and mixing, and taking pictures and reading data after standing.

[0016] The beneficial effects of the present invention are:

[0017] When there is an object to be detected in the sample, it is captured by the corresponding numbered capture microspheres, and then the detection microspheres identify the object to be detected on the numbered capture microspheres, and accumulate and color on their surface. The numbered capture microspheres form a single-layer microsphere arrangement with the numbered hemisphere at the bottom and the capture hemisphere at the top due to their own weight and the size of the detection dish, and then the naked eye observation or photo image analysis is performed. The present invention can complete the detection of multi-component samples in a convenient experiment. The kit has low production cost and simple structure. It can be operated without professional equipment and professional technicians. It is suitable for ordinary people and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the structure of a numbered capture microsphere in the present invention.

[0019] Figure 2 Schematic diagram of the microsphere capture state in an embodiment of the present invention.

[0020] Figure 3 It is a schematic diagram of the detection results of the present invention.

[0021] in:

[0022] 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 antibodies; 4. IgE in samples; 5. Allergen proteins; 6. Test dish; 61. Upper screw cap; 62. Filter; 63. Lower screw cap. DETAILED DESCRIPTION

[0023] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0024] It is to be understood that, unless otherwise specified, the materials and reagents used in the following examples are all existing materials and reagents, and the corresponding reactions can also be achieved by conventional technical means, which will not be described in detail in this application.

[0025] The multiple detection kit provided by the present invention mainly consists of numbered capture microspheres, detection microspheres and detection dishes; the numbered capture microspheres consist of numbered hemispheres (heavier) and capture hemispheres (lighter), the numbered hemispheres have coded information, the capture hemispheres are coated with proteins, and can specifically capture objects to be detected in samples; the detection microspheres are coupled with detection antibodies, and can identify the objects to be detected captured on the numbered capture microspheres; the detection dishes are composed of transparent glass or plastic, and their size only allows the numbered capture microspheres to be arranged in a single layer; when there are objects to be detected in the sample, they are captured by the corresponding numbered capture microspheres, and then the detection microspheres recognize the objects to be detected on the numbered capture microspheres and accumulate and color on their surfaces, and the numbered capture microspheres are arranged in a single layer with the numbered hemispheres at the bottom and the capture hemispheres at the top due to their own counterweight and the size of the detection dish, and then they are observed with the naked eye or analyzed by taking pictures.

[0026] Example

[0027] Preparation and use of allergen-specific IgE antibody detection kit (10 items in inhalation group)

[0028] 1. Preparation of numbered capture microsphere 1: The structure of numbered capture microsphere 1 is as follows Figure 1 As shown, the capture hemisphere 11 is composed of polystyrene material, with a diameter of 3mm, and a hemispherical cavity 12 with a diameter of 1.5mm is provided in the center of the hemisphere; the numbered hemisphere 13 is composed of polypropylene material, with a diameter of 3mm, and an iron bead with a diameter of 1mm is embedded in the bottom of the hemisphere as a weighted microsphere 14; the surface of the numbered hemisphere 13 has three equidistantly spaced 1mm*1mm coding areas, and the coding area has 3*3 squares, each square is black or white, forming 512 codes; the numbered capture microsphere can be suspended in the reaction solution, and the upper half is the capture hemisphere, and the lower half is the numbered hemisphere.

[0029] 2. Numbered capture microsphere 1 coupled with allergen protein 5: 12 different numbered capture microspheres were taken and coated with dust mites, mugwort, common ragweed, cockroaches, cat epithelium, dog epithelium, house dust, Alternaria, Mucor, willow, BSA (blank microsphere), and hemoglobin capture antibody (quality control microsphere); Take numbered microsphere 1 coated with dust mites as an example: 1000 number 1 capture microspheres were taken in a 500mL beaker, washed three times with 200ml 10mM pH 7.4 PBS buffer, 100ml 20mM pH 9.6 sodium bicarbonate buffer was added, 1mg of dust mite allergen was added under magnetic stirring, after magnetic stirring at room temperature for 2 hours, 10ml 10% BSA was added for blocking, magnetic stirring at room temperature for 4 hours, and then washed three times with 200ml 10mM pH 7.4 PBS buffer to obtain dust mite-numbered capture microspheres, such as Figure 2 shown.

[0030] 3. Preparation of detection microspheres: Take 100μl blue latex microspheres 2 and dissolve them in 1ml 10mM pH 7.4 PBS buffer by ultrasonic dispersion for 1 minute, add 10μl 50mg / ml EDC and 10μl 50mg / ml NHSS, mix well, and react at room temperature by shaking for 30min; remove the supernatant by magnetic washing, add 1ml 10mM pH 6.0 PB buffer for re-dissolution, then add 10μl 1mg / ml mouse anti-human IgE monoclonal antibody 3, mix well, and react at room temperature by shaking for 2 hours; add 100μl 10% BSA solution for blocking, and react at room temperature by shaking for 4 hours; magnetically wash the microspheres 3 times, and re-dissolve with 1ml PBST for use.

[0031] 4. Detection dish: The detection dish 6 is made of polycarbonate (PC), has an internal size of 35 mm*3.2 mm*45 mm, is transparent, and is provided with an upper screw cover 61 and a lower screw cover 63 , and a filter screen 62 is also provided in the lower screw cover 63 .

[0032] 5. Kit assembly: unscrew the upper screw cap 61 and add 1 of each of the 12 numbered 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 kit for one person.

[0033] 6. Test kit detection: Press the finger with a disposable peripheral blood collection device, squeeze the finger and use a disposable self-priming quantitative micro-blood collection pipette to draw 10μl of blood; turn the test dish upside down for 1 minute and then add all the blood in the pipette to the test dish; turn the test dish upside down for 1 minute and let it stand on a horizontal table at room temperature for 20 minutes; unscrew the upper screw cap 61 and the lower screw cap 63 to remove the reaction solution, rinse with clean water for 1 minute, then screw on the lower screw cap 63, fill with clean water and screw on the upper screw cap 61, turn it upside down to mix for 1 minute, then place it on a horizontal table and let it stand for 1 minute, open the APP to take a picture and analyze the results (regardless of whether there is analyte in the sample, the blank microspheres do not show color, and the quality control microspheres show color, otherwise the test results are invalid and the cause needs to be analyzed and retested).

[0034] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A convenient visual multiple detection kit, comprising numbered capture microspheres, detection microspheres and a detection dish, wherein the numbered capture microspheres comprise a capture hemisphere and a numbered hemisphere, wherein a cavity is provided in the capture hemisphere, wherein a weighted microsphere is provided in the numbered hemisphere, wherein the diameter range of the numbered capture microspheres is 1 to 5 mm, wherein the density of the numbered hemispheres is greater than the density of the capture hemispheres, wherein the total density of the numbered capture microspheres is ≥1, wherein the detection dish is made of a transparent material and wherein the width only allows a plurality of the numbered capture microspheres to be arranged in a single layer, wherein the width of the detection dish is 110% to 120% of the diameter of the numbered capture microspheres, wherein the numbered hemispheres are composed of an inert material that does not adsorb proteins and wherein the surface contains coding information, wherein the capture hemispheres are composed of an active material and wherein the surface can adsorb or couple proteins; wherein the detection microspheres are composed of an active material and wherein the surface can adsorb or couple proteins, wherein an upper screw cap is provided at the upper end of the detection dish, and a lower screw cap is provided at the lower end, wherein a filter screen is provided in the lower screw cap.

2. A convenient visual multiplex detection kit according to claim 1, characterized in that: The diameter range of the detection microspheres is 20-200 nm.

3. A convenient visual multiplex detection method, implemented by a convenient visual multiplex detection kit according to claim 1, comprising: Add the reaction solution, numbered capture microspheres, the sample to be tested and the detection microspheres into the detection dish, mix well, and react at room temperature; Remove the reaction solution, rinse with clean water, fill up with clean water and mix well, let it stand, then take a photo and read the data.

Images