Integrated ELISA (Enzyme-Linked Immuno Sorbent Assay) chip based on distance detection targets and detection method thereof

A distance detection and target technology, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of complex immune response operation, high cost of experimental methods, expensive equipment and other problems, and achieve reliable detection results, high sensitivity, and reaction process. simple effect

Inactive Publication Date: 2017-05-31
XIAMEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the shortcomings of the existing high-sensitivity quantitative detection and analysis method and its instruments and equipment, high c

Method used

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  • Integrated ELISA (Enzyme-Linked Immuno Sorbent Assay) chip based on distance detection targets and detection method thereof
  • Integrated ELISA (Enzyme-Linked Immuno Sorbent Assay) chip based on distance detection targets and detection method thereof
  • Integrated ELISA (Enzyme-Linked Immuno Sorbent Assay) chip based on distance detection targets and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Fabrication of chips

[0037] According to the chip design of Auto CAD in the computer, the required microstructures and microchannels are produced by laser etching on the PMMA material. The middle layer contains 11 circular holes, 9 elliptical holes, and air column channels. The upper layer is the water phase addition channel and the sample injection hole, and the bottom layer is the oil phase addition channel. The thickness of the three-layer chip is 2.2mm. The light-cut PMMA sheet is used to bond the three layers together by means of hot keys and summation. In order to prevent capillary action, the bonded chip channel was incubated with fluorine oil, and after the fluorine oil evaporated overnight, the chip was ready for use.

[0038] Specifically, see Figure 13 , the chip includes an upper layer 1, a middle layer 2 and a lower layer 3, wherein the middle layer 2 is provided with a row of cavities alternately arranged by circular holes and elliptical h...

Embodiment 2

[0043] Example 2: Synthesis and purification of nucleic acid molecules modified with biotin groups and sulfhydryl groups

[0044] Using ordinary CPG as a solid phase carrier, and using DNA monomer bases as raw materials, the sequence DNAs in Table 1 were synthesized from the 3' end to the 5' end on a DNA synthesizer. The specific synthesized sequences are shown in Table 1. Biotin-modified CPG is used as a solid-phase carrier, and DNA monomer bases are used as raw materials to synthesize from the 3' end to the 5' end on a DNA synthesizer, and finally the 5' end is modified to modify the sulfhydryl group. After the synthesis, the above-mentioned CPG was transferred to a 2 mL clean and sterilized Eppendorf tube, 0.5 mL of methylamine: ammonia water = 1:1 solution was added, and the DNA was cleaved from the CPG at 65° C. for 30 min by ammonolysis. After the aminolysis, the supernatant was extracted, and the CPG was washed with a small amount of ultrapure water, and the supernatant...

Embodiment 3

[0048] Example 3: Synthesis and modification of PtNPs

[0049] 100 μL of freshly prepared 0.4M ascorbic acid solution was added to 1 mL of chloroplatinic acid solution with a concentration of 1 mM, and quickly placed in a constant temperature dry bath at 80 °C for 30 min to obtain PtNPs with a size of 30 nm. In order to use PtNPs for subsequent immune responses, biomolecular modification is required. In this paper, SH-PEG-biotin linker was used to modify PtNPs to obtain biotinylated PtNPs (biotin-PtNPs). The specific operation is to add 10 μL of 1% Tween-20 and 5 μL of 100 μM mPEG-SH (MW ~ 5KD) to 1 mL of 4.5 nM PtNPs, vortex and mix, and then add 10 μL of 120 μM SH-PEG-biotinlinker and 50 μL 0.2 M H3PO4 solution, vortex mixed and react at 37°C for 1 h. After the reaction, wash with PBS solution (pH 7.4) containing 0.1% Tween-20 and 0.5% BSA at 13,000 rpm for 3 times, 4 min each time, and finally resuspend in 1 mL of the above solution to obtain a final concentration of 2.5 ...

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Abstract

The invention discloses an integrated ELISA (Enzyme-Linked Immuno Sorbent Assay) chip based on distance detection targets and a detection method thereof. The integrated ELISA chip is provided with a plurality of water-phase cavities and oil-phase cavities which are alternatively arranged into a line, and front cavity bodies are communicated with rear cavity bodies; water-phase inlets are formed in the water-phase cavities; oil-phase inlets are formed in the oil-phase cavities; the water-phase cavity in the leftmost side is connected with a dye cavity which is additionally provided with a third inlet; the dye cavity is communicated with a dye gas column channel; and scales are arranged beside the dye gas column channel. The integrated ELISA chip disclosed by the invention integrates fussy steps of immunological experiment, cleaning and distance signal output onto a microfluidic pneumatic chip, and can be used for high-sensitivity quantitative instantaneous detection of multiple targets such as protein and cells and the like.

Description

[0001] The invention relates to an integrated detection method integrating complicated immune experiment steps, cleaning steps and signal output on a microfluidic pneumatic chip, which is used for high-sensitivity quantitative real-time detection of various targets such as proteins and cells. Background technique [0002] Enzyme-Linked Immunosorbent Assay (ELISA) was first proposed in 1971 by Engvall and Perlmann. It is a method of adsorbing known antibodies or antigens on the surface of solid supports (such as polystyrene plates, microporous membranes, magnetic beads, etc.) and maintaining their immunogenicity. Labeling, a technique for qualitative or quantitative detection based on the degree of color development of an enzyme-catalyzed substrate. This method has the advantages of wide application range, high sensitivity, strong specificity, standardized operation and easy automation. At present, clinical diagnosis based on immune response mainly relies on optical, electrica...

Claims

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Application Information

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IPC IPC(8): G01N33/535G01N33/542G01N33/543
CPCG01N33/535G01N33/542G01N33/54326G01N33/54346
Inventor 杨朝勇刘丹周君恺李星锐李久兴朱志
Owner XIAMEN UNIV
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