ELISA detection chip based on nucleotide sequence coding and preparation and application thereof

A technology for detecting chips and nucleic acid sequences, which can be used in measurement devices, chemical instruments and methods, and containers for laboratories, etc., and can solve the problems of small number of codes, low detection throughput, and poor ease of operation.

Active Publication Date: 2016-07-27
崔玉峰
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Each of these techniques suffers from one or more limitations, such as low number of codes, poor ease of operation, and low throughput

Method used

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  • ELISA detection chip based on nucleotide sequence coding and preparation and application thereof
  • ELISA detection chip based on nucleotide sequence coding and preparation and application thereof
  • ELISA detection chip based on nucleotide sequence coding and preparation and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0067] This embodiment provides a preparation and application based on an ELISA chip, specifically related to the double-antibody sandwich method, multi-person, multi-marker detection, as follows:

[0068] Step 1, surface modification of micron magnetic beads: The surface of about 1.1 million magnetic beads with a diameter of 3.5 μm is coated with anti-streptavidin (SA for short) by biochemical methods, and the density of SA on the surface of the magnetic beads is about 4000 / μm 2 , Therefore, the total amount of the surface area SA of a magnetic bead with a diameter of 3.5 μm is about the product of its surface area S and density, that is, 4000 beads / μm 2 ×4πr 2 ≈150,000 SAs. Divide the magnetic beads into 10020 equal parts, each about 110 beads; repeat three times.

[0069] Step 2, microbead coding: take the first 10020 sequences from the primary coding library, design and synthesize the hairpin DNA with the coding sequence, and the top of the loop of the hairpin structure ...

Embodiment 2

[0080] This embodiment provides a preparation and application based on an ELISA chip, involving indirect method: known antigen-test antibody-detection secondary antibody, capture method: IgM anti-antibody-test IgM antibody-specific antigen-labeled antibody, single The comprehensive detection of markers in multiple populations is as follows:

[0081] Step 1, surface activation of polystyrene (PS) micro-beads: use chemical methods to coat the surface of about 710,000 PS beads with a diameter of 2 μm with glutaraldehyde, divide the beads into 7 groups, each group has 1002 parts, and each part has about 100 capsules; repeat three times.

[0082] Step 2, PS bead coding and capture molecule immobilization: Synthesize amino-modified coding DNA, which consists of two parts, with an amino-labeled primer pairing region at the 3' end, sequence 3'NH 2 -CAGCACTGACCCTTTTGGGACCGC-5', followed by the coding region, which was taken from the first 7014 sequences of the preferred coding library...

Embodiment 3

[0093] This example provides a preparation and application based on an ELISA chip, involving double-antibody sandwich method, double-antigen sandwich method and indirect method, multi-person multi-marker detection, details are as follows:

[0094] Step 1, surface activation of silicon nano-beads: Divide about 15 million silicon nano-beads with a diameter of 350 nm activated by SA on the surface into 36 groups, of which 12 groups of test beads each have about 1.2 million beads, and the other 24 groups of control beads have about 500 beads each , that is, 12 groups of positive and negative controls. Repeat 2 times.

[0095] Step 2, immobilization and DNA encoding of capture molecules on silica beads: 12 kinds of biotin-labeled capture molecules (Table 3) were mixed with the nanobeads used in each group for detection, positive control and negative control group, and immobilized on the surface of the nanobeads. The number of capture molecules is equivalent to about 1 / 3 of the num...

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Abstract

The invention discloses an ELISA detection chip based on nucleotide sequence coding and preparation and application thereof.The preparation of the chip includes the steps that coded nucleic acid and captured molecules are immobilized to the surfaces of micro-nano beads to form a micro-nano bead compound; targeted markers and marked detection molecules are immobilized to the surface of the obtained micro-nano bead compound to form a micro-nano bead complex; a substrate with distributed micro-nano pit arrays is loaded with the obtained micro-nano bead complex to prepare the ELISA detection chip; ELISA detection and coded nucleic acid decoding are carried out.Compared with the prior art, numerous samples and multiple markers can be detected at the same time on one chip, ELISA detection is carried out in micro-nano pits, the reaction volume can be below the 10-24 L level, and thus the reagent dosage can be greatly reduced.The ELISA detection chip can be applied to clinical medicine detection, blood donor detection, physical examinations, detection of toxins in agricultural and sideline products, detection of illegal food additives and environmental pollution detection.

Description

technical field [0001] The present invention relates to a kind of biological chip technology, specifically a kind of ELISA detection chip based on nucleic acid sequence coding and its preparation and application, that is to say, specifically the production and application of an enzyme-linked immunosorbent reaction (abbreviated as ELISA) chip coded by nucleic acid sequence The detection method can simultaneously detect multiple samples and multiple target markers on one chip, and the total number of detected samples × number of markers can be as high as one million or more. Not only the throughput is high, but also consumables can be greatly saved. In the field of medicine, it can realize early detection and diagnosis of diseases, provide enough time for prevention and treatment, and can also be used for environmental pollution detection and detection of pesticide residues, veterinary drug residues, biological toxins and illegal food additives in agricultural and sideline produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543B01L3/00
CPCB01L3/5027G01N33/54346
Inventor 王志民侯彩玲何珊珊王媛媛
Owner 崔玉峰
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