Paper-based micro-fluidic chip enhancement type chemiluminescence gene sensing method

A microfluidic chip, chemiluminescence technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as unclean washing, cross-contamination of reagents, narrow application range, etc., to enhance detection sensitivity, The hydrophilic reaction area is fine and the screen printing effect is good.

Active Publication Date: 2014-08-27
SOUTH CHINA NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] (1) The target DNA length is less than 50bp; the longer the target DNA length, the wider the application range, and the application range is obviously narrower than 50bp;
[0009] (2) The heating process in the wax screen printing method is 130°C (150s). If the temperature is too high and the time is too long, the properties of the paper will change, which is not conducive to the subsequent detection reaction;
[0010] (3) Rinsing is carried out in the reaction pool, which will lead to unclean rinsing, and multiple rinsing will lead to cross-contamination of reagents;
[0011] (4) The signal substance used - nanoporous gold adsorbed by carbon quantum dots, its synthesis process is relatively complicated, and the process of marking the signal probe with the signal substance is complicated and takes a long time;
[0012] (5) A weak luminescence analyzer is used to collect and analyze chemiluminescence signals. Although it can achieve low sensitivity detection, the analyzer is expensive, resulting in high detection costs, and does not have the ability to detect high-throughput

Method used

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  • Paper-based micro-fluidic chip enhancement type chemiluminescence gene sensing method
  • Paper-based micro-fluidic chip enhancement type chemiluminescence gene sensing method
  • Paper-based micro-fluidic chip enhancement type chemiluminescence gene sensing method

Examples

Experimental program
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Effect test

Embodiment 1

[0056] A method for paper-based microfluidic chip-enhanced chemiluminescence detection of hlyA gene, comprising the following steps:

[0057] (1) Fabrication of paper-based microfluidic chips

[0058] like figure 1 As shown, press a 200-mesh nylon screen printing plate (diameter 4mm, wash channel length 4mm, width 2mm) on 200mm×200mm Whatman No. 1 chromatography paper (the two are close to each other), and then apply wax on the screen printing plate, wax Infiltrate the chromatography paper through the screen printing plate; then, place the screen printing plate with the chromatography paper on the heating plate for heating (heating at 100°C for 5s), with the side with the chromatography paper facing the heating plate; after heating, the chromatography paper The paper falls off naturally from the screen printing plate, and is cooled to dry at room temperature. The prepared paper-based microfluidic chip will have reaction pools one by one on it.

[0059] (2) Design capture pr...

Embodiment 2

[0068] A paper-based microfluidic chip enhanced chemiluminescence detection method for hlyA gene, the steps and materials are the same as in Example 1. Several experimental groups were set up, and the concentrations of p-iodophenol in the bottom solution were 1×10 -2 mol / L, 1×10 -3 mol / L, 4×10 -4 mol / L, 1×10 -4 mol / L, 1×10 -5 mol / L.

[0069] The chemiluminescence intensity values ​​of each experimental group are as follows: image 3 shown.

[0070] It can be seen that there is a threshold value for the enhancing effect of p-iodophenol. When the concentration of p-iodophenol is low, there is almost no enhancement effect on luminescence, and when it is higher than a threshold (1×10 -4 mol / L), the enhanced luminescence begins; in a certain concentration range (1×10 -4 mol / L~1×10 -3 mol / L), the luminescence tends to be stable, continue to increase the concentration to a larger threshold (1×10 -3 mol / L), the luminescence was significantly weakened. The possible reason fo...

Embodiment 3

[0073] A paper-based microfluidic chip enhanced chemiluminescence detection method for hlyA gene, the steps and materials are the same as in Example 1.

[0074] Since the optimal pH value of horseradish peroxidase catalysis is neutral or weakly acidic, and the quantum yield of luminol chemiluminescence is the highest when the pH value is about 10.0, it is necessary to choose a pH value that can balance the relationship between the two. pH.

[0075] Several experimental groups were set up, and the pH values ​​of the bottom liquid were 7.0, 7.5, 8.0, 8.2, 8.5, 9.0, and 10.0, respectively.

[0076] The chemiluminescence intensity values ​​of each experimental group are as follows: Figure 4 shown.

[0077] It can be seen that the acceptable pH value is from 7.5 to 9.0, and those less than 7.0 and greater than 10.0 basically do not emit light. The chemiluminescence intensity was the largest and relatively stable when the pH value of the bottom solution was 8.2.

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Abstract

The invention discloses a paper-based micro-fluidic chip enhancement type chemiluminescence hlyA gene detection method. The method comprises the steps that (1) a screen printing board is pressed on chromatography paper, coated with wax and heated, and the chromatography paper is naturally disengaged from the screen printing board and aired; (2) a catching probe and a signal probe are designed; (3) a paper-based micro-fluidic chip is pretreated; (4) a DNA sample to be tested and the catching probe are incubated, the signal probe is added to conduct a hybridization reaction with the DNA sample to be tested, and HRP-SA is added for incubation; finally a base solution is added to trigger enhancement type chemiluminescence, and an luminescence signal is collected by a CCD digital imaging device in an imaging mode. Compared with an expensive and complex optical imaging system, the simple CCD device is adopted for imaging detection, an enhancement type chemiluminescence system is combined with a biotin-streptavidin affine magnification system, the detection flexibility of paper-based micro-fluidic chip enhancement type chemiluminescence is greatly improved, and the detection limit can reach 6.3*10<-2>pmol/L.

Description

technical field [0001] The invention belongs to the field of gene detection, and in particular relates to a paper-based microfluidic chip-enhanced chemiluminescent method for detecting Listeria monocytogenes hlyA gene. Background technique [0002] A gene is a functional DNA molecular fragment, the basic unit of genetic information, and the most basic determinant of all species. Genetic detection refers to a technology that detects target nucleic acid molecules through certain detection methods, and analyzes the target nucleic acid molecule disease-causing genes, disease susceptibility genes, etc. Listeria monocytogenes (abbreviated as "Listeria monocytogenes" in some places in this paper), as an important food-borne pathogen, can cause human and animal infection diseases, and it is composed of a variety of virulence factors. Since Listeria lysin was reported by the Parrisius group in 1985, there has been much evidence that Listeria monocytogenes LLO, which is encoded by th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/682C12Q1/6837C12Q2563/131C12Q2563/101C12Q2565/629
Inventor 章春笋刘菲菲
Owner SOUTH CHINA NORMAL UNIVERSITY
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