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Ultrahigh-sensitivity protease digital detection method

A digital detection and protease technology, applied in the field of enzyme detection, can solve the problems of limited sensitivity and false negative of protease

Pending Publication Date: 2021-07-30
SHENZHEN TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The operation process is simple, and it can quickly find whether there is protease in the detection sample, but the detection sensitivity of protease is limited, and when the concentration of protease in the sample is low, false negative results are prone to occur

Method used

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  • Ultrahigh-sensitivity protease digital detection method

Examples

Experimental program
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Embodiment 1

[0073] In this embodiment, a microarray microfluidic chip is prepared, and the chip consists of 2014 3D CAD software design, consisting of a microarray layer and a cover sheet layer, the microarray layer contains 1 million circular reaction microwells, each microwell can only accommodate one magnetic bead, and the volume of the microwell is fL level, the microarray The material of the layer and the cover sheet layer is a mixed silica gel (polydimethylsiloxane (PDMS) and tetraethyl orthosilicate, the mass ratio is 10:1), which is produced by injection molding process.

[0074] The specific production process includes:

[0075] (1) Weigh PDMS and tetraethyl orthosilicate according to the mass ratio of 10:1, after fully stirring and mixing, carry out degassing and defoaming treatment in a vacuum device;

[0076] (2) Inject the degassed and defoamed mixture into the microarray layer mold, and cure at 80° C. for 30 minutes;

[0077] (3) peel off the formed silica gel chip from t...

Embodiment 2

[0082] This embodiment prepares the magnetic bead-antibody complex, and the preparation method of the magnetic bead-antibody complex comprises the following steps:

[0083] (1) Take 50 μL carboxy-modified magnetic beads (5 mg / mL), wash them twice with PBS, add pre-cooled 20 μL EDC (20 mg / mL) and 20 μL NHS (20 mg / mL) respectively, and use MES buffer for EDC and NHS Dissolve (20mM, pH 6), shake at room temperature for 30 minutes to activate the carboxyl group, and use a magnetic rack to enrich the magnetic beads;

[0084] (2) Use pre-cooled MES buffer to wash the magnetic beads enriched in step (1) 3 times, add 20 μg anti-3CL protease antibody, shake and react at room temperature for 2 hours, use a magnetic rack to enrich the magnetic beads, and discard the supernatant , wash the magnetic beads 3 times with pre-cooled MES buffer, add Tris-BSA buffer (25mM Tris-HCl, 0.1% Tween-20, 0.5% BSA, pH 7.4), shake at room temperature for 30min, and block the excess activated carboxyl grou...

Embodiment 3

[0086] This embodiment takes the 3CL protease of coronavirus as an example to detect.

[0087] The schematic diagram of the detection is as figure 2 As shown, it specifically includes the following steps: (1) react the magnetic bead-antibody complex prepared in Example 2 with 3CL protease in PBS buffer at 4°C for 60 minutes to form a magnetic bead / antibody / 3CL protease complex, and enrich the Magnetic beads, discard the supernatant, add pre-cooled PBS to wash 3 times, and use Tris-HCl buffer (20mM, pH7.0) to resuspend;

[0088] (2) Add fluorescent polypeptide substrate (sequence is Dabcyl-TSAVLQSGFRVM-FITC, wherein Dabcyl is fluorescent quenching group succinimidyl ester; FITC is fluorescein isothiocyanate) to step (1) resuspension liquid The final volume is 50 μL, and then the liquid is injected from the injection port of the microarray microfluidic chip prepared in Example 1. After standing at room temperature for 5 minutes, fluorine oil is added from the injection port, a...

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Abstract

The invention relates to an ultrahigh-sensitivity protease digital detection method. The method comprises the following steps: (1) mixing a protease standard substance with a magnetic bead antibody compound, carrying out a reaction, collecting a reactant, and mixing the reactant with polypeptide to obtain a mixed solution; (2) injecting the mixed solution into a microarray micro-fluidic chip, performing incubating, and detecting the percentage of the fluorescent dots in the total particles; (3) constructing a mathematical relationship between the percentage and the protease concentration; and (4) calculating the protease concentration in the sample by using the mathematical relationship in the step (3) according to the percentage of the fluorescent dots of the detection sample in the total particles in the step (1) and the step (2). The method can realize high-sensitivity detection of the protease, is simple to operate and high in standard curve fitting degree, and can accurately calculate the concentration of the protease in the sample.

Description

technical field [0001] The invention belongs to the technical field of enzyme detection and relates to an ultrahigh-sensitivity digital detection method for protease. Background technique [0002] Protease (Protease) is the general term for a class of enzymes that hydrolyze protein peptide bonds. According to the way they hydrolyze polypeptides, they can be divided into two types: endopeptidases and exopeptidases. Endopeptidases cut proteins or polypeptide substrate molecules internal peptides The exopeptidase hydrolyzes the peptide bonds one by one from the end of the free amino group or carboxyl group of the protein molecule; according to its active center, proteases can be divided into serine proteases, sulfhydryl proteases, metalloproteases and aspartic acid proteases; according to their reaction The optimum pH value is divided into acid protease, neutral protease and alkaline protease. [0003] Proteases widely exist in animals, plants, microorganisms and viruses. Pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N33/543G01N33/573
CPCG01N21/6428G01N21/6486G01N33/573G01N33/54326G01N2021/6432G01N2333/95
Inventor 黎荣松葛晨晨刘敬欣查玲
Owner SHENZHEN TECH UNIV
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