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A detection method for single molecular protein

A detection method and single-molecule technology, applied in the field of protein detection, can solve the problems of inability to meet the requirements for detection of proteins with low expression abundance and low detection sensitivity.

Active Publication Date: 2022-02-08
PILOT GENE TECH HANGZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of this, the purpose of the present invention is to provide a single-molecule protein detection method for the problems of low detection sensitivity in the prior art and the inability to meet the detection requirements for proteins with low expression abundance

Method used

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  • A detection method for single molecular protein
  • A detection method for single molecular protein
  • A detection method for single molecular protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] 1. Laser confocal detection system ( figure 1 with figure 2 )

[0081] The laser confocal detection system includes a microfluidic chip A, a point light source B, an imaging unit C, a scanning lens D and a three-dimensional droplet imaging unit.

[0082] The point light source B adopts a laser or an LED lamp to form a point light source and project it onto the droplet.

[0083] The imaging unit C is an EMCCD photon detector, and the imaging unit C collects and detects the fluorescent signals processed by the four-channel fluorescent filter F and the lens G, and can use different fluorescent channels to independently synthesize the characteristics of the droplet containing the fluorescent signal Numerical imaging of droplet shape and fluorescence intensity °C.

[0084] 2. Microfluidic chip

[0085] The preparation of the hot-pressing mold, using the nickel mold to prepare the upper structure, see the detailed structure figure 1, wherein the width of the droplet for...

Embodiment 2

[0094] Example 2: Detection of GPC1 by direct fluorescence method

[0095] Preparation of multilayer droplets:

[0096] (1) Prepare the oil phase. Prepare the oil phase according to the following proportions (both by mass): 50g mineral oil + 50g n-tetradecane + 1.5g emulsifier EM90 + 1.5g TritonX-100, and use it for later use after ultrasonic degassing.

[0097] (2) Prepare the water phase. Prepare different concentrations of GPC1 protein standards (abcam, ab114484), that is, use the standard diluent to dilute the original GPC1 protein standard, and the final GPC1 protein concentrations are 0, 0.1, 0.25, 0.5, 1.0, 2.5, 5 and 10PM, respectively. , the final volume is not less than 200 μl. Use Merck’s magnetic beads to couple GPC1 capture antibody (abcam, ab55971) 6 μL GPC1 protein 200 μl in the capture solution, incubate at 37 ° C for 1 h, wash the supernatant, and then add FITC-labeled detection antibody (Invitrogen, Cat#62-8411) 200 μl , the detection antibody was diluted...

Embodiment 3

[0101] Example 3: Detection of GPC1 by HRP enzyme labeling method

[0102] Preparation of multilayer droplets:

[0103] (1) Prepare the oil phase. Prepare the oil phase according to the following proportions (both by mass): 50g mineral oil + 50g n-tetradecane + 1.5g emulsifier EM90 + 1.5g TritonX-100, and use it for later use after ultrasonic degassing.

[0104] (2) Prepare the water phase. Prepare different concentrations of GPC1 protein standards (abcam, ab114484), that is, use the standard diluent to serially dilute the original GPC1 protein standard, to the final GPC1 protein concentration of 0, 0.1, 0.25, 0.5, 1.0, 2.5, 5 and 10pM The final volume is not less than 200 μl. 200 μl of GPC1 protein in 6 μl capture solution of Merck’s magnetic bead-coupled GPC1 capture antibody (abcam, ab55971), incubated at 37°C for 1 h, washed the supernatant, and then added 200 μl of HRP-labeled detection antibody (Invitrogen, Cat#31430). The detection antibody was diluted with PBS at a...

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Abstract

The invention belongs to the field of protein detection, and discloses a detection method of single-molecule protein. The protein to be tested is mixed with the target protein antibody to form a complex of the protein to be tested and the target protein antibody, which is mixed with the detection antibody marked with a marker, dissociated and collected to be tested. The complex of the protein to be tested and the detection antibody; the label is a fluorescein label or an enzyme label; the complex of the protein to be tested and the detection antibody is directly dispersed on the microfluidic chip or the protein to be tested is first The complex with the detection antibody and the substrate corresponding to the enzyme label are mixed and reacted, and then the reaction solution is dispersed in the microfluidic chip; the laser confocal detection system is used for fluorescent signal scanning and single molecule counting. The detection method of the invention can quickly perform quantitative detection on single-molecule proteins, has high detection sensitivity, and is suitable for proteins whose expression abundance is too low. The detection method of the invention has the advantages of convenient operation, accurate detection result, small deviation of CV value, high throughput, and can detect multiple samples at one time.

Description

technical field [0001] The invention belongs to the field of protein detection, and in particular relates to a single-molecular protein detection method. Background technique [0002] Protein, as the most important component of the body, interprets the growth, development and change of every point of the individual life. Since the 1940s, with the development of classical protein detection techniques such as immunohistochemistry, the value of protein as a biomarker has gradually been valued by people. Although immunohistochemistry, ELISA, Luminex and other protein detection technologies have realized the detection of thousands of protein biomarkers, the development speed of protein biomarkers is still slow: only 1-2 new biomarkers per year into practical clinical applications. According to statistics, among more than 400,000 known human proteins, about 300,000 cannot be detected by traditional methods due to their low expression abundance. Of the approximately 100,000 prot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N21/64
CPCG01N21/6428G01N33/68
Inventor 夏江宋小慧位玉玲
Owner PILOT GENE TECH HANGZHOU CO LTD
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