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Single cell transfected protein analysis chip with high flux and high sensitivity

A high-sensitivity, analytical chip technology, used in material analysis by electromagnetic means, material analysis by optical means, and material analysis, etc., can solve problems such as low sensitivity, inactivation of protein antigen sites, and false negatives, and achieve functional The effect of good stability, good storage stability and high portability

Active Publication Date: 2021-06-15
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limitation of photosensitive reagents in the gel, the efficiency of photosensitive immobilization is low, and the lower limit of detection is 27,000 protein molecules in a single cell, which is difficult to apply to the detection of low-abundance proteins, and there is still a lot of room for improvement, and scWB is effective in detecting High background fluorescence for proteins and low sensitivity, which is not satisfactory for detecting low-abundance proteins
In addition, shorter excitation wavelengths (UVB, 280-320nm) may cause inactivation of protein antigenic sites, resulting in false negative effects
And equally difficult to apply to single-cell protein analysis of rare cell populations

Method used

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  • Single cell transfected protein analysis chip with high flux and high sensitivity
  • Single cell transfected protein analysis chip with high flux and high sensitivity
  • Single cell transfected protein analysis chip with high flux and high sensitivity

Examples

Experimental program
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Effect test

preparation example Construction

[0051] Step 3. Preparation of single-cell Western blot chip: treat the standard glass slide with silylating reagent (175528, J&K) for 30 minutes, then wash twice with methanol and deionized water, and dry with nitrogen; from 30% stock solution Prepare 12% gel solution and add Tris-HCl, SDS, Tritonx-100, ddH 2 O; APS and TEMED were added to initiate polymerization; the prepared solution was spread on a silica chip template and then covered with a silanized glass slide; the slide was lifted after 20 min and kept in a humid box at 4 °C;

[0052] After the gel has formed, gently lift the chip out of the mold. The chip is covered with a layer of template, and due to the design of the template, there is no gel in the isolated area. Hydrophobic barrier pens create hydrophobic regions along the barrier. To accurately locate transfected cells, we assigned trap coordinates to each cell. Different transfected cells were loaded onto the chip at the same time, and in addition to the cyl...

Embodiment 1

[0055] Example 1. Synthesis of light-sensitive protein immobilization gel N-(3-methacrylamidopropyl)-2-(1-methyl-1H-pyrrol-2-yl)-2H-tetrazole-5- Formamide

[0056] Step 1, synthetic 2-(1-methyl-1H-pyrrol-2-yl)-2H-tetrazolium-5-carboxylic acid ethyl ester, comprises the following steps:

[0057] 1.1. Dissolve methyl-1 hydrogen-pyrrole (5g, 12.5mmol) in 30mL trifluoroethanol;

[0058] 1.2. At -40°C, add iodobenzene diacetate (20g, 62.5mmol) to the solution in 1.1;

[0059] 1.3. Under nitrogen protection, stir for 2 hours at -40°C;

[0060] 1.4. Concentrate the product to black oil and dissolve in dichloromethane;

[0061] 1.5. Add 5-ethyl tetrazolium carboxylate (3g, 21.6mmol), copper(II) trifluoromethanesulfonate (3.1g, 8.6mmol), and triethylamine (16ml, 108mmol) to the solution in 1.4;

[0062] 1.6. Under nitrogen protection, stir at room temperature for 24 hours;

[0063] 1.7. After the reaction, wash the product with saturated ammonium chloride and brine, dry over anhydro...

Embodiment 2

[0076] Embodiment 2, different concentrations of light-sensitive protein immobilization gels to the immobilization efficiency of bovine serum albumin BSA

[0077] 1. The product III in Example 1 was dissolved in DMSO to form a storage solution S1 with a concentration of 100 mM;

[0078] 2. In a 1.5ml Ep tube, add 25μL 1.5M pH8.8 Tris-Hcl, 166.7μL 30% acrylamide / methylene bisacrylamide (29:1), 265.3μL ddHO, and add 15μL (3%), 7.5 μL (1.5%), 3.75 μL (0.75%), 0 μL (0%) of S1, configured as a gel precursor;

[0079] 3. Add 10 μl 5% SDS, 10 μl 5% TritonX-100, 4 μl APS, 4 μl TEMED, shake gently to prepare S1 solutions with different concentrations. Add the solution dropwise on the porous microarray mold, cover the glass slide gently to avoid the generation of air bubbles, let it stand for 20 minutes, wait for the gel to solidify, peel off the mold, and make a porous microarray gel;

[0080] 4. According to 0.5% SDS, 0.1% v / v Triton X-100, 0.25% sodium deoxycholate, 12.5mM Tris, 96...

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Abstract

The invention discloses a single cell transfected protein analysis chip with high flux and high sensitivity, which relates to the field of microchip analysis platforms. The chip is composed of a carrier and a light-sensitive protein fixed gel coating, the coating is divided into more than one detection area by a hydrophobic separation belt, and cell wells are arranged in the detection areas. Polyacrylamide gel with tetrazole as a photosensitive group is selected as light-sensitive protein fixing gel, is easily crosslinked with protein through ultraviolet irradiation, has a sensitive and accurate detection effect on low-abundance protein, and can really realize gene expression detection on transfected single cells and rare cells; and due to the regionalized microchip design, simultaneous detection of multiple targets and multiple targets can be realized, and the method has important significance in analysis of exogenous gene expression and evaluation research of transfection conditions.

Description

technical field [0001] The invention relates to the field of microchip analysis platforms, in particular to a high-throughput and high-sensitivity single-cell transfection protein analysis chip. Background technique [0002] Complex signal transduction and expression of protein modifications underlie many functions of organisms. Biologists routinely introduce foreign DNA, RNA and proteins into cells to alter their signaling and behavior (1) . Transfection is the process of introducing foreign nucleic acids into cells to produce transgenic cells, which are powerful analytical tools for studying gene function and regulation, as well as protein function (2) . Transfection can be divided into two categories: transient transfection and stable transfection. Stable transfection generally means that the introduced genetic material (transgene) with a selectable marker gene is integrated into the host genome such that transgene expression can be maintained even after host cell rep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/543G01N21/31G01N21/64G01N27/447
CPCG01N33/6803G01N33/54393G01N21/6458G01N21/6428G01N21/31G01N27/447G01N2021/6439
Inventor 丁显廷谢海洋李山鹤
Owner SHANGHAI JIAO TONG UNIV
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