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Cell surface glycan in-situ electrogenerated fluorescence imaging analysis method based on a bipolar electrode

A cell surface and fluorescence imaging technology, applied in the field of biosensing, can solve the problems of insufficient sensitivity, difficult to obtain raw materials, difficult to analyze glycans, etc., and achieve the effects of avoiding mutual interference, reducing consumption and high sensitivity

Pending Publication Date: 2020-06-23
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Mass spectrometry is a powerful tool for carbohydrate analysis, but it is difficult to analyze glycans on the surface of living cells due to its destructive nature
Although lectins can recognize highly specific carbohydrates on the cell surface, the detection process usually involves fluorescently labeling the cells, resulting in insufficient sensitivity due to the presence of autofluorescence
Bioorthogonal metabolic labeling has high reaction kinetics but requires copper as a catalyst, which is cytotoxic and whose starting materials are difficult to obtain

Method used

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  • Cell surface glycan in-situ electrogenerated fluorescence imaging analysis method based on a bipolar electrode
  • Cell surface glycan in-situ electrogenerated fluorescence imaging analysis method based on a bipolar electrode
  • Cell surface glycan in-situ electrogenerated fluorescence imaging analysis method based on a bipolar electrode

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: the synthesis of nanoprobe

[0041] The specific steps for the synthesis of 13nm AuNPs are as follows: the magnetron, two-neck flask and condenser tube used in the experiment were soaked in an alkali tank overnight, soaked in aqua regia for 24 hours, and then rinsed with a large amount of primary water and secondary water before use. to boiling HAuCl 4 solution (1 mM, 50 mL), quickly added trisodium citrate solution (38.8 mM, 5 mL), stirred continuously and kept boiling for 15 minutes, while the color of the solution changed from clear to black, purple, and finally to Deep red, the solution was cooled to room temperature. Finally, the prepared AuNPs solution was stored at 4 °C for later use.

[0042] The specific steps of Bio-DNA-AuNPs-Fc-DNA synthesis were as follows: 1 mg BSPP was added to the above-prepared AuNPs aqueous solution (10 nM, 1.5 mL) to replace the citrate ligand to protect AuNPs. To increase the stability of AuNPs-DNA complexes, incubate...

Embodiment 2

[0046] Example 2: Principle Verification 1

[0047]1) We first measured the cyclic voltammetry curve of the ITO electrode of the cultured cells in 0.1 M PBS solution, and no obvious redox peak appeared; -1 GAO, 10 mM aniline, a mixture of 100 μM BH and 5% FBS, 0.1 mg mL −1 The CV curves of avidin and 4 nM Bio-DNA-AuNPs-Fc-DNA, the CV curves of the gradually modified electrode showed obvious redox peaks at 0.106 V and 0.045 V, and the detection results showed that using this method, the electrode The active molecule Fc can be successfully modified, see image 3 a.

[0048] 2) Secondly, the saturation N of the ITO electrode was determined 2 In the cyclic voltammetry curves of 0.1 M PBS solution and 50 μM resazurin in the PBS solution in the atmosphere, there is no obvious redox peak in the CV curve containing only PBS, but it can be seen in the CV curve of resazurin Resazurin reduction peak appeared at -0.579 V. Experimental results show that resazurin can be reduced.

Embodiment 3

[0049] Example 3: Principle Verification 2

[0050] HepG2 cells were inoculated at a certain concentration in the anode microfluidic channel of the bipolar electrode and incubated for 6 hours. After the cells adhered to the wall, galactose oxidase was added to oxidize the hydroxyl on the galactose on the cell surface to aldehyde groups, and aniline catalyzed biotin The hydrazide reacts to form a hydrazone complex, thus introducing the biotin target on the cell surface, using avidin as a bridge, and binding to the Bio-DNA-AuNPs-Fc-DNA nanoprobe. Resazurin molecules are added to the cathode pool of the bipolar electrode. Under a certain external potential, the Fc introduced by the anode is oxidized, and the non-fluorescent resazurin at the cathode is reduced to highly fluorescent resorufin, which is quantified by the fluorescence intensity of the cathode. The expression level of galactose on the surface of anode cells was detected.

[0051] at 1.0 x 10 6 cells mL -1 , in the ...

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Abstract

The invention discloses a cell surface glycan in-situ electrogenerated fluorescence imaging analysis method based on a bipolar electrode. The method comprises the following steps: 1) preparing a bipolar electrode micro-fluidic chip; (2) preparing a Bio-DNA (deoxyribonucleic acid)-AuNPs (gold nanoparticles)-Fc-DNA (deoxyribonucleic acid) nanoprobe; 3, incubating HepG2 cells in situ in an anode channel of the bipolar electrode, adding galactose oxidase to oxidize hydroxyl on galactose on the surfaces of the cells into aldehyde groups, aniline catalyzes a biotin hydrazide reaction to form a hydrazone compound, avidin serves as a bridge, and the hydrazone compound is combined with the Bio-DNA-AuNPs-Fc-DNA nanoprobe; 4) adding azure molecules into a cathode pool of the bipolar electrode, oxidizing Fc introduced by the anode under the potential of 2.4 V, reducing azure of the cathode into high-fluorescence resorufin, and quantitatively detecting the galactose expression level on the surfaceof the anode cell through the fluorescence intensity of the cathode; according to the method, the damage of fluorescence labeling or mass spectrometry to cells is avoided by utilizing a mild enzymaticoxidation method, and a sensitive electrochemical signal is converted into a visual optical signal by combining the bipolar electrode, so that high-resolution fluorescence imaging is realized.

Description

technical field [0001] The invention belongs to the technical field of biosensing, and relates to a technique for quantitatively detecting polysaccharides on the surface of living cells by electrofluorescence, in particular to an in-situ electroluminescent imaging analysis method for polysaccharides on the surface of cells based on bipolar electrodes. Background technique [0002] At present, there are mainly mass spectrometry, lectin method and bioorthogonal labeling strategies for the detection of cell surface glycans. Mass spectrometry is a powerful tool for carbohydrate analysis, but it is difficult to analyze glycans on the surface of living cells due to its destructive nature. Although lectins can recognize highly specific carbohydrates on the cell surface, the detection process usually involves fluorescent labeling of cells, resulting in insufficient sensitivity due to the presence of autofluorescence. Bioorthogonal metabolic labeling has high reaction kinetics, but ...

Claims

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Application Information

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IPC IPC(8): G01N27/26G01N27/30G01N21/64
CPCG01N27/26G01N27/30G01N21/6402G01N21/6486
Inventor 刘松琴吴亚锋田召燕
Owner SOUTHEAST UNIV
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