Radio chemistry visual analysis method for antigen in single cell

An analysis method and internal antigen technology, applied in the field of electrochemiluminescence, can solve the problems of poor prognosis, difficult operation, and hindering cell differentiation of acute myeloid leukemia

Active Publication Date: 2021-11-02
NANJING UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

However, BPE has a physical limitation: the shorter the conductor, the greater the voltage that needs to be applied to polarize it and induce the ECL reaction
According to theoretical calculations, in order to induce BPE-ECL reaction on a submicron conductor, a voltage of tens of thousands of volts or even higher must be applied, which is not easy to operate
In addition, this high voltage can cause vigorous water electrolysis, forming a large number of air bubbles in the cell medium and affecting the imaging process.
[0006] High expression of lysine-specific demethylase 1 (also known as KDM1 / LSD1) hinders cell differentiation and leads to poor prognosis in acute myeloid leukemia

Method used

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  • Radio chemistry visual analysis method for antigen in single cell
  • Radio chemistry visual analysis method for antigen in single cell
  • Radio chemistry visual analysis method for antigen in single cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Visualization experiment of single-armed carbon nanotubes in hydrogel

[0034] For initial visualization of individual SWCNTs using BPE-ECL, agarose containing SWCNTs-COOH (0.005% wt), L012 (luminol analog) (1 mM), and 10 mM phosphate buffered saline (PBS, pH 7.4) were hydrolyzed The glue is placed in a capillary (id. 0.6 mm). Through transmission electron microscopy, it is found that the length of a single SWCNTs-COOH is less than 500nm, and the width is less than 10nm. After the gel was formed in the capillary, the SEM image showed that the gel was a porous structure with a pore size of less than 70 μm, which was suitable for retaining a cell.

[0035] According to the results of this example, when only an electric field of 1kV / cm is applied, a resolvable ECL tube sheet can be observed in the capillary, see figure 2 b. As the electric field gradually increases from 1kV / cm to 1.2kV / cm, the ECL intensity of these points gradually increases. The dependence...

Embodiment 2

[0036] Example 2 Visual labeling of carcinoembryonic antigen on a single cell membrane

[0037] Nanotubes for Single-Cell BPE-ECL Imaging to Display Antigens on Cell Membranes. Carcinoembryonic antigen antibody (anti-CEA Ab) was labeled on SWCNTs to form anti-CEA Ab-SWCNTs complex, and then incubated with fixed MCF 7 cells (the cell membrane highly expresses CEA antigen), and then It was embedded in agarose hydrogel containing L012, and a voltage of 1000V was applied to the two feeding electrodes while recording ECL images. from image 3A-C It can be observed that the entire surface of a single cell produces a visible ECL spot, which overlaps well with the bright field cell. Furthermore, in order to rule out possible non-specific adsorption of SWCNTs, cells were incubated with SWCNTs without antibody, and ECL images were collected. The results showed that almost no ECL spots were observed on the cells, proving that the nonspecific adsorption of SWCNTs in cells was negligibl...

Embodiment 3

[0038] Example 3 Visual labeling of KDM1 / LSD1 protein on the nucleus

[0039] The present application further carried out an experiment of visually analyzing antigens in cells, namely: lysine-specific demethylase 1 (also known as KDM1 / LSD1). High expression of KDM1 / LSD1 can hinder cell differentiation and lead to poor prognosis in acute myeloid leukemia. First, cells were co-incubated with KDM1 / LSD1 antibody-modified SWCNTs, and then fixed with paraformaldehyde. Finally, the cells were permeabilized with Triton X-100 to facilitate the entry of L012 and buffer. Then, a voltage of 1 kV / cm was applied to the feed electrode, and an ECL image was recorded. like Figure 4 Overlay of brightfield and ECL images shown in A and B ( Figure 4 C) shows that ECL emission is concentrated near the nucleus in the cell, and is significantly different from the ECL visualization distribution of membrane proteins ( image 3 C). Fluorescent characterization of the position of the SWCNTs modi...

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Abstract

Electrochemical microscopes gradually develop in decades in the past and are used for imaging single-cell biomolecules; however, it is difficult to achieve electrochemical visualization of intracellular proteins in a single cell. According to the application, a model protein (KDM1/LSD1 antigen) in a cell nucleus of an MCF 7 cell is observed by using a wireless bipolar electrochemiluminescence technology (BPE-ECL) for the first time. The submicron-sized single-walled carbon nanotube is connected with the antibody and is used for identifying the corresponding KDM1/LSD1 antigen. Under a low electric field of 1000V/cm, L012 (luminol analogue) is subjected to electrochemical oxidation at the anode end of the nanotube, and an ECL signal is emitted, so that wireless visualization of the position of the nanotube is realized. Under the condition that an externally applied voltage which is low enough is used, bipolar ECL emission on a cell nucleus in a single cell can be observed, and electrochemical imaging of a KDM1/LSD1 antigen in the single cell is supported.

Description

technical field [0001] The application belongs to the field of electrochemiluminescence, and in particular relates to a radiochemical visualization analysis method of single-cell intracellular antigens. Background technique [0002] The specific detection of proteins in single cells is of great significance for elucidating molecular mechanisms of cell biology and disease diagnosis. [0003] Fluorescence imaging is a commonly used visualization method for detecting proteins in the plasma membrane and inside living cells. However, the presence of background fluorescence interferes with quantitative analysis of these proteins. Electrochemiluminescence (ECL), as a nearly zero-background optical technique, is widely used in the sensitive and quantitative detection of various antigens in serum and cells. During this process, luminescent groups (such as Ru(bpy) 3 2+ ) is labeled on the antibody that specifically binds to the corresponding antigen to form an immune complex. Und...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/58G01N33/573G01N27/327G01N27/49
CPCG01N33/57415G01N33/57473G01N33/57496G01N33/583G01N33/573G01N27/3278G01N27/49G01N2333/90245Y02A50/30
Inventor 江德臣王玉玲徐静娟陈洪渊
Owner NANJING UNIV
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