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Reagent-free electrochemiluminescence detection method for cell imaging

A detection method and cell imaging technology, applied in chemiluminescence/bioluminescence, analysis by chemical reaction of materials, measurement devices, etc. and other problems, to achieve the effect of uniform particle size, good dispersion and good stability

Pending Publication Date: 2022-03-08
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technique cannot directly enable imaging of the entire cell bottom due to the requirement for high concentrations of co-reactants and the short lifetime of co-reactant radicals.
If the cells are permeabilized with surfactants, the detection of living cells cannot be achieved
In addition, the introduction of high concentrations of co-reactants can also cause severe cell damage

Method used

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  • Reagent-free electrochemiluminescence detection method for cell imaging
  • Reagent-free electrochemiluminescence detection method for cell imaging

Examples

Experimental program
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Effect test

Embodiment 1

[0017] Example 1: Combining figure 2 , to synthesize polymer dots coupled with triethylamine.

[0018] M1 (100mg), M2 (59.7mg), tetrakis(triphenylphosphine) palladium (17.3mg) and potassium carbonate (207.3mg) were mixed in toluene / water (4mL / 1mL) and placed under argon atmosphere In a Shrek tube, after stirring at 100°C for 72 hours, precipitate twice in methanol to obtain a light orange solid P1. Then P1 (100mg) was mixed with diethylamine (1.17g) and potassium carbonate (221.1mg) in tetrahydrofuran / dimethylformamide (16mL: 4mL), placed in a Shrek tube under an argon atmosphere, 80 After stirring for 96 h at °C, TEA-PFBT was precipitated twice in methanol to obtain TEA-PFBT as an orange solid.

[0019] TEA-PFBT (100μL, 1mg mL -1 THF solution) and PSMA (20μL, 1mg mL -1 THF solution) was added into 1.88 mL THF, ultrasonically degassed for 20 minutes, then the mixture was quickly injected into 1 mL of water, and ultrasonic treatment was continued for 4 minutes. After conc...

Embodiment 2

[0020] Example 2: Combining figure 1 , TEA-Pdots were coupled to streptavidin.

[0021] TEA-Pdots (1mL, 50μg mL -1 ), 4-hydroxyethylpiperazineethanesulfonic acid (20μL, 1M) and polyethylene glycol (20μL, 5wt%, M w =3350) after mixing, adjust the pH of the solution to about 7.0. Subsequently, freshly prepared EDC (20 μL, 5 mg mL -1 ) and streptavidin (20 μL, 1 mg mL -1 ) were sequentially added to the mixture, and stirred at room temperature for 5 hours. Then, bovine serum albumin (20 μL, 10 wt%) was added to the solution and stirring was continued for 30 minutes to prevent non-specific adsorption. Finally, streptavidin-coupled TEA-Pots (SA@TEA-Pots) were isolated by ultrafiltration three times with Triton X-100 (0.5 wt%).

Embodiment 3

[0022] Example 3: Binding figure 1 , reagent-free ECL imaging detection of analyte molecules on the surface of living cells using SA@TEA-Pots

[0023] at 5% CO 2 In a humid atmosphere at 37 °C, SK-BR-3 cells were incubated with 10% fetal bovine serum and 100 μg mL -1 Penicillin-streptomycin McCoy's 5A culture medium to obtain a concentration of 5 × 10 5 -2×10 6 cells / mL of cell dispersion. Take 200μL of this dispersion liquid and biotinylated antibody (1μL, 1mg mL -1 ) in the dark at room temperature for 45 minutes with rotation and shaking, and then washed three times with phosphate buffered saline (PBS). Subsequently, cells were incubated with 200 μL SA@TEA-Pots for another 45 minutes in the dark at room temperature, washed three times with PBS and transferred to 2.5cm×2.5cm ITO conductive slides at 37°C, 5% CO 2 Cells were immobilized on the surface of ITO conductive slides by incubating in medium for 8 hours.

[0024] The ECL imaging process was performed on a self-...

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Abstract

The invention relates to a reagent-free electrochemiluminescence (ECL) detection method for cell imaging. According to the method, a designed and synthesized triethylamine group (TEA) coupling polymerization point (TEA-Pdots) is used as a luminous body, and in-situ reagent-free ECL imaging detection is carried out on molecules to be detected on the surface of a living cell in the absence of an additional co-reactant. The imaging method comprises the following steps: firstly, coupling TEA-Pdots with streptavidin (SA), and then labeling the obtained SA-coated TEA-Pdots onto a to-be-detected molecule on the surface of a cell through the double recognition effect of a biotin labeled antibody, the corresponding to-be-detected molecule and SA; furthermore, the cells are fixed on the surface of an ITO conductive slide through incubation, and ECL signals are obtained through cyclic potential scanning. By taking human epidermal growth factor receptor-2 (HER2) as an example, ECL imaging detection of HER2 on the surface of a living cell is realized. Based on the ECL of the intramolecular double electron transfer enhanced polymer point, the ECL imaging detection of the molecules to be detected on the surface of the living cell in the absence of an additional co-reactant is realized for the first time, and a simple and reliable biological imaging method is provided for the in-situ detection of the related molecules on the surface of the living cell.

Description

1. Technical field [0001] The invention relates to a reagent-free electrochemiluminescence detection method for cell imaging. 2. Background technology [0002] Due to its extremely low background and excellent controllability, electrochemiluminescence (ECL) microscopy is ideal for studying various cellular properties, such as the chemical composition of the cell surface and interior. However, due to the requirement for high concentrations of co-reactants and the short lifetime of co-reactant radicals, this technique cannot directly enable imaging of the entire cell bottom. If the cells are permeabilized with surfactants, the detection of living cells cannot be realized. In addition, the introduction of high concentrations of co-reactants can also cause severe cell damage. Therefore, in live cell sensing and microscopic ECL imaging, the development of ECL luminophores without additional co-reactants is an urgent need in this field. [0003] In order to realize the reagent-...

Claims

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Application Information

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IPC IPC(8): G01N21/76G01N33/569
CPCG01N21/76G01N33/56966
Inventor 鞠熀先王宁宁
Owner NANJING UNIV
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