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Method for detecting induced pluripotent stem cell

A technology of pluripotent stem cells and silicon spheres, applied in the field of bioengineering, can solve the problems of low fluorescence intensity of organic fluorescent dyes, high toxicity of organic fluorescent dyes, and difficult judgment results, achieving good biological safety and low possibility of interference , the effect of low detection cost

Inactive Publication Date: 2010-08-11
SHANGHAI JIAO TONG UNIV
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Problems solved by technology

[0004] Found that Yamanaka et al published "Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors" on pages 861 to 872 of the 131st issue of "cell" (cell) magazine in 2007 through a literature search of the prior art (through specific transcription factors Inducing the recombination of human fibroblasts into pluripotent stem cells), this paper proposes to identify specific markers on the surface of ips cells by immunofluorescence technology. The sensitivity is also large, and the judgment result is sometimes difficult, the operation is cumbersome, there are many controls, and the time is long; the cost of antibodies with fluorescent dyes is high; the fluorescence intensity of organic fluorescent dyes is low; the fluorescence will be quenched in a short time, and organic fluorescent dyes are highly toxic

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  • Method for detecting induced pluripotent stem cell
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  • Method for detecting induced pluripotent stem cell

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Embodiment 1

[0025] Detection of specific oct4, sox2, nanog, and AP antigens expressed on the surface of iPS cells

[0026] The detection method is to analyze the expression of oct4, sox2, nanog, and AP antigens in ips cells. Human oct4, sox2, nanog, and AP monoclonal antibodies are used as detection methods, anti-BSA monoclonal antibody is used as a negative control, and anti-beta-tubulin monoclonal antibody is used as a negative control. positive control.

[0027] Step 1, weigh 1.35g (0.005mol) FeCl 3 ·6H 2 O and 0.69 (0.0025mol) FeSO 4 ·7H 2 O was dissolved in 50ml of deionized water, and 20ml of 1.5M NaOH was added under nitrogen protection at 80°C. After vigorously stirring for 1 hour, the black precipitate was separated by magnetic separation, and washed three times with deionized water. Obtain magnetic nanoparticles, then disperse the magnetic nanoparticles in 20ml deionized water to form a solution with a concentration of 8mg / ml;

[0028] Step 2, take 1.1417g (5mmol) CdCl 2 2.5...

Embodiment 2

[0034] Detection of specific SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 antigens expressed on the surface of ips cells

[0035] The detection method is to analyze the expression of SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 antigen in ips cells, select anti-SSEA-3, SSEA-4, TRA-1-60, TRA-1- 81 monoclonal antibody was used as a detection method, anti-BSA monoclonal antibody was used as a negative control, and anti-GAPDH monoclonal antibody was used as a positive control;

[0036] Step 1, weigh 1.35g (0.005mol) FeCl 3 ·6H 2 O and 0.69 (0.0025mol) FeSO 4 ·7H 2 O was dissolved in 50ml of deionized water, and 20ml of 1.5M NaOH was added under nitrogen protection at 80°C. After vigorously stirring for 1 hour, the black precipitate was separated by magnetic separation, and washed three times with deionized water. Obtain magnetic nanoparticles, and finally disperse the magnetic nanoparticles in 20ml deionized water to form a solution with a concentration of 8mg / ml;

[0037] Step 2, 1.1417g (5mmol...

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Abstract

The invention relates to a method for detecting an induced pluripotent stem cell in the technical field of detection, which comprises the following steps of: preparing magnetic nano-particles and water-soluble quantum dots; generating silicon dioxide microspheres by hydrolyzing tetraethyl orthosilicate and preparing fluorescent magnetic nano silicon spheres by adopting an inverse microemulsion method; carrying out surface modification on the fluorescent magnetic nano silicon spheres and then coupling an antibody to obtain the fluorescent magnetic nano silicon spheres coupled with the antibody; preparing a microarray chip by using internal control protein as a positive control, using bovine serum albumin as a negative control and utilizing the antibody for resisting a specific marker expressed on the surface of the induced pluripotent stem cell; and detecting a sample by utilizing the microarray chip and then carrying out fluorescence labeling on the microarray chip by utilizing the fluorescent magnetic nano silicon spheres coupled with the antibody to obtain a detecting result. The method of the invention can realize the simultaneous detection of a plurality of specific molecules on a single sample and has little sample usage quantity, high sensitivity and specificity, low detecting cost, simple operation, high automation degree and high detecting speed.

Description

technical field [0001] The invention relates to a detection method in the technical field of bioengineering, in particular to a detection method for induced pluripotent stem cells. Background technique [0002] Quantum dots combined with substances such as antibodies, antigens or DNA are called biological probes or fluorescent probes. However, such fluorescent probes can only perform qualitative or quantitative analysis on the labeled biomolecules, and do not have the function of collecting or separating the biomolecules to be qualitatively or quantitatively analyzed. Magnetic nanoparticles have attracted much attention because of their good superparamagnetism and easy surface functionalization, which can complex antibodies, antigens or immunoglobulins. Combined with the target to be separated in vivo, immunomagnetic separation is performed under the action of a magnetic field, but it does not have the function of labeling itself. Labeling and separation have become import...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N33/533
Inventor 阮静崔大祥贺蓉吉佳佳
Owner SHANGHAI JIAO TONG UNIV
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