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Composition and method for detecting cells

A composition and cell technology, applied in the measurement of color/spectral characteristics, etc., can solve the problems of clinical application limitations and insufficient sensitivity

Active Publication Date: 2021-08-20
章毅 +7
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Common DNA-assembled gold nanocomposites often achieve stable binding through Au-S bonds. However, due to insufficient sensitivity and other reasons, this method is limited in clinical applications.

Method used

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  • Composition and method for detecting cells
  • Composition and method for detecting cells
  • Composition and method for detecting cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Construction of the mesenchymal stem cell colorimetric system, the specific steps are as follows:

[0044]Synthesis of silver nanocuscriminals as follows, 3 to 5 μl, 100 ~ 150 μm S1 and 100 ~ 150 μL, 100 to 150 μm agno 3 Mix, at 4 ° C reaction 20 to 30 min; add 3 ~ 5 μL, 1 to 2 mm NABH 4 After a dramatic shock of 1 to 2 min, the reaction was reacted at 4 ° C for 2 to 3 hours; the centrifugal polynucleotide-Ag nanocarcrumus (DNA-AGNCs) was filtered from 30 k centrifugal column, and the centrifugal polynucleotide-Ag nanocolus (DNA-AGNCs) was collected. Take 5 ~ 8 μL, 10 ~ 15 μm 4- (N-maleimide methyl) cyclohexane-1-carboxylate sulfonic acid group succinamide sodium salt (SMCC) and 3 to 5 μl concentration 1.3 ~ 2 μm of CD44 antibody mixed, 25 ° C reacted 2 to 3 h activating antibody amino groups. The activated antibody was added to the supernatant, and the reaction was reacted at 25 ° C for 2 to 3 h to obtain a crosslinked DNA-antibody complex.

[0045] Next, the ad...

Embodiment 2

[0051] Example 2 Quantitative detection of mesenchymal stem cells

[0052] According to the method in Example 1, 100 μL of mesenchymal stem cells containing different concentrations (0 cells / ml, 10) were added to the system in the system. 2 Cell / ml, 10 3 Cell / ml, 10 4 Cell / ml, 5 × 10 4 Cell / ml, 10 5 A cell / mL) solution for ultraviolet detection. Figure 3 The ultraviolet-visible absorbing spectrum corresponding to different concentrations of mesenchymal stem cells and a color picture corresponding to the concentration of gold nanoparticles with mesenchymal stem cells. Figure 4 The ratio of the ultraviolet absorption value of Jin nanoparticles at 620 nm and 520 nm is shown in the detection system, and the concentration of mixed stem cells is changed. With the increase of mesenchymal stem cell concentration, the peak value of gold nanoparticles in the final detection system gradually decreased, and the peak at 620 nm gradually increased, which indicates that more and more...

Embodiment 3

[0053] Example 3 Specific Study

[0054] Cells (HepG2 and L02) expressing low expression using CD44 were used as a control, according to the above method, respectively, respectively, respectively, respectively. 5 Cells / ml cells for ultraviolet - visible absorption light detection. As can be seen from the figure, the A620 / A520 value of the final reaction system is weaker in the case where there is no cellular presence; when it is detected that the CD44 highly expressed mesenchymal stem cells, the A620 / A520 value obtained by the reaction system is obvious. The increase in non-specific cells of CD44 low expression, the A620 / A520 value of the final reaction system is basically the same as the result obtained in the blank control. The above results have been effectively proved that the mesenchymal stem cell colorimetric detection method based on single-stranded DNA functionalized gold nanoparticles constructed in this example is specific.

[0055] Thereby, the specific antigen ...

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Abstract

A composition for detecting cells, comprising a polynucleotide with a sulfhydryl group at the 5' end, attached to the metal silver of the polynucleotide to form a polynucleotide-Ag nanocluster; and the polynucleotide Conjugated primary antibody for binding to target cells and immobilizing polynucleotide‑Ag nanoclusters on target cells; secondary antibody coupled to magnetic particles for binding to target cells; oligonucleotides The acid, when in solution, attaches to the surface of the metal nanoparticles. The detection composition of the present invention adopts the combination of single-stranded polynucleotides and single-stranded oligonucleotides, utilizes the photoelectric properties of different metals, establishes the correlation between spectral absorption peaks and target cells, and realizes detection of The target cells in the sample are qualitatively and quantitatively detected.

Description

Technical field [0001] The present invention relates to a method of qualitatively and quantifying detection in samples in samples, and more particularly to a method and composition of the cells that have a photocylocell in the sample to perform qualitative and quantitative detection of the cells in the sample. Background technique [0002] Mesenchymal Stem Cells, MSCs is a class of stem cells with self-renewal, multi-directional potential and low immunogenicity. Relevant research shows that MSCs can use direct contact and byeprocess to inhibit excessive immune responses, maintain local micro-environment steady states, promote the repair of tissue, and treat inflammatory related diseases and autoimmune diseases Come to the new direction. Sensitive detection of mesenchymal stem cells can provide many important information for clinical diagnosis and treatment of these diseases. [0003] Gold nanoparticles are highly received in the field of life sciences due to their unique physical...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/33
Inventor 章毅赵婧伍婷曹亚朱小立
Owner 章毅