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Transcription factor fluorescence detection of based on DNA-silver nanocluster allosteric probe

A technology of transcription factors and silver nanoclusters is applied in the field of analysis and detection, which can solve the problems of low sensitivity of allosteric probes, and achieve the effects of convenient detection methods, good application prospects and strong fluorescence.

Active Publication Date: 2019-01-15
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the construction of most fluorescent allosteric probes still requires the modification of relatively expensive fluorescent chemical groups, and a few label-free allosteric probes have low sensitivity and can only achieve μM level detection.

Method used

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  • Transcription factor fluorescence detection of based on DNA-silver nanocluster allosteric probe
  • Transcription factor fluorescence detection of based on DNA-silver nanocluster allosteric probe
  • Transcription factor fluorescence detection of based on DNA-silver nanocluster allosteric probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The detection method for illustrating the transcription factor of the present invention comprises the following steps:

[0042] 1) Referring to existing literature reports (Journal of the American Chemical Society, 2013, 135(32), 11832-11839), and making improvements, DNA silver nanoclusters were synthesized in situ on DNA templates. Dissolve 1OD of DNA template in 900 μL of buffer 1 (10 mM citrate buffer, pH 6.8), heat to 90 °C, maintain for 15 min, and then cool down to 37 °C stepwise for 45 min, then add 15 μL, 10 mM freshly prepared Silver nitrate solution, after vortex mixing, react in the ice-water mixture for 30min in the dark, then, quickly add 20μL, 10mM freshly prepared sodium borohydride solution (sodium borohydride solution needs to be prepared with ice water), vigorously vortex on the vortex mixer Vortex for 30s, immerse in ice-water mixture for 10s, and vortex for 30s, repeat at least three times. Then react overnight in an ice-water mixture protected fro...

Embodiment 2

[0047] Selective experiment of detection method of the present invention

[0048] Repeat steps 1)-2) in Example 1, replace the transcription factor solution in step 2) with interfering substances such as BSA, HAS, Humanα-thrombin, p53, etc. of corresponding concentrations, and mix the interfering substances to prepare a mixed interfering solution , other conditions remain unchanged, detect fluorescence, and obtain the selectivity result of detecting target p50 by the method of the present invention, such as image 3 shown. from image 3 It can be seen that the method of the present invention has good selectivity to the target transcription factor.

Embodiment 3

[0050] The recovery rate experiment of detection method of the present invention

[0051] Repeat steps 1)-4) in Example 1, in step 4), carry out sample addition detection, add the p50 of 15nM, 25nM, 40nM, 50nM, 200nM, 500nM respectively, detect, obtain the method of the present invention in practice The recovery rate in the sample detection, the results are shown in Table 1.

[0052] Table 1 The inventive method detects the recovery rate of p50 in the DLD-1 cell extract

[0053]

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Abstract

The invention belongs to the field of analysis and detection, and relates to a transcription factor fluorescence detection of based on DNA-silver nanocluster allosteric probe. The method includes a) using hairpin DNA as template, adding silver nitrate and sodium borohydride, and reducing that silver nitrate and sodium borohydride to generate DNA-silver nanocluster allosteric probe AgSwitch in situ; B) incubating a plurality of transcription factor solutions of different concentrations with AgSwitch obtained in step a); C) measuring the fluorescence value of the system, obtaining a linear relationship between the concentration of the transcription factor and the fluorescence intensity, and drawing a standard curve; D) measuring fluorescence intensity of the sample to be tested under the same conditions as the step c), and obtaining the concentration of transcription factors in the sample to be tested according to the standard curve of the step c). The method has the advantages of label-free, enzyme-free and high sensitivity, and can realize efficient detection of the transcription factors in the biological sample.

Description

technical field [0001] The invention belongs to the field of analysis and detection, and more specifically relates to a transcription factor fluorescence detection method based on a DNA-silver nano cluster allosteric probe. Background technique [0002] Transcription factors (Transcription factors, TFs) are DNA-binding proteins in cells, which play a vital role in regulating cell proliferation, apoptosis, transformation and so on. When a cell receives a signal from inside or outside the cell, the expression of various signaling molecules in the signaling pathway changes, and finally these signaling molecules transmit information step by step to transcription factors that are in a resting state outside the nucleus. The factor is activated and enters the nucleus, binds to the transcription factor binding region on the target gene, and initiates the transcription process. The transcriptional process plays a role in regulating the expression of cellular proteins and can change ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6816
CPCC12Q1/6816C12Q2525/301C12Q2563/107C12Q2563/137
Inventor 周学敏李昺之陈月王晶卢巧云洪俊丽朱婉莹
Owner NANJING MEDICAL UNIV
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