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Application of gold nano-cluster in detection of protein amyloid fibrosis in biological sample and screening of inhibitor

A gold nanocluster and biological sample technology, which is applied in the application field of inhibitors, can solve the problems of cumbersome synthesis methods, and achieve the effects of high sensitivity, large Stocks shift, and low toxicity

Active Publication Date: 2020-05-08
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

When they are combined with protein fibers, their fluorescence intensity will increase, and with the increase of protein incubation time, the fluorescence intensity will tend to remain unchanged until the end of the fibrosis process, but these methods are still Small molecule probes, their synthesis methods are relatively cumbersome

Method used

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  • Application of gold nano-cluster in detection of protein amyloid fibrosis in biological sample and screening of inhibitor
  • Application of gold nano-cluster in detection of protein amyloid fibrosis in biological sample and screening of inhibitor
  • Application of gold nano-cluster in detection of protein amyloid fibrosis in biological sample and screening of inhibitor

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Application of gold nanoclusters to detect protein amyloid fibrillation in biological samples

[0028] Taking lysozyme as a model protein, the specific detection method is as follows:

[0029] 1. Accurately weigh 2mg of lysozyme and 0.0176g of NaCl in a centrifuge tube, then add 2mL of pH=2 hydrochloric acid solution to it and shake to dissolve it fully, then add 20μL of 5mM NaN to it 3 Aqueous solution, make lysozyme, NaCl, NaN 3 The final concentrations were 1 mg / mL, 150 mM, and 0.05 mM, respectively. Finally, the centrifuge tube was sealed with a parafilm, placed in a shaking water bath at 65° C. at a shaking speed of 60 rpm, and incubated for 120 hours to completely fibrillate the lysozyme. After the lysozyme fiberization process was completed, the lysozyme fiber samples were taken out and stored in a refrigerator at 4°C.

[0030] To demonstrate that gold nanoclusters can be used to detect fibrosis of lysozyme, a control experiment was first performed. Sample 1 ...

Embodiment 2

[0041] Application of gold nanoclusters in screening inhibitors of protein amyloid fibrillation

[0042] Using lysozyme as a model protein to study the inhibitory effect of ascorbic acid on protein fibrosis, the specific method is as follows:

[0043] 1. Incubate the lysozyme according to the method in step 1 of the above-mentioned Example 1, and take 40 μL of samples and add them to 410 μL of ultrapure water at 0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, 108h, and 120h respectively 50 μL of gold nanoclusters were added to the sample. The excitation wavelength is set to 365nm, and the scanning band is 500-700nm to detect the fluorescence of each sample. The fibrosis process of lysozyme was detected by the fluorescence response of gold nanoclusters to lysozyme incubated for different times. Taking the incubation time as the abscissa and the fluorescence intensity of the gold nanoclusters as the ordinate, the growth curve of the fluorescence intensity changing with the incubat...

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Abstract

The invention discloses application of gold a nano-cluster in detection of protein amyloid fibrosis in a biological sample and screening of inhibitors. According to the invention, lysozyme is used asa model protein, research finds that the fluorescence intensity of a gold nano-cluster is enhanced along with the increase of the lysozyme fiber concentration, and the lysozyme fiber concentration andthe fluorescence intensity are in a linear relationship within a certain concentration range, so that the gold nano-cluster can be applied to the detection of a protein amyloid fibrosis process in abiological sample. Compared with a fluorescence method using traditional organic dye thionein T (ThT), the method has the advantages that the sensitivity is high, the gold nano-cluster has the advantages of low toxicity and large Stocks displacement, and the fluorescence signal of the gold nano-cluster is sensitive to change along with the degree of protein fibrosis, so that the change of the protein from the initial stage of fibrosis to the logarithmic phase can be detected more sensitively, the process and mechanism of protein amyloid fibrosis can be studied more effectively, and the gold nano-cluster can be used for screening inhibitors of protein amyloid fibrosis.

Description

technical field [0001] The invention belongs to the technical field of protein amyloid fibrillation detection, and specifically relates to the application of gold nanoclusters in detecting protein amyloid fibrillation in biological samples, and the application of gold nanoclusters in screening inhibitors of protein amyloid fibrosis. Background technique [0002] Elderly diseases such as Alzheimer's disease and Huntington's disease have attracted more and more attention. Clinical studies have shown that a very important cause of these senile diseases is the denaturation and aggregation of proteins in the body, that is, protein amyloid fibrosis. For example, when insulin denatures and accumulates in the body, it will lead to the occurrence of type 2 diabetes. Therefore, studying the mechanism of protein fibrosis and screening inhibitors of protein amyloid fibrosis are of great significance in the treatment and prevention of diseases. However, the formation mechanism of amylo...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6428
Inventor 张耀东郭英马雪娟夏梦凡蒲莉孙鹏跃
Owner SHAANXI NORMAL UNIV
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