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Biosensor for detecting peptidylarginine deiminases, and preparation method and application thereof

A peptidyl arginine deiminase and biosensor technology, which can be applied to instruments, measuring devices, scientific instruments, etc., can solve the problems of low detection sensitivity, difficult operation and complicated operation, and easy to be interfered by the external environment. Sensitive, efficient, selective, easy to operate, and highly selective

Inactive Publication Date: 2017-10-20
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the fluorescent labeling process is very complicated and is easily disturbed by the external environment. These methods are also difficult to operate and complicated, and the detection sensitivity is not high, so it is difficult to be used for real-time detection and analysis.

Method used

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  • Biosensor for detecting peptidylarginine deiminases, and preparation method and application thereof
  • Biosensor for detecting peptidylarginine deiminases, and preparation method and application thereof
  • Biosensor for detecting peptidylarginine deiminases, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Reaction under different modification conditions

[0030] First, gold electrodes were polished with 1 μm, 0.3 μm and 0.05 μm aluminum powder. Then, the gold electrode was sonicated in ethanol and double-distilled water for 5 minutes each. Then the piranha (H 2 SO 4 :H 2 o 2 =3︰1) Purify for 5 minutes, rinse with double distilled water and use 0.5 M H2 SO 4 Electrochemical purification to remove any remaining impurities. After drying with nitrogen, the gold electrode was used to modify the polypeptide chain 1. React at 4°C for 16-18h; each 100μL modified reaction solution consisted of 50μL, 10μM polypeptide 1, 30μL, 10mM PBS and 20μL, 50mM TCEP, and mixed them evenly. The electrodes are submerged in the solution. At this time, polypeptide chain 2 is also activated and modified with silver nanoparticles. 4 Reaction 16-18h. 60 μL of 5 μM polypeptide chain 2 was added to 1440 μL of the prepared silver nanoparticle solution.

[0031] Cucurbituril 8 can c...

Embodiment 2

[0032] Example 2: Detection of different concentrations of PAD4

[0033] Add different concentrations of PAD4 to the reaction system (0.005nM, 0.01nM, 0.05nM, 0.1nM, 0.5nM, 1nM, 5nM, 10nM, 50nM, 100nM, 200nM ) The results are as follows image 3 shown. The activity of PAD4 to catalyze the citrullination of arginine increases with the increase of its concentration, and the peak current value also increases. image 3 The inset plot further illustrates the linearity of PAD4 from 0.005 nM to 200 nM. The linear equation is I (10 -5 A)=2.94233+1.12126lgC PAD4 (nM), R 2 =0.993. A detection limit of 0.79 pM was obtained at three times the noise ratio.

Embodiment 3

[0034] Example 3: Research on the Effect of PAD4 Inhibitors

[0035] For the research on PAD4 inhibitors, it is generally the research on the mechanism of action of chloramidine on its inhibitors. Chloramidine is an irreversible PAD4 inactivator, and the combination of the inhibitor will destroy the structure of the active site of PAD4, thereby greatly reducing the activity of PAD4. Figure 4 The effect of different concentrations of inhibitors on PAD4 activity is shown. The activity of PAD4 was greatly reduced with increasing inhibitor concentration. The inset plots represent the concentration of the inhibitor chloramidine versus the rate of inhibition.

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Abstract

The invention relates to a biosensor for detecting peptidylarginine deiminases, and a preparation method and application thereof. The sensor is characterized in that a polypeptide chain 1 (FGGGRGAC) is modified on the surface of a gold electrode; the sulfydryl of the cysteine at the end C of the polypeptide chain fixes the polypeptide chain 1 onto the surface of the electrode through a gold-sulfur bond; polypeptide chain 2 (FGGGGGC) functional silver nanometer particles are connected with silver nanometer particles through the sulfydryl of cysteine of the polypeptide chain 2. Under the assistance of the mutual action of supermolecule cucurbituril 8(CB[8]) assisted subject and object, the functional silver nanometer particles can be recognized with FGG at the end N of the polypeptide chain 1 and FGG at the end N of the polypeptide chain 2 due to CB[B]. The biosensor uses a super molecular chemistry assistance signal marking method for improving the experiment sensitivity; the linear range of the PAD4 can be detected to be 0.005 nM to 200 nM; the detection limit is 0.79 nM; the specificity is very high; the target and other reference protein can be effectively distinguished; the goal of detecting the activity of the endogenous PAD4 in the HL-60 lysates can be achieved.

Description

technical field [0001] The invention relates to a biosensor for detecting peptidyl arginine deiminase, a preparation method and application thereof. Background technique [0002] Peptidylarginine deiminase is a calcium-dependent posttranslational family of enzymes that catalyze the conversion of peptidylarginine residues to peptidylcitrulline residues. PAD isoforms are PAD1, PAD2, PAD3, PAD4, and PAD6, and PAD4 is the only enzyme of these isoforms located in the nucleus. PAD4 can catalyze the citrullination of arginine residues in histones H2A, H3 and H4, thereby regulating the transcription, differentiation and pluripotency of downstream genes. Recent studies have found that the abnormal expression of PAD4 can lead to the occurrence of rheumatoid arthritis. Through the process of protein citrullination, high levels of PAD4 activity in cells are related to the production of autoantibodies in disease progression. In addition, a large amount of overexpression of PAD4 is also...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327
CPCG01N27/3271
Inventor 李根喜赵婧杨莉莉吕赟
Owner SHANGHAI UNIV
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