Electrochemical method for detecting activity of protein acetyltransferase

An acetyltransferase, electrochemical technology, applied in the direction of electrochemical variables of materials, measuring devices, scientific instruments, etc., can solve the problems of high cost of antibody labeling, complex probe preparation, differences between antibody batches, etc., and achieve a high degree of design. Flexibility, selectivity and repeatability, enhanced selectivity effect

Active Publication Date: 2019-01-11
SUZHOU CHIEN SHIUNG INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method based on enzyme-linked immunosorbent immunoassay and acetylation antibody recognition is the dominant tool for the detection of acetylation-related ...

Method used

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  • Electrochemical method for detecting activity of protein acetyltransferase
  • Electrochemical method for detecting activity of protein acetyltransferase
  • Electrochemical method for detecting activity of protein acetyltransferase

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Embodiment 1

[0043] Technical solution and working principle

[0044] An electrochemical method for detecting protein acetyltransferase activity according to the present invention comprises the following steps: adding P2 templated silver nanoparticles (P2-AgNPs) to the detection system of P1 modified gold electrode (P1 / AuE) and p300 protein ) and cucurbit[8] urea (CB[8]), the activity of p300 protein was detected by electrochemical measurement, so as to obtain the activity of protein transferase; the amino acid sequence of P1 is: 11-mercaptoundecanoic acid (MUA) -GGGFRGKGGKGLGKGGAKA; the amino acid sequence of P2 is: FGGGASLWWSEKL.

[0045] The working principle of the present invention, such as figure 1 As shown, the present invention provides a polypeptide probe whose amino acid sequence is P1: 11-mercaptoundecanoic acid (MUA)-GGGFRGKGGKGLGKGGAKA, wherein cysteine ​​C can realize the polypeptide probe on the surface of the gold electrode through the Au-S bond The self-assembly of...

Embodiment 2

[0047] Synthesis of P2-templated silver nanoparticles (P2-AgNPs)

[0048] The synthesis method of P2-templated silver nanoparticles (P2-AgNPs), including the following steps: 0.2 mM AgNO 3 Mix with 100ng / μL P2 in 5mL aqueous solution, and magnetically stir for 20min. NaBH4 (2 mM) was slowly added to the mixed solution, and magnetically stirred for 10 min. The formation of P2-AgNPs was indicated when the final solution appeared a stable yellow color.

[0049] like figure 2 As shown, the formation and availability of P2-AgNPs were first investigated, and in the UV-vis spectra, the characteristic peak at 405 nm confirmed the presence of Ag nanoparticles. By TEM analysis, most of the particles are dispersed with a particle size of about 10 nm (inset).

[0050] In the control experiment, the control polypeptide P3 with the phenylalanine residue removed was used instead of P2 for template synthesis of silver nanoparticles. Wherein, the amino acid sequence of P3 is: GGGA...

Embodiment 3

[0053] Preparation of P1 modified gold electrode (P1 / AuE)

[0054] Before modification, the gold electrode first needs to be polished into a mirror surface with 1 μm, 0.3 μm and 0.05 μm alumina suspension on the suede. Then, with piranha solution (H 2 SO 4 : H 2 o 2 = 3: 1) Soak and wash for 5 minutes to remove organic matter adsorbed on the electrode. Electrodes were ultrasonically cleaned in double distilled water and ethanol, respectively. The electrode was soaked in 50% nitric acid for 30min, then in 0.5MH 2 SO 4 In electrochemical cleaning, the scanning voltage was from 0 V to 1.5 V, and the cycle was 30 times. Blow dry in a nitrogen atmosphere, and immerse the treated gold electrode in 100 μL, 0.5 μM P1 solution. Trichloroethyl phosphate (TCEP) was added to the P1 solution to prevent the MUA end groups of P1 from forming disulfide bonds. After soaking for 16 hours, the gold electrodes were incubated in 1 mM MCH for 30 minutes, washed with double distilled ...

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Abstract

The invention discloses an electrochemical method for detecting the activity of protein acetyltransferase. P2 templated silver nanoparticles and cucurbit [8] urea are added into a P1 modified gold electrode and a detection system of a p300 protein, the activity of the p300 protein is detected by electrochemical measurement, and the protein transferase activity is obtained; the amino acid sequenceof P1 is 11-mercaptoundecanoic acid (MUA)-GGGFRGKGGKGLGKGGAKA; and the amino acid sequence of P2 is FGGGASLWWSEKL. p300 is used as a model, and the silver nanoparticles are synthesized by a signal template P2, and assembled with a sensing template P1 through a cavity, so that the sensitivity of detection is ensured, high design flexibility is achieved, and meanwhile, the selectivity of HATs is enhanced. The development of the simple and practical electrochemical method capable of detecting HATs activity is expected to help early diagnosis of major diseases.

Description

technical field [0001] The invention belongs to the technical field of bioelectrochemical detection, and in particular relates to an electrochemical method for detecting protein acetyltransferase activity. Background technique [0002] In 1931, Dr. Warburg won the Nobel Prize in Physiology and Medicine for his major discovery that "cancer is a metabolic disease". Studies by many universities in the United States have also confirmed that genes are not the fundamental factor of cancer, and it is the cytoplasm that controls cancer. The difference between normal cytoplasm and cancer cell cytoplasm is the difference in metabolism. Therefore, metabolic disorders are the root of cell canceration. According to relevant standards, about 1 / 4 of the world's population suffers from metabolic syndrome (MS) in 2015. The prevalence rate in my country is 16.5%, and the age-standardized prevalence rates for men and women are 10.0% and 23.3%, respectively. %, the prevalence rate in the north...

Claims

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Application Information

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IPC IPC(8): G01N27/48G01N27/327
CPCG01N27/3277G01N27/48
Inventor 苗向阳吴昀王杨郁惠珍顾准
Owner SUZHOU CHIEN SHIUNG INST OF TECH
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