Hydroxyapatite-based electrochemical probe construction method and method for determining activity and inhibition property of BACE1

A hydroxyapatite, chemical probe technology, applied in the field of biosensing, can solve the problems of low sensitivity and complicated operation

Inactive Publication Date: 2016-11-09
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the traditional method still has problems

Method used

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  • Hydroxyapatite-based electrochemical probe construction method and method for determining activity and inhibition property of BACE1
  • Hydroxyapatite-based electrochemical probe construction method and method for determining activity and inhibition property of BACE1
  • Hydroxyapatite-based electrochemical probe construction method and method for determining activity and inhibition property of BACE1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] A gold electrode with a diameter of 2 mm was placed in a layer containing 0.05 μm Al 2 o 3 Polish on a polishing cloth, rinse the polished electrode with secondary water, and then ultrasonically clean it with absolute ethanol and secondary water for 5 minutes to remove the polishing powder attached to the surface of the electrode, and then dry it with nitrogen. Put the treated gold electrode, Ag / AgCl reference electrode and platinum wire counter electrode together into the reaction cell containing 5mM potassium ferricyanide solution, and perform cyclic voltammetry scanning at a voltage of -0.1-0.6V. Set the scanning speed to 0.1V s -1 ,

[0066] The potential difference was 85mV, indicating that the surface of the gold electrode was cleaned. Take 5 μL of the polypeptide (CKTEEISEVNLDAEFRHDSGY) with a concentration of 4 μM and drop it on the surface of the electrode to react for 12 hours.

[0067] After the electrode is washed, take 5 μL of the peptide with a concent...

Embodiment 2

[0070] Because hydroxyapatite contains a large amount of phosphate, and alkaline phosphatase can hydrolyze sodium pyrophosphate into phosphate, and the phosphate will react with Na 2 MoO 4 The formation of phosphomolybdate precipitation in an acidic environment produces dual electrochemical signals, so the dual-signal mode was first tested. Drop 1mg / mL hydroxyapatite solution on the surface of the electrode with 6mM Na 2 MoO 4 reaction, followed by 0.5M H 2 SO 4 It is an electrolyte, within the range of 0.1 ~ 0.5V, the frequency of 15HZ sweeps the square wave voltammetry curve. Take 100μM sodium pyrophosphate solution and drop it on the surface of the electrode with Na 2 MoO 4 reaction, and reacting sodium pyrophosphate first with alkaline phosphatase and then with Na 2 MoO 4 reaction, and then likewise in 0.5M H 2 SO 4 Sweep square wave voltammetry curve in solution. Finally, the prepared electrochemical probe was mixed with Na 2 MoO 4 reaction, also at 0.5M H 2...

Embodiment 3

[0072]Put the blank gold electrode and the gold electrode modified by the peptide in the 5mM potassium ferricyanide solution reaction cell, and perform cyclic voltammetry scanning at the voltage of -0.1-0.6V, and characterize the modification of the peptide on the electrode surface by the change of the electrode peak current . Add 0 and 2.5 U / mL BACE1 solutions dropwise on the surface of the two polypeptide-modified electrodes to react for 1 hour, rinse the electrodes and then drop the hydroxyapatite probe solution on the electrode surface to react for 1 hour; then add 100 μM pyrophosphate dropwise The sodium solution was reacted for 40 minutes; finally, Na was added dropwise on the surface of the electrode 2 MoO 4 , reacted for 40 minutes, and then in 0.5M H 2 SO 4 Sweep the square wave voltammetry curve in the solution, detect it by voltammetry, and test the feasibility of the method according to the obtained corresponding peak current value.

[0073] Similarly, 0 and 2....

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Abstract

The invention discloses a hydroxyapatite-based electrochemical probe and a construction method thereof, and a method for determining the activity and the inhibition property of BACE1. According to the present invention, an A[beta] antibody and alkaline phosphatase are co-modified on a hydroxyapatite matrix to form the hydroxyapatite-based electrochemical probe; the preparation method comprises: sequentially placing hydroxyapatite nanoparticles in a polyethylenimine solution and carrying out a reaction, placing in a glutaraldehyde solution and carrying out a reaction, and placing in a solution containing an A[beta] antibody and alkaline phosphatase and carrying out a reaction so as to obtain the probe; and the method for determining the activity and the inhibition property of BACE1 by using the hydroxyapatite-based electrochemical probe has advantages of simpleness, rapidness, high sensitivity and wide detection range, and can be widely promoted and applied.

Description

technical field [0001] The present invention relates to an electrochemical probe, in particular to a molecular probe for measuring the activity and inhibition of BACE1, in particular to a method for constructing a hydroxyapatite-based electrochemical probe and a hydroxyapatite-based electrochemical probe. A chemical probe is used to determine the activity and inhibition of BACE1, which belongs to the technical field of biosensing. Background technique [0002] In recent years, studies on Alzheimer's disease have shown that the accumulation of β-amyloid peptide (Aβ peptide) in the brain is one of the main causes of Alzheimer's disease. Aβ peptides are produced in vivo mainly through the hydrolysis of β-like precursor protein (APP) by β-secretase (such as pre-amyloid β-decomposing enzyme, BACE1) and γ-secretase. In vivo, the activity of β-secretase plays a decisive role in the production of Aβ peptide. Inhibiting the activity of β-secretase can reduce the production of Aβ pe...

Claims

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Application Information

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IPC IPC(8): G01N27/327G01N27/48G01N33/68G01N33/543
CPCG01N27/3277G01N27/48G01N33/5438G01N33/6896
Inventor 阳明辉
Owner CENT SOUTH UNIV
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