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Electrochemical immunosensor electric signal marker for prostate specific antigen detection and detection method

A prostate-specific, immunosensor technology, applied in the detection of prostate-specific antigen, electrochemical immunosensor signal markers for prostate-specific antigen detection and the detection field, can solve the problems of high cost, instability, difficult preparation, etc., and reduce the threshold of entities , convenient detection, obvious effect of electrical signal

Inactive Publication Date: 2019-03-15
ANYANG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these natural enzymes have some potential disadvantages, such as poor stability, high cost, and difficult preparation
In addition, the metal catalytic center of catalase and glucose oxidase is buried in the middle of the protein, and it is not easy to directly transfer electrons on the surface of the electrode, and a special enzyme catalytic substrate (such as hydrogen peroxide, glucose, etc.) is required; alkaline phosphoric acid The catalyzed product of monoesterase has poor oxidation resistance, is unstable in the air, has a weak electrical signal, and easily forms polymers to passivate the electrode
These factors severely limit the practical application of electrochemical immunosensors.

Method used

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  • Electrochemical immunosensor electric signal marker for prostate specific antigen detection and detection method
  • Electrochemical immunosensor electric signal marker for prostate specific antigen detection and detection method
  • Electrochemical immunosensor electric signal marker for prostate specific antigen detection and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Preparation of secondary antibody / carbon nanotube / protease complex

[0032] A1: Pretreatment of carbon nanotubes;

[0033] 20 mg of multi-walled carbon nanotubes were boiled with 50 mL of 2 M nitric acid for 12 hours, cooled and washed by centrifugation three times at 14,000 rpm for 5 minutes each time. Then heat the centrifuged multi-walled carbon nanotubes in 50 mL of nitric acid / sulfuric acid (volume ratio 1:3) mixed solution for 12 hours at a temperature of 60-120°C, then centrifuge and wash several times until the pH is close to 7, and finally The centrifuge is vacuum-dried to obtain shorter carbon nanotubes;

[0034] A2: Preparation of secondary antibody / carbon nanotube / protease complex;

[0035] Add the carbon nanotubes (1.5 mg) obtained in A1 into 1 mL of MES buffer solution, sonicate for 30 minutes, then add 1 mL of MES (pH 7.4, 10 mM) buffer solution containing 0.4 M EDC and 0.1 M NHS, and centrifuge. Discard the excess EDC and NHS, add 150 μL of...

Embodiment 2

[0036] Example 2: Activity Analysis of Secondary Antibody / Carbon Nanotube / Protease Complex

[0037] Mix 5 μL of the secondary antibody / carbon nanotube / protease complex obtained in step A2 with 100 μL of PBS buffer solution (pH 7.4, 0.2 M) containing 2 mM polypeptide GARGGH, and react at room temperature for 1 hour, then wash the reaction solution with PBS without 0.5 mM copper ions was diluted to 500 μL, and then centrifuged at 14,000 rpm. The supernatant was tested by mass spectrometry. The test model of mass spectrometry was negative ion model. From figure 2 It can be seen that the synthesized secondary antibody / carbon nanotube / protease complex can catalyze the hydrolysis of the polypeptide GARGGH, and the main peak at 268.1035 Da is the mass spectrum peak of the hydrolyzed product GGH ( figure 2 ), which indicated that trypsin modified to carbon nanotubes could catalyze the hydrolysis of polypeptide GARGGH; when copper ions were added, a new mass spectrum peak appeared a...

Embodiment 3

[0038] Example 3: Sensor Response to Prostate Specific Antigen

[0039] B: Preparation of working electrode

[0040] B1: Carboxylated graphene was modified on the surface of the glassy carbon electrode, that is, 5 μL of 0.5 mg / mL carboxylated graphene solution was dropped onto the surface of the glassy carbon electrode with a diameter of 3 mm, and dried naturally;

[0041] B2: Preparation of the primary antibody-modified electrode, that is, soak the electrode obtained in B1 in PBS buffer solution containing 0.4 M EDC and 0.1 M NHS for 15 minutes, rinse the surface of the electrode with secondary water, and dry it with nitrogen; then 10 μL The primary antibody was dripped onto the surface of the electrode, and after reacting for 2 hours, rinse the electrode with secondary water, then soak the electrode in PBS solution containing 0.1% BSA (w / v) for 30 minutes, take out the electrode, and rinse the electrode surface with secondary water, Blow dry with nitrogen;

[0042] B3: To ...

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Abstract

The invention discloses an electrochemical immunosensor electric signal marker for prostate specific antigen detection. The electric signal marker is protease, and a catalytic substrate of the protease is polypeptide with the sequence characteristic that the third amino acid from a cleavage site to a carbon end is histidine H. A detection method for prostate specific antigen detection adopts the electrochemical immunosensor electric signal marker for prostate specific antigen detection. The method comprises the steps as follows: A, preparing a secondary antibody / carbon nanotube / protease compound; B, preparing a working electrode; C, soaking the working electrode in a PBS solution containing copper ions and the polypeptide substrate for 2 h, and conducting an electrochemical test with linear sweep voltammetry. The electrical signal marker has obvious electrical signals, can meet the requirement of detection of concentration of prostate specific antigens in human serum and is harmless toworkers and environments due to the fact that no biphenyl substances are used in the catalytic substrate.

Description

technical field [0001] The invention relates to a detection method for prostate specific antigen, in particular to an electrochemical immunosensor signal marker and a detection method for detection of prostate specific antigen, and belongs to the technical field of biochemistry. Background technique [0002] Prostate specific antigen is a biomarker of prostate diseases, and its concentration in serum is closely related to the occurrence of prostate cancer. For example, the concentration of PSA in normal human serum is lower than 4 ng / mL, but when the concentration is greater than 10 ng / mL, the prostate patient is very likely to suffer from prostate cancer. Therefore, the detection of PSA is of great significance in the early diagnosis of prostatic diseases. At present, the method used for the clinical detection of prostate specific antigen is mainly enzyme-linked immunosorbent assay, but this method requires the use of special instruments, is expensive, the detection proced...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327G01N27/48G01N33/68G01N33/574
CPCG01N27/308G01N27/3275G01N27/3277G01N27/48G01N33/57434G01N33/57484G01N33/689G01N2800/342G01N2800/7028
Inventor 夏宁刘林杨素玲邓德华
Owner ANYANG NORMAL UNIV
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