Method for detecting thrombin by electrochemical biosensor on basis of magnetic biocomposite and lead ion-dependent DNase

A biosensor and composite material technology, applied in the field of electrochemical biosensor detection of thrombin, can solve the problems of cumbersome steps, low sensitivity, and time-consuming detection, and achieve the effects of simple operation, improved sensitivity, and simple and portable equipment

Inactive Publication Date: 2018-11-13
NANJING MEDICAL UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

[0006] The purpose of the present invention is to provide a method for detecting thrombin based on an electrochemical biosensor based on magnetic biocomposite materials and lead ion-dependent DNA enzymes, which can detect thrombin quickly, with high sensitivity and high selectivity, and overcomes the existing detection methods. The shortcomings of low sensitivity, time-consuming detection, high cost and cumbersome steps

Method used

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  • Method for detecting thrombin by electrochemical biosensor on basis of magnetic biocomposite and lead ion-dependent DNase
  • Method for detecting thrombin by electrochemical biosensor on basis of magnetic biocomposite and lead ion-dependent DNase
  • Method for detecting thrombin by electrochemical biosensor on basis of magnetic biocomposite and lead ion-dependent DNase

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Experimental program
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Effect test

Embodiment 1

[0036] a) Construction of magnetic biocomposite material Fe 3 o 4 @Au-S1 / S2: According to Fe 3 o 4 with HAuCl 4 The mass ratio is 1:6 to synthesize Fe 3 o 4 @Au nanoparticles, 5 μL primer strand S1 modified to 0.8 mg magnetic Fe 3 o 4 @Au nanoparticles, add 5 μL thrombin aptamer S2 and incubate at 37°C for 40 minutes;

[0037] b) Digestion assistance by lead ion-dependent DNase: Add 500 μL of 5pM, 10pM, 25pM, 50pM, 100pM, 500pM, 1000pM, 5000pM thrombin solutions to the magnetic material constructed in step a), Co-incubate at 37°C for 30min with the assistance of DNase S3 and lead ions;

[0038] c) Construction of an electrochemical biosensor: Add methylene blue MB to the reactant obtained in step b), incubate the reaction to obtain a sample, and the sample is magnetically induced to adsorb to the surface of a magnetic glassy carbon electrode, and the MB is measured at -0.6-0.2V DPV signal, establish a standard curve;

[0039] d) Sample detection: the thrombin sample ...

Embodiment 2

[0042] a) Construction of magnetic biocomposite material Fe 3 o 4 @Au-S1 / S2: According to Fe 3 o 4 with HAuCl 4 The mass ratio is 1:5 to synthesize Fe 3 o 4 @Au nanoparticles, 4 μL primer strand S1 modified to 0.8 mg magnetic Fe 3 o 4 @Au nanoparticles, add 4μL thrombin aptamer S2 and incubate at 37°C for 30min;

[0043] b) Digestion assistance by lead ion-dependent DNase: Add 500 μL of 5pM, 10pM, 25pM, 50pM, 100pM, 500pM, 1000pM, 5000pM thrombin solutions to the magnetic material constructed in step a), Co-incubate at 37°C for 20min with the assistance of DNase S3 and lead ions;

[0044] c) Construction of an electrochemical biosensor: Add methylene blue MB to the reactant obtained in step b), incubate the reaction to obtain a sample, and the sample is magnetically induced to adsorb to the surface of a magnetic glassy carbon electrode, and the MB is measured at -0.6-0.2V DPV signal, establish a standard curve;

[0045] d) Sample detection: the thrombin sample of unk...

Embodiment 3

[0048] a) Construction of magnetic biocomposite material Fe 3 o 4 @Au-S1 / S2: According to Fe 3 o 4 with HAuCl 4 The mass ratio is 1:4 to synthesize Fe 3 o 4 @Au nanoparticles, 3 μL primer strand S1 modified to 0.8 mg magnetic Fe 3 o 4 @Au nanoparticles, add 3μL thrombin aptamer S2 and incubate at 37°C for 20min;

[0049] b) Digestion assistance by lead ion-dependent DNase: Add 500 μL of 5pM, 10pM, 25pM, 50pM, 100pM, 500pM, 1000pM, 5000pM thrombin solutions to the magnetic material constructed in step a), Co-incubate at 37°C for 10 min with the assistance of DNase S3 and lead ions;

[0050] c) Construction of an electrochemical biosensor: Add methylene blue MB to the reactant obtained in step b), incubate the reaction to obtain a sample, and the sample is magnetically induced to adsorb to the surface of a magnetic glassy carbon electrode, and the MB is measured at -0.6-0.2V DPV signal, establish a standard curve;

[0051] d) Sample detection: the thrombin sample of un...

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Abstract

The invention belongs to the technical field of analysis and detection and particularly relates to a method for detecting thrombin by an electrochemical biosensor on the basis of a magnetic biocomposite and lead ion-dependent DNase. The method comprises the following steps: a) construction of the magnetic biocomposite Fe3O4@Au-S1/S2; b) enzyme digestion assistance of lead ion-dependent DNase; c) construction of the electrochemical biosensor: adding MB (methylene blue) to an obtained reactant in step b), performing an incubation reaction to obtain a sample, adsorbing the sample to the surface of a magnetic glassy carbon electrode by magnetic induction, measuring a DPV signal of MB and establishing a standard curve; d) sample detection: detecting a thrombin sample with the method, measuringthe DPV signal, and substituting the signal into the standard curve to obtain thrombin concentration of the sample. With adoption of the method, thrombin can be detected rapidly with high sensitivityand high selectivity, and the defects of low sensitivity, long time consumption for detection, too high cost and troublesome step of the conventional detection method are overcome.

Description

technical field [0001] The invention belongs to the technical field of analysis and detection, and more specifically relates to a method for detecting thrombin with an electrochemical biosensor based on a magnetic biocomposite material and a lead ion-dependent DNA enzyme. Background technique [0002] Thrombin is a serine protease, which is converted from prothrombin under the action of factor Xa, converts fibrinogen into fibrin, and plays an important role in the coagulation cascade mechanism. Studies have shown that thrombin is closely related to many diseases such as thromboembolic diseases, central nervous diseases, kidney diseases, etc., and changes in its concentration will lead to functional changes. For example, in kidney tissue, low concentrations of thrombin have a cytoprotective effect, while high concentrations of thrombin can cause kidney damage. Therefore, quantitative detection of thrombin content in complex biological samples is of great significance for cli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327
CPCG01N27/3271G01N27/3275
Inventor 周学敏朱春红朱婉莹徐磊
Owner NANJING MEDICAL UNIV
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