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Bioelectrochemical sensor for detecting thrombin and its preparation method and application

A technology of bioelectrochemistry and thrombin, applied in the direction of material electrochemical variables, scientific instruments, biological testing, etc., can solve the problems of reducing detection sensitivity, affecting the binding efficiency of aptamer chain and thrombin, and achieving the effect of wide application prospects

Inactive Publication Date: 2016-05-25
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, among the electrochemical sensors for detecting thrombin, the most common design is still based on the combination of thrombin aptamer and thrombin. Factors such as these often affect the binding efficiency of the aptamer chain and thrombin, thereby reducing the sensitivity of the detection

Method used

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  • Bioelectrochemical sensor for detecting thrombin and its preparation method and application
  • Bioelectrochemical sensor for detecting thrombin and its preparation method and application
  • Bioelectrochemical sensor for detecting thrombin and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Preparation of bioelectrochemical aptasensor for detecting thrombin

[0027] The pyrolytic graphite electrodes were sequentially ground on 800-mesh and 3000-mesh fine corundum sandpaper, and then polished to a mirror surface on silk with a mixture of alumina powder and water with a particle size of 0.05 μm. They were then ultrasonically cleaned in absolute ethanol and ultrapure water for 5 min, rinsed and dry-cleaned with ultrapure water after removal, and dried with nitrogen. Then take 20 μL of 3.5 mg / mL PDDA solution (containing 0.05 M NaCl) to soak the electrode for 20 min, then rinse with ultrapure water and blow dry with nitrogen. Then 40 μL of AuNPs with a diameter of 7 nm was soaked in the electrode for 1 h, then rinsed with water and blown dry with nitrogen. Finally, 40 μL of 1 mMCB[7] was taken to soak the electrode for 60 min, then rinsed with ultrapure water, and then dried with nitrogen, and 20 μL of the mixed solution after the cycle reaction wa...

Embodiment 2

[0030] Embodiment 2: Detection method of bioelectrochemical aptasensor for detecting thrombin

[0031] Add an appropriate amount (about 5mL) of 10mM Tris-HCl (pH7.4) solution into the electrolytic cell, and then pass high-purity nitrogen into it and maintain it for at least 10 minutes; immerse the three-electrode system in the Tris-HCl solution, and continue to maintain a certain Flow rate of nitrogen until the end of the test. Connect the three-electrode system to the electrochemical workstation (model: chi660c; China Shanghai Chenhua Instrument Co., Ltd.), turn on the electrochemical workstation and the test computer, start the control software of the electrochemical workstation, select the detection method as square wave voltammetry, and set The detection parameters are as follows: the potential scanning range is 0~-0.6V, the potential increment is 4mV, the amplitude is 25mV, and the frequency is 15Hz; run the program to start detection, and the corresponding data map will ...

Embodiment 3

[0033] Example 3: Detection of different concentrations of thrombin by bioelectrochemical aptasensor for detection of thrombin

[0034] First, add 5 μL of thrombin solution of different concentrations and 5 μL of 10 nM aptamer DNA chain to 22.5 μL of thrombin binding buffer (10 mM Tris-HCl, 100 mM NaCl, 5 mM KCl, pH7.4), react at 37 ° C for 1 h, and then add 10 nM 5 μL of extended strand, 5 μL of 1 μM probe strand, 10× enzyme reaction buffer (100 mM Bispropane-HCl, 100 mM MgCl 2 , 10mMATP, 10mMDTT, pH 7.0) 5μL and 400unit / μL T4 DNA ligase 0.2μL, mix well, react at 16°C for 0.5h, and then react at 65°C for 10min to inactivate the ligase. Add 0.2 μL of 100 unit / μL ExoIII to the solution, react at 37° C. for 20 minutes, and then react at 70° C. for 20 minutes to inactivate ExoIII. After the reaction, 20 μL of the reacted mixed solution was taken and added dropwise to the surface of the functionalized pyrolytic graphite electrode, incubated at room temperature for 1 h, and then e...

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Abstract

The invention relates to a bio-electrochemical sensor for detecting thrombin as well as a preparation method and application of the bio-electrochemical sensor. The sensor is a three-electrode system sensor, wherein a counter electrode is a platinum electrode, a reference electrode is a saturated calomel electrode and a working electrode is a functionally-modified pyrolytic graphite electrode; the functionally-modified pyrolytic graphite electrode is obtained by sequentially modifying poly(diallyldimethylammonium chloride), gold nanoparticles, cucurbit(7)uril and a circulating mixed solution on the surface of a pyrolytic graphite electrode; the circulating mixed solution contains 100nM of MB marked probe DNA chain, 1nM of extended DNA chain, 1nM of thrombin aptamer chain, 1.6 unit / microliter of T4DNA ligase and 0.4 unit / microliter of ExoIII. According to the bio-electrochemical sensor, specific combining capacity of the thrombin and aptamer is utilized, ExoIII circulating cutting reaction is triggered by a ligase auxiliary effect and the rapid and sensitive detection advantages of electrochemistry are combined, so that the high-sensitivity detection of the thrombin is realized.

Description

technical field [0001] The invention relates to a bioelectrochemical aptamer sensor and its preparation method and application, in particular to a bioelectrochemical sensor capable of highly sensitive detection of thrombin and its preparation method and application. Background technique [0002] Thrombin is an extracellular serine protease that plays an important role in the blood coagulation cascade reaction. It catalyzes the conversion of fibrinogen into fibrin to promote blood coagulation. It is the drug of choice for local hemostasis. It also plays an important regulatory role in many physiological processes such as inflammation and tissue repair of blood vessel walls. In addition, it has hormone-like regulatory effects in the process of thrombus formation and platelet activation. At the same time, thrombin is positively correlated with osteopontin-positive hepatocellular carcinoma, which can be used as a therapeutic target. Therefore, efficient, sensitive and specific...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N27/48G01N2333/974
Inventor 李根喜赵婧吴吉光仲卫冬辛美玲
Owner SHANGHAI UNIV
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