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Method for detecting thrombin in ultra-sensitive mode by utilizing competitive type nano sensor

A nano-sensor, thrombin technology, applied in instruments, measuring devices, scientific instruments, etc., can solve the problems of complex sample processing and insufficient sensitivity, and achieve the effects of good detection stability, high detection sensitivity and high sensitivity

Active Publication Date: 2018-08-14
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention realizes rapid and high-sensitivity quantitative analysis of thrombin in samples through the immune recognition of Raman signal probes and magnetic capture probes and the competitive reaction of thrombin, and solves the complex and sensitive sample processing in the prior art. not enough

Method used

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  • Method for detecting thrombin in ultra-sensitive mode by utilizing competitive type nano sensor
  • Method for detecting thrombin in ultra-sensitive mode by utilizing competitive type nano sensor
  • Method for detecting thrombin in ultra-sensitive mode by utilizing competitive type nano sensor

Examples

Experimental program
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Effect test

Embodiment 1

[0057] (1), preparation of gold nanoparticles

[0058] Under constant stirring, 100 mL of 1 mM chloroauric acid (HAuCl 4 ) solution was heated to boiling, and then 6 mL of 38.8 mM trisodium citrate aqueous solution was added. At this time, the color change of the solution is: light yellow-colorless-black-purple-dark red, wait for the solution to turn dark red and continue to heat and reflux for 15-20min. Finally, it was cooled to normal temperature to prepare 30nm gold nanoparticles. figure 2 A is gold nanoparticles obtained under ideal conditions.

[0059] (2), preparation of gold dimer nanoparticles

[0060] Centrifuge the prepared gold nanoparticles (concentration is about 1 mmol / L) once (8000rpm, 10min), then resuspend in three-distilled water (pH=7) for later use; take the above 1mL gold nanoparticles and react with 30μL 1mM BPE About 10min, add 20μL 2mM silver nitrate solution to terminate the reaction (10min) before the color change of the gold nanoparticles, centr...

Embodiment 2

[0073] In order to illustrate the specificity of the present invention, respectively prepared mouse IgG antibody standard solution, bovine serum albumin BSA, Lysozyme (abbreviated Lyso) standard solution and hemoglobin Hemoglobin (abbreviated Hb) standard solution, specific implementation steps with reference to Example 1 ; The obtained signal strength can be referred to Figure 5 , indicating that the present invention has strong specificity.

[0074] The mouse IgG antibody standard solution and bovine serum albumin BSA were purchased from Sangon Bioengineering (Shanghai) Co., Ltd., and the Lysozyme standard solution and hemoglobin Hemoglobin standard solution were purchased from Shanghai Yisheng Biotechnology Co., Ltd.

Embodiment 3

[0076] In order to test the application effect of this sensor in real samples, we added thrombin to human serum samples as a simulation of real samples. Prior to this, a series of treatments (filtration, centrifugation, dilution) should be performed on the serum to eliminate the possible influence of the matrix effect on the experimental results. Subsequently, different concentrations of thrombin were added to the treated serum. These samples were used for detection and analysis respectively, and the specific implementation steps refer to Example 1; the obtained results are shown in Table 1, these results show that this method can be well used for the detection of thrombin in serum.

[0077] Table 1 utilizes the nanosensor system of the present invention to measure the result of thrombin in simulated serum

[0078]

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Abstract

The invention discloses a method for detecting thrombin in an ultra-sensitive mode by utilizing a competitive type nano sensor. The method is characterized in that BPE and a gold nanoparticle are combined to form gold dimer which reacts with an aptamer-1, which in complementary pairing with TBA, to obtain a Raman signal probe; the Raman signal probe performs mixing reaction with a magnetic nanoparticle modified with the thrombin aptamer TBA to obtain a nano sensor system; when to-be-detected thrombin exists, the structure of the nano sensor system is broken by competition, so that changes of aRaman signal of liquid supernatant in the system are caused, and detection for content of thrombin is realized. The method is simple to operate, is good in detecting stability and is high in detecting sensitivity; while operates, the nano sensor system which is prepared in advance is sequentially added into a to-be-detected sample to react, and detection can be immediately performed through magnetic separation after reaction is ended, so that quick and quantitative detection is realized; and requirements of food safety and environmental monitoring department can be met, and practicability iswide.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for ultrasensitive detection of thrombin by using a competitive nanometer sensor based on a gold dimer substrate. Background technique [0002] Thrombin is an important serine proteolytic enzyme, which consists of two peptide chains connected by disulfide bonds. Thrombin converts fibrinogen in blood into fibrin, which has the functions of regulating blood coagulation and promoting blood circulation. As an important multifunctional active protein, thrombin plays a key role in thrombosis, angiogenesis, tumor growth and metastasis, etc., and thrombin is often used as a therapeutic agent and biomarker. At present, the methods for detecting thrombin mainly include colorimetric method, fluorescence method and electrochemical method, but these methods have more or less shortcomings, such as poor sensitivity, complicated operation, and failure to meet t...

Claims

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Application Information

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IPC IPC(8): G01N21/65
CPCG01N21/658
Inventor 胡勇军朱挺锋江宁静余萌张文建王棋
Owner SOUTH CHINA NORMAL UNIVERSITY
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