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Photoisomerization-based biosensor and application thereof in thrombin detection

A biosensor and photoisomerization technology, applied in the field of analysis and detection, can solve the problems of false positive test results, background fluorescence interference, complicated use of separation process, etc., achieve simple preparation and operation, realize recycling and regeneration, and have important academic and application prospects. Effect

Pending Publication Date: 2021-11-16
GUANGDONG MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing fluorescent aptasensors often face the background fluorescence interference of the matrix in the actual sample, which easily leads to false positive signals in the detection results, and the separation process in the sensing process is relatively too complicated, which limits its use.

Method used

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  • Photoisomerization-based biosensor and application thereof in thrombin detection
  • Photoisomerization-based biosensor and application thereof in thrombin detection
  • Photoisomerization-based biosensor and application thereof in thrombin detection

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preparation example Construction

[0051] The preparation method of the above-mentioned photoisomerization-based biosensor comprises the following steps:

[0052] (A1) Select a stilbene compound with an active reactive group as a fluorescent signal molecule, and react with a thrombin aptamer modified by an active reactive group in a solution to form a fluorescent aptamer sensor to obtain a fluorescent aptamer sensor products;

[0053] (A2) Separating and purifying the fluorescent aptasensor solution in step (A1) to obtain a purified fluorescent aptasensor product.

[0054] Further, in the step (A1), the reaction ratio of the fluorescent signal molecule to the thrombin aptamer is 5-15mM: 50-150μM, preferably 10mM: 100μM; the reaction solvent is sodium bicarbonate buffer, pH 8 -10, the reaction conditions are under the condition of avoiding light, magnetic stirring for 11-13h, preferably 12h; the reaction temperature is 15-30°C, the reaction temperature can be 15°C, 25°C and 30°C, more preferably 30°C.

[0055...

Embodiment 1

[0071] (1) Preparation of fluorescent aptasensors

[0072] Weigh an appropriate amount of 4-acetylamino-4'-stilbene-isothiocyanate-2,2'-disulfonic acid disodium salt (SITS) solid powder, add a certain amount of ultrapure water to dissolve it into a 100mM solution, as stock solution. Take 10 μL of 100 mM SITS solution and add it to 90 μL of sodium bicarbonate (0.1 M, pH=9.8) buffer solution to dilute it to 10 mM, and prepare it immediately for use. With an appropriate amount of ultrapure water, the sequence was 5'-NH 2 -(CH 2 ) 6 The amino-modified thrombin aptamer of -GTCCGTGGTAGG GCAGGTTGGGGTGAC-3' was dissolved and diluted to 100 μM, aliquoted into 100 μL tubes, and stored at -20°C. Take 100 μL each of the above 10 mM SITS and 100 μM nucleic acid aptamer solutions in the same 2 mL centrifuge tube, and react for 12 h at 30 ° C, protected from light, and magnetically stirred. Take out the reaction solution and use an ultrafiltration centrifuge tube to use 1M NaCl solution...

Embodiment 2

[0081] In order to illustrate the specificity of the present invention, cytochrome C (Cyt C), fibrinogen (FIB), lysozyme (Lys) and T4 polynucleotide kinase (T4 PNK) were selected as interfering proteins for parallel interference detection . Prepare the above-mentioned three proteins except T4 polynucleotide kinase and thrombin as standard solutions with concentrations of 2 μM and 5 μM, and T4 polynucleotide kinase to 35 U·mL -1 and 89U·mL -1 standard solution. Detect concrete steps with reference to embodiment one; The result is as follows Figure 4 As shown, the apparent fluorescence decay rate k of the sensor after the interfering protein was added app It was similar to that of the blank group, but had obvious changes in the group added with thrombin. This result shows that the present invention has good specificity and specificity.

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Abstract

The invention belongs to the technical field of analysis and detection, and relates to a photoisomerization-based biosensor and application thereof in thrombin detection. The biosensor is a fluorescent aptamer sensor, and the fluorescent aptamer sensor is formed by taking a stilbene compound as a fluorescent signal molecule to react with a thrombin aptamer in a covalent binding manner. The aptamer sensor is combined with thrombin, thrombin detection can be achieved, and the thrombin detection capacity of the aptamer is further confirmed by changing the sequence and modification sites of the aptamer; and the preparation operation is simple, interference of background fluorescence can be avoided theoretically, complex separation steps are not needed, analysis and detection of a sample can be completed within a short time, cyclic regeneration of the aptamer sensor can be achieved, the invention provides new inspiration for developing the design of biosensors for detecting biomacromolecules such as enzyme in the future, and has important academic and application prospects.

Description

technical field [0001] The invention relates to the technical field of analysis and detection, in particular to a biosensor based on photoisomerization and its application in thrombin detection. Background technique [0002] Thrombin is a sodium ion-activated serine proteolytic enzyme containing two polypeptide chains A and B. It is a biocatalyst produced by the organism itself and is also an important protein biomacromolecule. In organisms, it generally exists in the blood circulation in the form of prothrombin. When bleeding occurs in the organism, prothrombin is inactivated to produce thrombin, which promotes the cracking of fibrinogen in the blood into fibrin and forms coagulated blood clots. So as to achieve the effect of hemostasis, which is a self-protection mechanism of organisms. Thrombin is involved in the occurrence and development of various diseases in vivo, including the treatment of vascular diseases and tumors in the brain, and it can also affect the central...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6428G01N21/6408G01N21/64G01N2021/6417
Inventor 周郁斌陈慧芝周清徐晖
Owner GUANGDONG MEDICAL UNIV
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