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Universal fluorescent biosensor for detecting ATP, glutathione and Fpg glycosylase

A biosensor and glycosylase technology, applied in the field of general-purpose fluorescent biosensors, can solve the problems of complicated instrument operation and expensive equipment, and achieve the effects of good repeatability, fast response and fast speed

Active Publication Date: 2021-10-26
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims at the disadvantages of complex instrument operation, expensive equipment and professional operation in the existing detection method, and provides a detection method with high sensitivity, strong specificity and low cost A Universal Fluorescent Biosensor for the Detection of ATP, Glutathione and Fpg Glycosylase

Method used

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  • Universal fluorescent biosensor for detecting ATP, glutathione and Fpg glycosylase
  • Universal fluorescent biosensor for detecting ATP, glutathione and Fpg glycosylase
  • Universal fluorescent biosensor for detecting ATP, glutathione and Fpg glycosylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] A preparation method of the optical biosensor of the present invention, comprising the following steps:

[0063] a. Put 5 µL sgRNA (50nM), 5 µL ATP block (50nM), 5 µL 1×TAE in a 1.5mL centrifuge tube, vortex and mix well, put it in a water bath at 95°C for 5 minutes, and then cool to room temperature ;

[0064] b. Take 5 µL Apt1 (100nM), 5 µL Apt2 (100nM), 5 µL Cas12a protein (100nM), 5 µL S chain (50nM), 5 µL 10×Buffer2.1, 1 µL FQ chain (1µM), 4 Add µL sterile water, 5 µL ATP (0 µM, 100nM, 200nM, 500nM, 1µM, 2µM, 3µM, 4µM, 5µM, 6µM) to the 15µL solution obtained in the previous step;

[0065] c. Place the centrifuge tube in a 37°C water bath for 90 minutes;

[0066] d. Place the centrifuge tube in a 65°C water bath for 10 minutes to terminate the reaction;

[0067] e. The excitation wavelength is 485nm, the emission wavelength is 520nm, the voltage is 670V, and the target to be tested is detected through the change of fluorescence.

[0068] see results figure 2 ,...

Embodiment 2

[0070] A preparation method of the optical biosensor of the present invention, comprising the following steps:

[0071] a. Put 5 µL sgRNA (50nM), 5 µL GSH block (50nM), 5 µL 1×TAE in a 1.5mL centrifuge tube, vortex and mix well, put it in a water bath at 95°C for 5 minutes, and then cool to room temperature ;

[0072] b. Take 5 µL Cas12a protein (100nM), 5 µL S chain (50nM), 5 µL 10×Buffer2.1, 1 µL FQ chain (1 µM), 14 µL sterile water, 5 µL GSH (0 µM, 100nM, 200nM , 500nM, 1µM, 2µM, 4µM), added to the 15µL solution obtained in the previous step;

[0073] c. Place the centrifuge tube in a 37°C water bath for 90 minutes;

[0074] d. Place the centrifuge tube in a 65°C water bath for 10 minutes to terminate the reaction;

[0075] e. The excitation wavelength is 485nm, the emission wavelength is 520nm, the voltage is 670V, and the target to be tested is detected through the change of fluorescence.

[0076] see results image 3 ,from image 3 It can be seen in A that the dete...

Embodiment 3

[0078] A preparation method of the optical biosensor of the present invention, comprising the following steps:

[0079] a. Put 5 µL sgRNA (50nM), 5 µL Fpg block (50nM), 5 µL 1×TAE in a 1.5mL centrifuge tube, vortex and mix well, put it in a water bath at 95°C for 5 minutes, and then cool to room temperature ;

[0080] b. Take 5 µL Cas12a protein (100nM), 5 µL S chain (50nM), 5 µL 10×Buffer2.1, 1 µL FQ chain (1 µM), 14 µL sterilized water, 5 µL Fpg (0 µM, 50nM, 100nM , 200nM, 500nM, 1µM), added to the 15µL solution obtained in the previous step;

[0081] c. Place the centrifuge tube in a 37°C water bath for 90 minutes;

[0082] d. Place the centrifuge tube in a 65°C water bath for 10 minutes to terminate the reaction;

[0083] e. The excitation wavelength is 485nm, the emission wavelength is 520nm, the voltage is 670V, and the target to be tested is detected through the change of fluorescence.

[0084] see results Figure 4 . from Figure 4 It can be seen in A that the d...

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Abstract

The invention relates to the technical field of biosensors, in particular to a universal fluorescent biosensor for detecting ATP, GSH and Fpq glycosylase, which aims to solve the problems of low specificity and sensitivity and high cost of a detection method in the prior art. A universal fluorescent biosensor for detecting ATP, GSH (glutathione) and Fpq glycosylase is designed, according to the property of a target object, the ATP is combined with an aptamer to mediate strand displacement, GSH breaks a disulfide bond and Fpg glycosylase breaks 8oxo-G, so that closed sgRNA is mediated to restore conformation, Cas12a protein is combined, single-stranded DNA can be cut in a non-specific manner, a fluorescent signal is generated, and quantification of the target object is realized. The sensor has the advantages of high detection speed, low detection limit, high specificity and the like.

Description

technical field [0001] The invention belongs to the technical field of fluorescent biosensors, in particular to a general fluorescent biosensor for detecting ATP, glutathione and Fpg glycosylase. Background technique [0002] Adenosine triphosphate (ATP) is an important biomolecule in organisms and an essential energy source for cell synthesis. Abnormal expression of ATP is closely related to various diseases and is a reliable indicator for disease detection. [0003] Glutathione (GSH) is a thiol-containing tripeptide substance abundant in mammals. It is composed of glutamic acid, cysteine ​​and glycine. It has anti-oxidation, scavenging free radicals and regulating important functions capacity for physiological processes within cells. Since GSH is an important antioxidant substance, its abnormal content in the body is closely related to many diseases. [0004] A large amount of DNA damage is spontaneously generated in organisms every day, which induces gene mutation and i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6402G01N21/6486
Inventor 黄加栋朱志学王玉刘素王业茹孙文玉张曼茹江龙李静静张清心徐婉晴
Owner UNIV OF JINAN
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