Fluorescent biosensor of beta-glucosyltransferase, detection method and application

Abstract

The invention discloses a fluorescent biosensor of beta-glucosyltransferase as well as a detection method and application. The fluorescent biosensor comprises a detection probe, incision enzyme, excision enzyme, helicase, polymerase, primer DNA and SYBR dye; the detection probe is of a hairpin structure formed by single-stranded DNA, and has a recognition site capable of being recognized and cut by the incision enzyme, the recognition site contains 5-hydroxymethyl cytosine, and the two ends of the single-stranded DNA are modified with phosphate ester; the detection probe can be further cut bythe excision enzyme after being recognized and cut by the incision enzyme; the helicase can be used for untwisting the stem part of the hairpin structure; after the primer DNA and the single-strandedDNA are complementary, double-stranded DNA is formed under the action of the polymerase; and the SYBR dye can be specifically combined with the double-stranded DNA. The fluorescent biosensor providedby the invention has the advantages of being high in sensitivity to the beta-glucosyltransferase, good in specificity and short in detection time.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, and relates to a fluorescent biosensor of β-glucose transferase, a detection method and an application. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] β-glucosyltransferase (β-GT) is a DNA modifying enzyme that catalyzes the transfer of uridine diphosphate glucose (UDP-glucose) to the hydroxyl group of 5-hydroxymethylcytosine (5-hmC) to form β-glucosyl-5-hydroxymethylcytosine (5-ghmC). In phage, 5-hmC glycosylation catalyzed by β-GT is a key protective mechanism to prevent phage DNA from being digested by nucleases in host bacteria. In addition, 5-hmC glycosylation is also relate...

AI Technical Summary

Problems solved by technology

[0004] Traditional β-GT detection methods include capillary zone electrophoresis and radioactive isotope labeling (UDP-[ 14 C]-glucose or UDP-[ 3 H]-glucose), they are used for the detection of β-GT activity by directly monitoring the reaction product UDP or 5-ghmC of β-GTase, but the disadvantages such as various procedures, relatively expensive instruments, and radioactive hazards of the substrate are greatly improved. limit their wide application

Recently, novel β-GT biosensors based on electrochemical, photoelectrochemical, and electrochemiluminescent detection have been developed, which have the advantages of low detection cost, good reproducibility, and high selectivity; however, the inventors found that these biological Most of the sensors require relatively complicated fixation, washing and separation steps, and the process of preparing and functionalizing nanomaterials (including magnetic particles, tungsten disulfide nanosheets, gold nanoparticles and gold nanoclusters, etc.) is also cumbersome; in addition, due to these The high background caused by the method itself and the lack of proper signal amplification strategies lead to relatively low sensitivity of these biosensors

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