Nano-sensor for detecting O-acetylglucosamine transferase and detection method of nano-sensor

An acetylglucosamine and nano-sensor technology, applied in biochemical equipment and methods, instruments, measuring devices, etc., can solve the problems of enzyme activity loss, poor sensitivity, low efficiency, etc., and achieve low cost, high sensitivity and high efficiency. Effect

Inactive Publication Date: 2018-02-23
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are complex due to the complex chemistry required to transfer solution-phase enzyme reaction products to microwell plates, specific antibodies (e.g., specific glycopeptide monoclonal antibodies, anti-tetramethylrhodamine antibodies), and fluorescent UDP-GlcNAc analogues. Synthesis and design of complex FRET dyes, so the efficiency is low and the sensitivity is relatively poor
In addition, OGT is a temperature-sensitive enzyme, and the loss of enzyme activity may occur during enzyme purification, so quantitative detection of intracellular OGT activity in real samples is a great challenge.

Method used

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  • Nano-sensor for detecting O-acetylglucosamine transferase and detection method of nano-sensor
  • Nano-sensor for detecting O-acetylglucosamine transferase and detection method of nano-sensor
  • Nano-sensor for detecting O-acetylglucosamine transferase and detection method of nano-sensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 A nanosensor for detecting O-acetylglucosamine transferase based on a single quantum dot

[0041] The nanosensor for detecting O-acetylglucosamine transferase based on a single quantum dot includes: cyanine 5 / biotin-modified peptide chain; non-radioactive uridine-acetylglucosamine, proteinase K and streptavidin-coated quantum dots. Wherein, the peptide chain modified by cyanine 5 / biotin is: Biotin-Ser-Thr-Pro-Val-Ser-Arg-Ala-Asn-Met-Lys-Cy5.

Embodiment 2

[0042] Example 2 A detection method of a nanosensor based on a single quantum dot to detect O-acetylglucosamine transferase

[0043] The detection method of described O-acetylglucosamine transferase comprises the following steps:

[0044] S1. Contains 2-3 μmol per liter of cyanine 5 / biotin-modified peptide, 90 μmol per liter of uridine-acetylglucosamine, 0.01 unit of alkaline phosphatase, OGT to be tested and 10× reaction buffer Solution, the total volume of the composition is 20 microliters of the reaction solution and incubated at 37 ° C for 1 hour; Calcium chloride, composed of 0.5% Tween-20 and 0.5% NP-40, pH value is 7.8;

[0045] S2. Add 1 nanogram of proteinase K to the glycosylation reaction product in a system with a total reaction volume of 30 microliters, and react at 50° C. for 1 hour;

[0046] S3. Take 20 microliters of the reaction product of step S2 and react with 5 nanomoles per liter of quantum dots in 100 microliters of incubation buffer at room temperature...

Embodiment 3

[0048] Example 3 A detection method of a nanosensor based on a single quantum dot to detect O-acetylglucosamine transferase

[0049] The detection method of described O-acetylglucosamine transferase comprises the following steps:

[0050] S1. Contains 2-3 micromoles per liter of cyanine 5 / biotin-modified peptide, 100 micromoles per liter of uridine-acetylglucosamine, 0.1 unit of alkaline phosphatase, OGT to be tested and 10× reaction buffer Solution, the total volume of the composition is 20 microliters of the reaction solution and incubated at 37 ° C for 1 hour; Calcium chloride, composed of 0.5% Tween-20 and 0.5% NP-40, pH value is 7.8;

[0051] S2. Add 2 nanograms of proteinase K to the glycosylation reaction product in a system with a total reaction volume of 30 microliters, and react at 60° C. for 3 hours;

[0052] S3. Take 40 microliters of the reaction product of step S2 and react with 15 nanomoles per liter of quantum dots in 100 microliters of incubation buffer at r...

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Abstract

The invention belongs to the technical fields of biological analysis and detection and discloses a nano-sensor for detecting O-acetylglucosamine transferase based on a single quantum dot and a detection method of the nano-sensor. The nano-sensor comprises a cyanine 5/biotin modified peptide chain, non-radioactive uridine-acetylglucosamine and the quantum dot coated with streptavidin, wherein a cyanine 5/biotin modified peptide chain is Biotin-Ser-Thr-Pro-Val-Ser-Arg-Ala-Asn-Met-Lys-Cy5. The detection method of the nano-sensor is very simple, enzyme purification is not required, a radioactive isotope labeled sugar donor, a specific antibody and a fluorescence UDP-GlcNAc analogue do not need to be synthesized, the detection limit can be decreased to 3.47*10<-13> mol/L, and the sensitivity isvery high. The nano-sensor can be further applied to the measurement of enzyme kinetics parameters, the screening of an OGT inhibitor and the quantitative determination of OGT activity of cells.

Description

technical field [0001] The invention belongs to the technical field of biological analysis and detection, and in particular relates to a nanosensor for detecting O-acetylglucosamine transferase based on a single quantum dot and a detection method thereof. Background technique [0002] Protein glycosylation is a ubiquitous post-translational modification in which proteins are modified by an oxygen-linked N-acetylglucosamine (O-GlcNAc) monomer at a serine or threonine residue, known as the oxygen-linked Type acetylglucosamine glycosylation. O-GlcNAc plays an important role in a variety of cellular processes, including signal transduction, gene expression, protein degradation, metabolism, circadian rhythms, and neurodegenerative diseases. Human oxygen-linked acetylglucosamine transferase (OGT) is a cellular endogenous enzyme that catalyzes the transfer of the GlcNAc monomer from uridine-acetylglucosamine (UDP-GlcNAc) to the GlcNAc formed by serine or threonine residues of prot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48G01N21/64
CPCC12Q1/48G01N21/6486G01N2333/91102
Inventor 张春阳胡娟李玥颖
Owner SHANDONG NORMAL UNIV
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