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Biosensor for detecting uracil-DNA glycosylase, and preparation method thereof

A biosensor and glycosylase technology, applied in the field of biosensors, can solve the problems of complexity, difficulty in generalization, time-consuming sensitivity, etc., and achieve the effects of low detection limit, reduced complexity and high sensitivity

Active Publication Date: 2019-05-14
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditional UDG activity detection methods include gel electrophoresis, nucleic acid labeling and other methods, however, they require complex nucleic acid labeling and gel electrophoresis procedures, are time-consuming and have poor sensitivity, and are difficult to generalize
In order to overcome the above defects, some UDG activity detection methods based on colorimetry and fluorescence have been developed. These new technologies have brought great progress to the detection of UDG activity; however, further improvement is needed to achieve more sensitive and specific detection of UDG

Method used

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  • Biosensor for detecting uracil-DNA glycosylase, and preparation method thereof
  • Biosensor for detecting uracil-DNA glycosylase, and preparation method thereof
  • Biosensor for detecting uracil-DNA glycosylase, and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The steps for modifying Hairpin Probe and Track DNA to the surface of gold nanoparticles are as follows:

[0046] a. Take 1 mL nano-gold solution in a centrifuge tube, centrifuge for 15 min, and centrifuge the two tubes at the same time for later use. Centrifuge until the supernatant is colorless and transparent, remove the supernatant, and add 300 μL of sterile water to concentrate the nano-gold solution to 1 nM. Transfer to a 1 mL glass bottle, seal it with tinfoil, and add different volumes of Raman dyes (the final concentrations of 4NTP are 0.25uM, 0.5 uM, 1 uM, 2 uM and 3 uM, respectively).

[0047] b. After standing at room temperature for 30 min, add 150 μL of -SH-modified substrate probe (Hairpin Probe and Track DNA) with a concentration of 10 μM, mix well, and place at 4 °C for 24 h.

[0048] c. Slowly add 50 μL of PBS buffer several times, add magnets (soaked in aqua regia the day before) and stir for 10 min, then continue to add 27 μL of PBS buffer. Take ou...

Embodiment 2

[0055] The steps for modifying Hairpin Probe and Track DNA to the surface of gold nanoparticles are as follows:

[0056] a. Take 1 mL nano-gold solution in a centrifuge tube, centrifuge for 15 min, and centrifuge the two tubes at the same time for later use. Centrifuge until the supernatant is colorless and transparent, remove the supernatant, and add 300 μL of sterile water to concentrate the nano-gold solution to 1 nM. Transfer to a 1 mL glass bottle, seal it with tin foil, and add Raman dye (4NTP final concentration is 2 μM).

[0057] b. After standing at room temperature for 30 min, add 150 μL of -SH-modified substrate probe (Hairpin Probe and Track DNA) with a concentration of 10 μM, mix well, and place at 4 °C for 24 h.

[0058] c. Slowly add 50 μL of PBS buffer several times, add magnets (soaked in aqua regia the day before) and stir for 10 min, then continue to add 27 μL of PBS buffer. Take out the magnet, and place it at 4°C for 48 h.

[0059] d. After 48 hours, sl...

Embodiment 3

[0066] The steps for modifying Hairpin Probe and Track DNA to the surface of gold nanoparticles are as follows:

[0067] a. Take 1 mL nano-gold solution in a centrifuge tube, centrifuge for 15 min, and centrifuge the two tubes at the same time for later use. Centrifuge until the supernatant is colorless and transparent, remove the supernatant, and add 300 μL of sterile water to concentrate the nano-gold solution to 1 nM. Transfer to a 1 mL glass bottle, seal it with tin foil, and add Raman dye (4NTP final concentration is 2 μM).

[0068] b. After standing at room temperature for 30 min, add 150 μL of -SH-modified substrate probe (Hairpin Probe and Track DNA) with a concentration of 10 μM, mix well, and place at 4 °C for 24 h.

[0069] c. Slowly add 50 μL of PBS buffer several times, add magnets (soaked in aqua regia the day before) and stir for 10 min, then continue to add 27 μL of PBS buffer. Take out the magnet, and place it at 4°C for 48 h.

[0070] d. After 48 hours, sl...

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Abstract

The invention relates to the technical field of biosensors, and in particular to a biosensor based on a nanogold DNA molecular machine and surface enhanced Raman scattering, in order to solve the problems of relatively low method specificity and sensitivity and high cost in the method for detecting UDG activity in the prior art. A biosensor based on surface enhanced Raman scattering of a DNA molecular machine for detecting UDG activity combines the nanogold molecular machine with the surface enhanced Raman scattering to obtain homogeneous reaction mixed liquor. The preparation method comprisesthe following steps: synthesizing gold nanoparticles; modifying Hairpin Probe,Track DNA and Raman dye onto the surfaces of the gold nanoparticles; and mixing a labeled gold nano solution with a homogeneous reaction solution. The opening of the DNA molecular machine is realized by the specific cutting of incision enzyme IV, and high-sensitivity detection is realized by the surface enhanced Raman scattering; the circulation of the DNA molecular machine is achieved by using excision enzyme III, and a signal amplification role is achieved.

Description

technical field [0001] The invention belongs to the technical field of biosensors, in particular to a biosensor for detecting uracil-DNA glycosylase activity based on surface-enhanced Raman scattering of DNA molecular machines, and also relates to a preparation method thereof. Background technique [0002] The genome consists of specific pairings of DNA bases, the stability and accuracy of which are prerequisites for all living organisms. However, the structural properties of DNA bases can be disrupted by various environmental factors and endogenous reactive oxygen species, leading to genome instability and induction of carcinogenesis. Uracil is a common damaged base in DNA. During DNA replication, dUTP is misincorporated or cytosine is hydrolyzed and deaminated, resulting in the conversion of G:C base pairs into A:U base pairs, which eventually leads to gene mutation. Uracil in DNA is usually repaired by uracil-DNA glycosylase (UDG) in the base excision repair system. Ur...

Claims

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Application Information

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IPC IPC(8): G01N21/65
Inventor 王玉李莎莎刘素黄加栋张儒峰赵一菡瞿晓南孙文玉王业茹张曼如
Owner UNIV OF JINAN
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