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Biosensor for detecting hOGG1 activity and use thereof

A biosensor and activity technology, applied in the direction of instruments, measuring devices, scientific instruments, etc., can solve the problems of low sensitivity and specificity, achieve high detection sensitivity, sensitive detection, and improve the effect of reaction speed

Active Publication Date: 2018-06-15
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problems of low sensitivity and specificity in detecting hOGG1 activity in the prior art, the present invention provides a biosensor based on exonuclease III and DNA molecular machine isothermal amplification technology, which realizes the cyclic amplification of the target object, and uses Raman Spectroscopy Technology Realizes Precise Detection

Method used

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  • Biosensor for detecting hOGG1 activity and use thereof
  • Biosensor for detecting hOGG1 activity and use thereof
  • Biosensor for detecting hOGG1 activity and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of gold nanometers.

[0037] (1) Add 200ml of ultrapure water into the three-neck flask;

[0038] (2) Take 500uL of HAuCl with a concentration of 0.04g / mL 4 In the centrifuge tube, add 200ml ultrapure water, stir and heat to boil, stirring speed 450rpm;

[0039] (3) Under stirring conditions, take 3ml of 1% trisodium citrate solution and quickly add it to the solution in step (2). The color of the solution changes from light yellow to wine red. After continuing to heat for 15 minutes, remove the heat source and Slowly cool to room temperature, and store at 4°C for later use.

[0040] According to the absorbance value at 530 nm, the concentration of gold nanoparticles in the above solution is about 0.3 nM.

Embodiment 2

[0041] Example 2 Modification of gold nanometers.

[0042] (1) Walker DNA and Protect DNA were denatured at 90°C, annealed and hybridized at 55°C to form hybrid strands, and stored at 4°C for later use;

[0043] (2) Take 1 mL of the gold nano solution prepared in Example 1 in a centrifuge tube, centrifuge at 13000 rpm for 10 min. Centrifuge until the supernatant is colorless and transparent, remove the supernatant, and add 300 μL of sterilized water to make the concentration of the nano-gold solution to 3 nM. Transfer it to a 1mL glass bottle, seal it with tinfoil, and then add 12 μL of DTNB solution (final concentration: 2 μM) while stirring.

[0044] (3) After standing at room temperature for 30 min, add 150 μL of Walker DNA and Track DNA at a concentration of 30 μM, mix well, and place at 4 °C for 24 h.

[0045] (4) Slowly add 50 μL of PBS buffer several times, and add magnetic particles (ddH after soaking in aqua regia 24 hours in advance 2 O washed to neutral) after st...

Embodiment 3

[0048] Example 3 Effects of different concentrations of Raman dyes on hOGG1 glycosidase.

[0049](1) Add 2 μL of ExoⅢ (30U / mL) and 2 μL of hairpin probe (1U / mL) to 6 centrifuge tubes respectively, and add 2 μL of hOGG1 solution (1600U / ml) to 5 test tubes respectively, and 1 test tube Add an equal amount of PBS solution; shake the 6 test tubes for 30 seconds, and put them in a water bath at 37°C for 30 minutes;

[0050] (2) The above reaction solution was added to 20 μL of the surface-modified gold nano-solutions S2-S6 (1 nM) in Example 2, and placed in a water bath at 37°C for 120 min.

[0051] (3) Use a Raman spectrometer to detect the SERS spectrum of the reaction solution obtained in step (2).

[0052] According to the obtained spectra, the peak area ratios of the characteristic peaks of the SERS spectra of the positive samples and the blank samples were calculated respectively, and the signal-to-background ratios corresponding to the gold nanoparticle probes with differen...

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Abstract

The invention provides a biosensor for detecting 8-hydroxylaguanine DNA glycosidase activity. The biosensor comprises an exonuclease III, hairpin probes and gold nanoparticles modified by DNA on the surfaces. Through surface-enhanced Raman detection, the 8-hydroxylaguanine DNA glycosidase activity is detected. The biosensor has mild reaction conditions and a fast reaction rate. A detection methodusing the biosensor is easy to operate, has a short detection period and high detection sensitivity. The main processes based on the detection principle are carried out in a homogeneous phase so thatthe reaction speed is increased and the complexity of operation is reduced. The biosensor realizes fast, simple and sensitive detection of a target. The biosensor realizes a low preparation cost.

Description

technical field [0001] The invention relates to a biosensor for detecting hOGG1 activity based on surface-enhanced Raman scattering of DNA molecular machines and an application thereof, belonging to the field of nucleic acid aptamer biosensors. Background technique [0002] DNA molecules are particularly sensitive to reactive oxygen species and are vulnerable to reactive oxygen species (ROS) attacks, resulting in oxidative DNA damage. The major oxidation product of DNA molecules, 8-oxo-guanine (8-oxo-Gua), is regarded as a biomarker of DNA damage. 8-oxo-Gua is highly mutagenic, producing G:C-T:A transversion mutations when replicated by DNA polymerase. In mammalian cells, 8-oxo-Gua is primarily cleared by the 8-hydroxyguanine DNA glycosidase (hOGG1)-mediated base excision repair pathway. hOGG1 has DNA glycosylase and apurinic / apyrimidinic lyase activities, which can specifically recognize and excise 8-oxo-Gua produced by oxidative damage in DNA double strands, thereby rest...

Claims

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Application Information

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IPC IPC(8): G01N21/65
CPCG01N21/658
Inventor 王玉王海旺刘素黄加栋
Owner UNIV OF JINAN
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