Process for improving DNA detecting sensitivity

A technology of detection sensitivity and nano-metal particles, which is applied in the field of DNA detection, can solve the problems of DNA detection sensitivity decline and achieve the effect of increasing the degree of fixation and adsorption, and the same growth trend

Inactive Publication Date: 2003-12-31
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to overcome the problem that the DNA detection sensitivity decline caused by...

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  • Process for improving DNA detecting sensitivity
  • Process for improving DNA detecting sensitivity

Examples

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Embodiment 1

[0033] Example 1: 1,6-hexanedithiol was used as a linker to assemble 5 nm gold particles on the surface of QCM, and 20 nm gold particles were used as intensifying amplifiers for DNA detection

[0034] Take the AT-cut 9MHz QCM gold sheet, and use hot Piranha solution (98%H 2 SO 4 : 30%H 2 o 2 ) soaked for 3 minutes, fully rinsed with double distilled water, and measured its frequency with a quartz crystal microbalance. Modify the surface of QCM with 1,6-hexanedithiol, rinse with ethanol and twice double-distilled water after half an hour, and dry in the air. After QCM detection, add 20 microliters of 5nm gold sol dropwise, soak for 4 hours, rinse, and dry for measurement. frequency. Drop 2×10 -6 mol / L HS-DNA, after soaking for 1 hour, fully rinse and dry, then measure the frequency. Drop 2×10 -16 mol / L target DNA, after hybridization at 40°C for 2 hours, washed with phosphate buffer solution and twice redistilled water, and detected the frequency changes with a quartz cr...

Embodiment 2

[0035] Example 2: Use 1,6-hexanedithiol as linker to assemble 20nm gold particles on the surface of QCM and use 20nm gold particles as intensifying amplifiers for DNA detection

[0036] In a moist airtight chamber, drop 0.5% ethanol solution of 1,6-hexanedithiol onto the surface of QCM, rinse with ethanol and double-distilled water after half an hour, dry it, add dropwise after QCM detection 20 microliters of 20nm gold sol, soaked for 4 hours, rinsed, and dried for frequency measurement, there was a mass change of about 50ng. Drop 2×10 -6 mol / L HS-DNA, after soaking for 1 hour, fully rinsed and dried, and measured the frequency, the mass change was 107ng, while the mass change of HS-DNA immobilized directly on the bare QCM surface was 28ng. Drop 2×10 -16 mol / L target DNA, washed with phosphate buffer solution and double distilled water after hybridization at 40°C for 2 hours, the frequency change was 93ng. 20 microliters of DNA functionalized with 20nm gold sol was added dr...

Embodiment 3

[0038] Example 3: Using 1-amino 6-mercaptohexanethiol as linker to assemble 10nm gold particles on the surface of QCM and using 20nm gold particles as intensifying amplifiers for DNA detection

[0039] The surface of QCM was modified with 1-amino6-mercaptohexanol, and 20 microliters of 5nm gold sol was added dropwise after QCM detection, soaked for 4 hours, rinsed, and dried for frequency measurement. Drop 2×10 -6 mol / L HS-DNA, after soaking for 1 hour, fully rinse and dry, then measure the frequency. Drop 2×10 -16 mol / L target DNA, after hybridization at 40°C for 2 hours, washed with phosphate buffer solution and twice redistilled water, and detected the frequency changes with a quartz crystal microbalance. Hybridize with 20 microliters of DNA functionalized with 20nm gold sol, and use a quartz crystal microbalance to detect its frequency change and find that its detection sensitivity can reach 10 -16 mol / L.

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Abstract

The present invention relates to one kind of method of detecting DNA crossbreeding and mispairing in raised sensitivity based on DNA base pairing principle. Nano gold particles in different sizes are used separately as the surfactant of quartz crystal microbalance and distinguishing amplifier as DNA detector. By means of the decoration of the metal surface of the quartz crystal microbalance with double-functional radical connecting agent, nano metal particles are fixed to the quartz crystal microbalance. Single-strand DNA probe is then fixed onto the nano metal decorated surface, and the DNA probe and the target DNA single strand to be detected are made to crossbred. The relatively longer target DNA has one part complementary with the probe DNA via being fixed onto the quartz crystal microbalance and the other part paired with the complementary DNA single strand connected to nano metal particles to raise the detection sensitivity to 10E(-16).

Description

technical field [0001] The invention belongs to the field of DNA detection, in particular to a method for detecting DNA hybridization and mismatching with improved sensitivity based on the principle of DNA base pairing. Background technique [0002] In recent years, gene analysis has been widely used in many fields such as medicine, law, environment, etc., and has become a hot spot of scientific research. How to detect gene mutations at the molecular level, quickly and sensitively detect the number of gene mutations, and provide basic data for finding the correlation between gene mutations and lesions has always been the main topic of research. Therefore, it is very important and urgent to develop a simple and economical method for rapid detection of trace gene mutations. Quartz crystal microbalance (QCM) can detect DNA in a timely manner, and the method is quick and easy, but the sensitivity of this method is still relatively low and cannot reach the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 江龙唐季安刘涛
Owner INST OF CHEM CHINESE ACAD OF SCI
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