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Nucleic acid sequence detection method based on QCM

A detection method and nucleic acid sequence technology, applied in the field of nucleic acid sequence detection based on QCM, can solve problems such as dependence on enzyme activity, complicated steps, and long time consumption, and achieve high specificity, promote in-situ growth, and inhibit non-specific crystallization Effect

Pending Publication Date: 2020-08-14
LINYI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, each method has certain disadvantages, such as time-consuming, complicated steps, high cost or heavy dependence on enzyme activity, etc.

Method used

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  • Nucleic acid sequence detection method based on QCM
  • Nucleic acid sequence detection method based on QCM
  • Nucleic acid sequence detection method based on QCM

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 A QCM-based nucleic acid sequence detection method

[0045] (1) Cleaning of the wafer: Put the wafer (the quartz crystal microbalance sensor wafer is produced by Biolin Scientific in Sweden, its diameter is 14mm, the basic frequency is 5MHz) in piranha washing liquid (H 2 SO 4 : H 2 O 2 =3:1 volume ratio) soak for 10 minutes, then follow H 2 O: H 2 O 2 : NH 3 ·H 2 The lye is prepared with a volume ratio of O=5:1:1, the prepared lye is heated and boiled on an electric heating mantle, and the wafer is immersed in the boiling lye for 15 minutes; keep the lye in a boiling state during the soaking process. Rinse with ultrapure water and soak in ultrapure water for 5 minutes, then rinse with absolute ethanol and soak in absolute ethanol for 5 minutes; blow dry with nitrogen, and place the dried wafer in a clean bench under ultraviolet light for 20 Minutes to remove organic matter on the surface;

[0046] (2) Modification of the chip:

[0047] ① Spread the cleaned wafer fl...

Embodiment 2

[0065] Example 2 Verification of signal amplification ability

[0066] The capture of tDNA, the hybridization of pDNA and the in-situ growth of calcium carbonate crystals can all be monitored in real time by the quartz crystal microbalance sensor. The frequency of the whole process changes as figure 2 Shown. It can be seen from the inset that because the quality of DNA is very small, there is no obvious frequency change when the cDNA concentration is 1 nM, and there is no obvious frequency change after pDNA hybridization. But when ammonium carbonate is added, the crystallization process begins to occur, and a very obvious frequency change can be observed at this time. The frequency change exceeds 10000 Hz, which is about 1000 times the signal change when directly detecting DNA. This obvious frequency change indicates that crystallization is a good way to amplify the signal of a quartz crystal microbalance.

Embodiment 3

[0067] Example 3 Detection of DNA by selective crystallization method of quartz crystal Weitian plain

[0068] The in-situ selective crystalline quartz crystal microbalance response system is to detect and record a series of different concentrations of tDNA in real time. The results are as follows image 3 Shown. It was found that the higher the tDNA concentration, the greater the frequency change. The experiment of each concentration was repeated three times. The linear working curve between the quartz crystal microbalance signal response and the logarithm of the DNA concentration is as follows Figure 4 Shown. The linear regression satisfies the equation y=-21044.13-117073㏒c, and its correlation coefficient R 2 Is 0.997, where y is the frequency change of the quartz crystal microbalance sensor during the surface crystallization process, and c is the concentration of DNA. The linear range of DNA detection concentration is very wide, from 10aM to 1nM, and its minimum detection li...

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Abstract

The invention discloses a nucleic acid sequence detection method based on QCM, and belongs to the technical field of biological detection. By combining effective utilization of two functional groups -N(CH3)3 and -COOH, it is proved that a quartz crystal microbalance detection technology for in-situ crystallization has high specificity. A self-assembled film of the terminal functional group -N(CH3)3 is modified on the surface of a wafer, so that the wafer can be passivated, and non-specific crystallization can be effectively inhibited; and tDNA is hybridized with pDNA modified with the terminalfunctional group -COOH, so that the in-situ growth of calcium carbonate crystals on the surface of the wafer can be effectively promoted, and the purpose of detecting the tDNA is achieved.

Description

Technical field [0001] The present invention relates to the field of biological detection technology, and more specifically to a QCM-based nucleic acid sequence detection method. Background technique [0002] In the fields of clinical diagnosis, genetic analysis, and medical identification, it is of great significance to develop a DNA detection technology with high sensitivity and low cost. Many DNA detection technologies based on optical, electrochemical and mechanical instruments have been greatly developed. Quartz crystal microbalance (QCM) is a quality detection sensor, which has been widely used in many fields such as legal identification, air pollution monitoring, dynamic monitoring of biomolecules, and solubility determination. Recently, more and more researches have turned to quartz crystal microbalance sensors for DNA detection. In order to further expand the sensitivity of detection, researchers have made great efforts, mainly through mass amplification to improve sen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6825
CPCC12Q1/6825C12Q2565/607C12Q2561/113C12Q2563/167
Inventor 刘丽赏吴从从陈晋荣张振麟张书圣
Owner LINYI UNIVERSITY
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