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Method for testing ultratrace DNA by dual-amplifying technique and electrochemical quartz crystal microbalance

A dual amplification, quartz crystal technology, applied in the direction of material electrochemical variables, biochemical equipment and methods, measuring devices, etc., can solve the problem that the sensitivity cannot meet the requirements, and achieve high sensitivity, enhanced electrochemical signal, and improved sensitivity.

Inactive Publication Date: 2002-10-30
WUHAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although QCM is a sensitive mass detector that can detect sub-nanogram mass changes, in actual detection, the concentration of DNA is far below the range of direct detection by QCM, especially as a biological tool for detecting ultra-trace changes. sensor, its sensitivity can not meet the requirements

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Example 1: Confirmation and mass amplification of the formation of targeted double-stranded DNA molecules with doxorubicin (ADM)-modified nano-magnetic microspheres to detect ultra-trace target DNA.

[0012] 1. Preparation of doxorubicin-modified magnetic microspheres: prepare 2.0×10 -6 The aqueous solution of mol / L doxorubicin is used to prepare the cross-linking reaction between amino groups and aldehyde groups. After adding 0.02 g of aldehyde-based magnetic microspheres (commercially available products), the mixture is stirred at 4°C for reaction 4 Hours, the microspheres were washed 3 times with double distilled water on the magnetic stand, added 0.5 mL of double distilled water, and stored at 4°C for later use.

[0013] 2. Self-assembled α-lipoic acid monolayer film: The quartz wafer was immersed in anhydrous methanol solution for 30 minutes before use, then ultrasonically cleaned for 1 minute, washed 3 times with double distilled water, and dried in the air. The ...

Embodiment 2

[0018] Example 2: Nano-magnetic microspheres modified with actinomycin D (ActD) were used to confirm the formation of targeted double-stranded DNA molecules and perform mass amplification to detect ultra-trace target DNA.

[0019] Preparation of nano-magnetic microspheres modified by actinomycin D: preparation 1.0×10 -6 The acetone solution of mol / L actinomycin D was used to prepare the cross-linking reaction between amino groups and aldehyde groups. After adding 0.02 g of aldehyde-based magnetic microspheres, the mixture was stirred at 4°C and reacted for 4 hours. The ball was washed 3 times with acetone on the magnetic stand, added 0.5 mL of acetone, and stored at 4°C for later use. All the other steps are the same as in Example 1.

Embodiment 3

[0020] Example 3: Confirmation and mass amplification of the formation of targeted double-stranded DNA molecules by albumin nanospheres modified with mitoxantrone, and detection of ultra-trace target DNA.

[0021] Preparation of albumin nanospheres modified by mitoxantrone: preparation 2.0×10 -6 The mol / L mitoxantrone aqueous solution is used to prepare the cross-linking reaction between amino groups and aldehyde groups. After adding 0.02 g of aldehyde-based albumin microspheres, the mixture is stirred at 4°C and reacted for 4 hours, 12000 rpm Centrifuge at 1 / min, wash 3 times with double distilled water, add 0.5 mL of double distilled water, and store at 4°C for later use. All the other steps are the same as in Example 1.

[0022] The present invention can also be labeled with doxorubicin drugs such as doxorubicin, epirubicin and zorubicin, and other anthracycline antineoplastic drugs such as bisantrene, or other drugs or biological reagents that can selectively interact wit...

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PUM

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Abstract

A method for detecting ultratrace DNA by electrochemical quartz crystal microbalance and double amplifying technique includes such steps as plating a thin Au or Ag film or quartz crystal, selectivelyadsorbing DNA by quartz crystal oscillator, molecular hybridizing of DNA to obtain dual-chain DNa, amplifying the detected signals in mass and electrochemical areas by the nano microbead marked by chemical or biologic reagent which can selectively act on the dual chains of DNA and has electrochemical activity, and measuring the mass signal and electrochemical signal. Its advantages are high selectivity and high sensitivity.

Description

technical field [0001] The invention relates to a method for detecting ultra-trace DNA by an electrochemical quartz crystal microbalance with double amplification technology. Background technique [0002] Quartz crystal microbalance (QCM), also known as piezoelectric sensor, is based on the piezoelectric effect. The principle of the piezoelectric DNA biosensor is: first coat a thin layer of gold or silver film on the quartz wafer, and then try to make the quartz crystal oscillator selectively adsorb DNA, and hybridize DNA molecules to another DNA containing complementary base sequence. Recognize, combine into double-stranded DNA, and use frequency changes to reflect hybridization. Since QCM can have a frequency response to the increase of nanogram-level mass on the surface, it is a very sensitive mass detection device suitable for detecting the change of single-stranded DNA (ssDNA) before and after hybridization, so it can be applied to DNA biology. sensor research. The c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N27/30G01N27/327G01N33/50
Inventor 张盛龙丁虹胡先明王海涛罗顺德
Owner WUHAN UNIV
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