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Ultratrace DNA detecting method by means of nano microsphere amplificaltion technology and piezoelectric DNA biosensor

A biosensor, nano-microsphere technology, applied in biochemical equipment and methods, microbial determination/inspection, biological testing, etc., can solve the problem that the sensitivity cannot meet the requirements, and achieve the effect of high sensitivity and strong selectivity

Inactive Publication Date: 2002-10-23
WUHAN UNIV
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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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  • Ultratrace DNA detecting method by means of nano microsphere amplificaltion technology and piezoelectric DNA biosensor
  • Ultratrace DNA detecting method by means of nano microsphere amplificaltion technology and piezoelectric DNA biosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1: Nano-magnetic microspheres modified with actinomycin D were used to confirm the formation of targeted double-stranded DNA molecules and perform mass amplification to detect ultra-trace target DNA.

[0013] 1. Preparation of nano-magnetic microspheres modified by actinomycin D: prepare 1.0×10 -6 The acetone solution of mol / L actinomycin D (ActD) is used to prepare the cross-linking reaction between amino-aldehyde groups, after adding 0.02g aldehyde-based magnetic microspheres (available commercially available products), this mixture is placed in 4 Stir and react at °C for 4 hours, wash the microspheres with acetone three times on the magnetic stand, add 0.5 mL of acetone, and store at 4°C for later use.

[0014] 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. Quickly...

Embodiment 2

[0017] Example 2: Using doxorubicin-modified nano-magnetic microspheres to confirm the formation of targeted double-stranded DNA molecules and perform mass amplification to detect ultra-trace target DNA.

[0018] Preparation of doxorubicin-modified magnetic microspheres: prepare 2.0×10 -6 The aqueous solution of mol / L doxorubicin (ADM) is 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 is stirred at 4 °C and reacted for 4 hours. Wash the ball with double distilled water three times on the magnetic stand, 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.

[0019] Its result shows, compared with conventional piezoelectric DNA biosensor, the present invention has greatly improved the sensitivity of QCM (referring to accompanying drawing, among the figure abscissa is time Time / Sec, and ordinate is frequ...

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 0.5mL 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 with do...

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Abstract

On the quartz wafer a thin layer of gold film or silver film is plated, then the quartz wafer is vibrated to selectively adsorb DNA, and the hybridization of DNA molecule can be used to combine into double-chain DNA, then the nano microsphere of medicine of biological reagent which is marked to make selective action with double-chain DNA can be used to confirm target double-chain DNA molecule formation and make mass amplification so as to detect ultratrace target DNA. Its selectivity is strong and its sensitiveness is high.

Description

technical field [0001] The invention relates to a method for detecting ultra-trace DNA by a piezoelectric DNA biosensor with nanometer microsphere amplification technology. Background technique [0002] Piezoelectric sensors, also known as quartz crystal microbalances (QCM), are 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. [0...

Claims

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Application Information

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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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