Method for quantitative evaluating HP DNA hairpin configuration on substrate surface based on enzymic hydrolysis ability and background signal eliminating method based on enzymic hydrolysis ability

An evaluation method and background signal technology, applied in the field of DNA self-assembled membranes, can solve the problem of inability to distinguish the number and ratio of different configurations

Inactive Publication Date: 2018-06-22
BEIJING NORMAL UNIVERSITY
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Problems solved by technology

Electrochemical methods such as CV / CC can be used to measure the amount of DNA on the electrode surface through the labeling of electroactive substances, but the amount of electricity detected by the electrochemical method is the total number of mixed configurations of DNA on the electrode surface, and the number and ratio of different configurations cannot be distinguished

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  • Method for quantitative evaluating HP DNA hairpin configuration on substrate surface based on enzymic hydrolysis ability and background signal eliminating method based on enzymic hydrolysis ability
  • Method for quantitative evaluating HP DNA hairpin configuration on substrate surface based on enzymic hydrolysis ability and background signal eliminating method based on enzymic hydrolysis ability
  • Method for quantitative evaluating HP DNA hairpin configuration on substrate surface based on enzymic hydrolysis ability and background signal eliminating method based on enzymic hydrolysis ability

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Embodiment

[0052] 1. Removal of dimers in the Hairpin-16 / ssDNA self-assembled membrane

[0053] (1) Construction of Hairpin-16 / ssDNA self-assembled membranes (SAMs): 0-1 μmol Hairpin-16 and ssDNA were mixed and mixed with a vortex shaker to prepare Hairpin-16 / ssDNA mixed solutions with different ratios. Soak the activated gold electrode in the pre-configured Hairpin-16 / ssDNA mixed solution, and place it at room temperature for 4-24h to obtain the Hairpin-16 / ssDNA self-assembled membrane, as shown in figure 1 shown.

[0054] (2) Removal of dimers in the Hairpin-16 / ssDNA self-assembled membrane: Place the above-mentioned gold electrode in newly prepared hot water at 75-95°C for denaturation for 0.5-3 minutes, and then place the electrode statically in 10mM Tris-buffer (100mM~200mM Na + , 30mM~80mM Mg 2+ ) for 1 to 12 hours, the dimer configuration on the electrode surface can be removed, and the hairpin configuration can be refolded. Such as figure 2 As shown in the cyclic voltammogr...

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Abstract

The invention relates to the technical field of DNA self-assembled membranes, particularly to a method for quantitative evaluating the HP DNA hairpin configuration on a substrate surface based on enzymic hydrolysis ability and a background signal eliminating method based on enzymic hydrolysis ability. The specific steps comprise: removing dimers through high temperature denaturation; immersing a substrate in an exonuclease I buffer solution having an enzyme amount of 100-300 U, and carrying out hydrolysis; and determining the proportion of the HP DNA hairpin configuration on the surface of anelectrode by using an electricity integration technology. According to the present invention, exonuclease has difference between in hydrolysis of ssDNA and in hydrolysis of HP DNA hairpin configuration, and the electrochemical method is combined to quantitatively evaluate the proportion of the HP DNA hairpin configuration on the surface of the substrate; and with the exonuclease I, the backgroundsignal caused by non-hairpin configuration can be effectively eliminated so as to substantially improve the detection sensitivity of the Hairpin DNA-based biosensor.

Description

technical field [0001] The invention relates to the technical field of DNA self-assembled membranes, in particular to a method for quantitatively evaluating HPDNA configuration on a substrate surface based on enzyme hydrolysis ability and eliminating background signals. Background technique [0002] Hairpin DNA (HP DNA for short) is a single-stranded DNA that can form a "hairpin-like" configuration connected by a stem-loop (loop-stem). In recent years, HP DNA has been immobilized on the substrate as a probe to achieve rapid, high-sensitivity, high-throughput, label-free analysis and diagnosis of targets in the form of solid devices, and various types of HP DNA-based organisms have been produced. Chip / Sensor. [0003] However, when the HPD probes are immobilized on the substrate by conventional assembly means, the obtained probe film will be a mixed configuration including single-stranded coil configuration, hairpin configuration, and head-to-tail connected dimer configurati...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/48
CPCG01N27/48
Inventor 李运超高晓怡王杏林
Owner BEIJING NORMAL UNIVERSITY
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