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Nucleic acid detection method based on surface plasmon resonance technology

A technology of surface plasmon and detection method, which is applied in the field of nucleic acid analysis, can solve problems such as difficult widespread application, pollution, and unstable structure, and achieve the effects of easy popularization and application, good biocompatibility, and sensitive detection

Inactive Publication Date: 2015-10-07
NANJING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] (1) The sensitivity of the nucleic acid detection method is low, it is difficult to meet the requirements of clinical sample detection, and the structure is not stable enough and the repeatability is poor, such as literature 1
[0005] (2) Nanomaterials are expensive, special equipment is required for synthesis, biological functionalization is difficult, and nanogold production and nanomorphology control are complicated, which restricts its mass production and application, such as literature 2
[0006] (3) In the detection methods of the above-mentioned documents 1 and 2, the main focus is on synthesizing nanomaterials with specific morphology with metal elements such as gold and silver to enhance the steric effect enhancement signal of the gold film interface of the SPR chip, which is easy to cause pollution. And these methods are not universal, so it is difficult to widely apply

Method used

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  • Nucleic acid detection method based on surface plasmon resonance technology
  • Nucleic acid detection method based on surface plasmon resonance technology
  • Nucleic acid detection method based on surface plasmon resonance technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The SPR-based nucleic acid detection method uses a carboxylated SPR gold-plated chip as a carrier, EDC and NHS activate the carboxyl group, and then assembles the amino-terminated cDNA and ethanolamine onto the surface of the gold-plated chip through amidation reaction as a capture probe and seals the interface. The process is as follows:

[0042] (1) The bare SPR chip was prepared in piranha acid (concentrated H 2 SO 4 : Concentrated H 2 o 2 =1:3) After soaking for 10 minutes, then use the boiling mixture (concentrated ammonia water: concentrated H 2 o 2 : ultrapure water = 1:1:4) for 30 minutes, and finally washed twice with ultrapure water and ethanol 2 Blow dry; then, soak the treated gold flakes in 10-30mM MPA for 10-15 hours for pre-treatment, wash with ethanol and N 2 Blow dry and set aside.

[0043] (2) Soak the gold-plated sheet with (0.4M EDC / 0.1M NHS) for 30 minutes to activate the carboxyl groups on the surface of the gold sheet. Soak in 1-5 μM amino...

Embodiment 2

[0045] Generate long-chain dsDNA by hybridization chain cycle reaction, and verify its properties and structural characteristics:

[0046] Firstly, long-chain dsDNA is generated by hybridization chain cycle reaction, and the synthesis steps are as follows:

[0047] (1) Use buffer (0.5-1M dipotassium hydrogen phosphate and potassium dihydrogen phosphate to add 0.2-0.25M sodium chloride to adjust the pH to 7-7.5; the same below) to dissolve the cervical DNA (H1 and H2) solution.

[0048] (2) Dilute the neck loop DNA (H1, H2) to 10 μM, take 100 μL each and mix it, then quickly raise the temperature to 90-95°C, keep it for 5-10 minutes, take it out, and quickly cool it to room temperature in an ice-water bath to complete the hybrid chain cycle reaction to obtain long-chain dsDNA.

[0049] (3) Verify the HCR reaction product by agarose electrophoresis: prepare 2.5% agarose gel, add H1, H2 and HCR reaction product dsDNA into the electrophoresis tank respectively. The voltage was a...

Embodiment 3

[0052] Using SPR technology to characterize the HCR reaction process, the reaction process is as follows:

[0053] (1) Rinse the surface of the Au / cDNA assembly layer with 100uL buffer solution, and obtain the baseline a after equilibration; add 1uM target DNA solution diluted with 100uL buffer solution, and react with the Au / cDNA assembly layer for 1-3 hours to obtain Au / cDNA / target DNA Layers were assembled and flushed with buffer to reach equilibrium baseline b.

[0054] (2) Dilute the neck loop DNA (H1, H2) to 5-10 μM with buffer solution, take 100 μL each and mix, then quickly raise the temperature to 90-95 °C, keep it for 5-10 minutes, take it out, cool it to room temperature in an ice-water bath, and then add it quickly In the Au / cDNA / target DNA assembly layer, react for 10-12 hours to obtain the Au / cDNA / dsDNA assembly layer, wash with buffer to reach the equilibrium baseline c.

[0055] Such as image 3 As shown, the results show that after adding the target DNA, bec...

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Abstract

The invention discloses a nucleic acid detection method based on surface plasmon resonance technology, which belongs to the field of nucleic acid analysis. The nucleic acid detection method is combined by hybrid chain polymerization and a porphyrin groove embedded technology, so as to prepare a porphyrin-dsDNA long-chain polymerization nanoscale efficient biomimetic catalyst, and redox deposition reaction of 4-chlorine-1-naphthol and hydrogen peroxide is catalyzed by utilizing the catalyst, so as to obtain a significant SPR enhanced signal. The nucleic acid detection method has the advantages of high sensitivity, low price, simplicity in preparation, good stability and repeatability and the like when being used for detecting nucleic acid.

Description

technical field [0001] The invention belongs to the field of nucleic acid analysis, in particular to a nucleic acid detection method based on surface plasmon resonance technology. Background technique [0002] Cancer is the disease with the second highest mortality rate and the mortality rate is still increasing, but there is no specific treatment drug for this type of disease, so explore highly sensitive nucleic acid analysis strategies for early detection of oncogenes, especially for specific DNA / RNA sequences at low physiological levels analysis method is very necessary. Surface plasmon resonance (SPR) technology is a simple and direct sensing technology. It is a charge layer formed by surface plasmons on the interface between metal and dielectric. Under the excitation of electromagnetic waves, surface plasmons resonate. Since the technology was first used in the determination of the interaction between antibodies and their antigens in the 1980s, it has been widely used ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6825C12Q2565/628
Inventor 邓盛元袁培新辛鹏季旭波单丹
Owner NANJING UNIV OF SCI & TECH
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