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False complementary peptide nucleic acid probe biochip and detection method based on SPR principle

A biochip, peptide nucleic acid technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of reducing the universality and selectivity of PNA, reduce the cost of use and experimental conditions, and accurately detect and identify , high sensitivity and specificity

Inactive Publication Date: 2006-05-10
THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because traditional PNA can hybridize with single strand or dsDNA, but when combined with dsDNA in strand invasion mode, due to the strong binding force between PNA double strands, it can only be polyhomologous pyrimidine PNA (triple strand invasion, Triplex invasion) or polyhomopurine PNA (Double Strand Invasion, Duplex Invasion), which obviously reduces the generality and selectivity of PNA as a probe

Method used

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  • False complementary peptide nucleic acid probe biochip and detection method based on SPR principle
  • False complementary peptide nucleic acid probe biochip and detection method based on SPR principle
  • False complementary peptide nucleic acid probe biochip and detection method based on SPR principle

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Example 1 On a glass slide, a nano-gold film method with a surface plasmon resonance sensitivity of 50nm thickness is paved, and the steps are as follows:

[0056] (1), prepare gold sol by trisodium citrate reduction method (Frens method): get 100ml of 0.01% chloroauric acid aqueous solution and heat to boil, accurately add 0.75ml of 1% trisodium citrate aqueous solution under stirring, golden yellow chloroauric acid The aqueous solution turned purple within 2 minutes, continued to boil for 15 minutes, and returned to the original volume with distilled water after cooling.

[0057] (2), gold film paving:

[0058] A. After the slides are acid-washed and cleaned, put them in N-β-(aminoethyl)-γaminopropyl trivalent oxysilane (APTMS) / toluene solution and reflux for 12 hours, then wash them, take them out, and dry them with nitrogen , put in a clean and dry place for later use;

[0059] B. After soaking the treated glass slide in the nano-gold solution for 12 hours, immers...

Embodiment 2

[0060] Embodiment 2 The method of applying surface self-assembled monolayer technology to make pcPNAs probe array on the surface of gold film, the steps are as follows:

[0061] (1), prepared nano-gold film in piranha solution (30%H 2 o 2 : Concentrated H 2 SO 4 = 1: 3), then rinsed repeatedly with double distilled water, and dried with nitrogen;

[0062] (2), drop the SH-pcPNAs / PBS buffer solution of 500nM concentration and the self-assembly reaction on the nano-gold film, and react for 24 hours at 100% humidity to generate a self-assembled array of probes;

[0063] (3) Blot the buffer solution to dryness respectively, block with 6-mercaptohexanol, wash with double distilled water, and blow dry with nitrogen gas.

Embodiment 3

[0064] Example 3 One-step rapid processing of specimens with reagent Chelex-100 to extract bacterial DNA, the steps are as follows:

[0065] (1), collect one ring of pure culture colonies with a sterile inoculation loop, and place in a 1.5ml sterile Eppendorf tube;

[0066] (2) Add 50 μl of 5% Chelex suspension, mix well, add 20 mg / ml proteinase K, digest in a water bath at 56°C for more than 2 hours, then put it in a boiling water bath for 7.5 minutes, then immediately put it in an ice bath for 3 minutes, and centrifuge at 12000 r / min for 5 minutes , take the supernatant as a DNA sample.

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Abstract

This invention discloses a complementary peptidenucleic acid probe biological chip, wherein the solid-phase holder of the said chip joins with the complementary peptidenucleic acid probe. This invention also relates to the chip checking method basing on the plasma resonance SPR principle, which includes the crossing of the sample and the checking probes; clearing and wiping off the uncrossed samples; making the polarised light checking on each probe cross zone that is on the checking biological chip by using the plasma resonance equipment; using plasma resonance SPR principle to analyze the complementary peptidenucleic acid probe crossing reaction to obtain the result. This invention uses the pcPNAs as the cross probe, compraring to the ordinary DNA probe, it has higher sensitivity and especial charater, which not only avoids PCR reaction, but also delows the sample treatment request, simples the operation steps and shortens the checking time.

Description

technical field [0001] The invention relates to the field of biochips, in particular to a biochip using pseudo-complementary peptide nucleic acid as a probe and a detection method based on the principle of surface plasmon resonance. technical background [0002] With the wide application of molecular biology in the medical field, chip technology will surely become an indispensable new content in the future clinical disease diagnosis. Chip technology is produced along with the Human Genome Project. It is an important progress in molecular biology and medical diagnostic technology in recent years. Its outstanding features are high speed, high throughput, high parallelism, diversification, miniaturization, and automation. It has become a hotspot in the research of biomedical technology today, and has shown great development potential and application value in many fields such as genetic diagnosis, drug development, toxicity analysis, and pathogen detection. [0003] At present,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 顾大勇周元国石磊鲁卫平朱敏王华禹华伟梁冰张雅鸥
Owner THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA
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