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Nano-gold signal probe for DNA detection, production method and method for detecting DNA

A signal probe and nano-gold technology, which is applied in the field of nano-gold signal probe and its preparation, and the DNA detection field of nano-gold signal probe, can solve the problems of increasing the steps of the reaction, and achieve high-sensitivity detection, convenient use, and preparation simple effect

Inactive Publication Date: 2008-08-20
SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is very sensitive, but it needs to separate the DNA from the gold nano surface for detection, which increases the steps of the reaction

Method used

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  • Nano-gold signal probe for DNA detection, production method and method for detecting DNA
  • Nano-gold signal probe for DNA detection, production method and method for detecting DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation of nanozymes

[0052] The method for preparing nanozyme 1 is: adding 100 μL concentration of 1% (w / v) HRP (purchased from Sigma company) solution into 5 mL nano-gold solution (HRP: nano-gold molar ratio is 1250: 1), with NaOH solution Adjust the pH to 9.0. This was followed by incubation at 37°C for 30 minutes with gentle shaking (350 rpm). Then it was concentrated to 1mL by centrifugation (13,000rpm, 30min, 4°C) (nano-gold concentration was about 10nM). Then add thiol-modified DNA2 solution (final concentration 1.5 μM, molar number of thiol-modified signal probe is 300 times that of gold nanoparticles), and incubate at room temperature for 16 hours. Then, a certain amount of 1M PBS was added gradually until the final concentration of NaCl was 0.1M, and left at room temperature overnight. Afterwards, 100 μL of 10% (w / v) BSA solution was added to block the vacant sites on the surface of the gold particles, and blocked at room temperature for 30 m...

Embodiment 2

[0057] Embodiment 2 The influence of reaction temperature of the present invention on target gene detection

[0058] According to the product instructions, MMPs were first washed 3 times with 0.5×SSC buffer before use, then biotin (biotin)-labeled capture probe DNA 1 was added to MMPs containing TTL buffer, and mixed gently for about 10 minutes . The surface density of capture probes is about 4-6×10 11 chain / cm 2 . Then the complex of MMPs and capture probe (MMPs-capture probe, denoted as MMPs-cp) was washed twice with TTA buffer, suspended in the hybridization solution, and refrigerated at 4°C for use.

[0059] Add 50 μL of MMPs-cp into a 1.5 mL centrifuge tube, discard the supernatant by magnetic separation, then add 1 mL of hybridization buffer, and at the same time add specific targeting DNA3 at a concentration between 0 and 25 nM. °C, 800 rpm, and react for 1 hour. Then add 50 μL nanozyme working solution, and react at 37°C for another 1 hour. Finally, magnetic sepa...

Embodiment 3

[0061] Embodiment 3 The present invention is to the specificity of DNA detection

[0062] The experiment was carried out with reference to the conditions in Example 2, the reaction temperature was 37° C., and the specificity of the detection method of the present invention for DNA detection was analyzed. Specific targeting sequence DNA3 at a concentration of 250pM and non-homologous sequence DNA4 at a concentration of 250nM were tested.

[0063] The result is shown in Figure 3. It can be seen that even non-homologous sequences at concentrations as high as 1000-fold can be significantly distinguished from specific targets, indicating the high specificity of the method.

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Abstract

The invention discloses a nano-gold signal probe for DNA detection and the preparation method thereof, as well as a DNA detection method; the nano-gold signal probe is that the surface thereof is simultaneously assembled with proteins which can generate chromogenic or fluorescence signals and a sulfhydryl-modified signal probe DNA nano-gold-particle solution. A solid phase carrier is utilized in the DNA detection, and a sandwich complex of the solid phase carrier-target DNA-nano-gold signal probe is formed by DNA hybridization. As the surface of one nano-gold particle can be connected with a plurality of proteins which can generate chromogenic or fluorescence signals, one protein which can generate chromogenic or fluorescence signals, especially an enzyme catalyst, can catalyze a plurality of substrates to have chemical reactions, so as to amplify reaction signals, and further to significantly improve detection sensitivity. Therefore, the nano-gold signal probe, the preparation method and the DNA detection method have important significances in the biological detection, the early diagnosis and the treatment of diseases.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a nano-gold signal probe for DNA detection, a preparation method thereof, and a DNA detection method using the nano-gold signal probe. Background technique [0002] DNA detection is of great significance in the fields of clinical diagnosis, single nucleotide polymorphism analysis, DNA sequencing, forensic identification, organ transplantation, environmental analysis, and anti-terrorism. As people pay more and more attention to their own health, there is an urgent need for early diagnosis and treatment of various major diseases, which requires highly sensitive detection of disease-causing genes. However, since the content of the disease-causing gene in the body is very low in the early stage of the disease, it cannot be directly detected, and it is often necessary to amplify the gene or signal to be detected. At present, commonly used amplification methods include po...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/66C12Q1/28
Inventor 樊春海刘兴奋王丽华宋世平
Owner SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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