Small molecule probe based on nano-gold and aptamer and preparation method of small molecule probe

A technology of small molecule probes and aptamers, which is applied in the field of small molecule probes based on gold nanoparticles and aptamers and their preparation, can solve the problems of reduced sensitivity and specificity of molecular beacons, and reduce false positive results , high quenching ability, and the effect of reducing the probability of false positive results

Inactive Publication Date: 2012-09-19
华森新科(苏州)纳米技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The first problem to be solved by the present invention is to overcome the problems of molecular beacons in the prior art when detecting long-wavelength emission, the sensitivity and specificity decrease, and provide a method based on Small molecule probes of gold nanoparticles and aptamers

Method used

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  • Small molecule probe based on nano-gold and aptamer and preparation method of small molecule probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Preparation of nano gold-DNA fragments

[0057] 1.1 Preparation of gold nanoparticles

[0058] Synthesis of nano-gold, high concentration of nano-gold is prepared by citric acid reduction method and concentrated by centrifugal method (Hurst, S. J.; Lytton-Jean, A. K.; Mirkin, C. A., Anal Chem 2006, 8313-8.). Store at 4°C, and dilute or concentrate according to specific needs during use. The synthesized nano-gold is 13 nanometers.

[0059] Three sulfhydryl-modified DNA fragments complementary to adenosine, potassium and cocaine aptamers were prepared and obtained directly from commercial sources.

[0060] Assembling DNA fragments on the surface of gold nanoparticles through gold-sulfur bonds to obtain nano-gold-DNA fragments

[0061] Mix three sulfhydryl-modified DNAs complementary to the aptamers of adenosine, potassium ions, and cocaine, and thiol-modified 10 T DNAs (SH-T10) that are conducive to DNA hybridization and 10 nM gold nanoparticles. ...

Embodiment 2

[0062] Example 2 Preparation of fluorescent group-labeled nucleic acid aptamers and small molecule probes of gold nanoparticles and nucleic acid aptamers

[0063] 2.1 Prepare fluorophore-labeled adenosine, potassium ions, and cocaine aptamers, which are directly obtained from commercial sources. Any fluorophore that can be labeled on DNA is acceptable, but the excitation and emission peak positions of the three different fluorophores need to differ by more than 50 nm to avoid the superposition of emission spectra.

[0064] The aptamers labeled with fluorophores, adenosine, potassium ions and cocaine were mixed with the gold nano-solution of assembled DNA fragments in Example 1 in equimolar amounts, and left overnight at room temperature. Aptamers hybridize to the DNA fragments described above. Centrifuge to remove excess aptamers to obtain the desired small molecule probes.

Embodiment 3

[0065] Example 3 Detection of small molecule probes based on gold nanoparticles and aptamers of the present invention

[0066] Take the small molecule probe prepared in Example 2, add 10 mM A, 100 mM KCl and 1 mM cocaine at the same time, and let it stand at room temperature for 1 hour. fluorescent signal.

[0067] In order to investigate the specificity of the probe, analogs of various detection substances, such as analogs T, C, G, K of adenosine A, were added during the experiment. + analogs of Li + 、Na + As well as cocaine analogues ecgonine methyl ester (EME) and benzoic acid (BE), the respective concentrations are the same as their analogues, as shown in Figure 2-4, it can be seen that the selectivity of this probe is very good.

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Abstract

The invention discloses a small molecule probe based on nano-gold and aptamer and a preparation method of the small molecule probe. The small molecule probe comprises nano-gold particles, more than one type of sulfydryl modifying deoxyribonucleic acid (DNA) fragment assembled on the nano-gold particles and more than one type of aptamer which is marked by fluorescent groups, and at least a part of fragments of the more than one type of the aptamer are complemented and hybridized with the more than one type of sulfydryl modifying DNA group. The small molecule probe is short in detecting time, simple in operation, high in specificity, good in repeatability, high in accuracy, and the false positive result is effectively reduced. By means of the aptamer technology, a high detection sensibility is achieved, and various molecules can be simultaneously detected.

Description

technical field [0001] The invention belongs to the technical field of biological analysis and detection, and in particular relates to a small molecule probe based on nano-gold and an aptamer and a preparation method thereof. Background technique [0002] Nucleic acid aptamer (aptamer) is a kind of DNA or RNA oligonucleotide sequence artificially screened in vitro, which can efficiently and specifically recognize its ligand (A. D. Ellington, J. W. Szostak, Nature 1990, 346, 818-822) , has become an increasingly important molecular tool in the fields of clinical diagnosis and disease treatment. Nucleic acid aptamers are a series of single-stranded nucleic acid molecules that can bind to specific target molecules, as specific as antibodies, and have strict recognition ability and high affinity for binding ligands. The basis of the interaction between nucleic acid aptamers and target molecules is the complementarity of the spatial structure. Nucleic acid aptamers usually have ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68G01N21/64
Inventor 樊春海宋世平黄庆
Owner 华森新科(苏州)纳米技术有限公司
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