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Nucleic acid apt (aptamer) biosensor for rapidly detecting STX (saxitoxin) and preparation method of nucleic acid apt biosensor

A saxitoxin and biosensor technology, which is applied in the field of biomolecules to achieve important scientific significance, market value, and simple operation.

Active Publication Date: 2019-04-09
NINGBO UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Aiming at the defects and deficiencies of the prior art, the present invention provides a nucleic acid aptamer biosensor for rapid detection of saxitoxin to realize ultrasensitive, high specificity, rapid detection, The needs of on-site detection; the establishment of this method is based on the high selectivity of nucleic acid aptamers for specific binding targets and the signal amplification effect of polymerase chelate (PV) coupled colloidal gold nanoparticles, which can specifically and sensitively Rapid detection of saxitoxin in samples, this method has not been reported at home and abroad

Method used

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  • Nucleic acid apt (aptamer) biosensor for rapidly detecting STX (saxitoxin) and preparation method of nucleic acid apt biosensor
  • Nucleic acid apt (aptamer) biosensor for rapidly detecting STX (saxitoxin) and preparation method of nucleic acid apt biosensor
  • Nucleic acid apt (aptamer) biosensor for rapidly detecting STX (saxitoxin) and preparation method of nucleic acid apt biosensor

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Effect test

Embodiment 1

[0038] A rapid detection nucleic acid aptamer biosensor for saxitoxin, such as figure 1 As shown, its preparation principle is as follows:

[0039] 1. Preparation of signal probe (cDNA-AuNPs-PV) and capture probe conjugate (AuMNPs-Apt)

[0040] (1) Preparation of saxitoxin (STX) aptamer

[0041] According to the STX aptamer sequence, aptamer (Aptamer, Apt) and thiolated complementary DNA (cDNA) were designed.

[0042] (2) Preparation technology of signal probe (cDNA-AuNPs-PV)

[0043] Using PV as a signal amplification carrier, labeling thiolated complementary DNA strands (cDNA) and then coupling colloidal gold nanoparticles (AuNPs), synthesizing nano-peroxide complexes (cDNA-AuNPs-PV) as signal probes, and performing characterization analysis . At the same time, comparing different signaling probes in TMB-H 2 o 2 For the signal amplification effect in the solution, it is determined that the thiolated complementary DNA sequence synthesized in step (1) has the best amplif...

Embodiment 2

[0049] The STX aptamer used in the present invention is based on the STX aptamer sequence reported in the literature, truncated and modified by thiol modification, and a large number of aptamer sequences are synthesized. The aptamers before and after modification were used to verify the recovery rate of the detection method, as follows: using the modified STX aptamer, the addition recovery rate was determined within the range of 0.1-5.0 ng / mL of STX spiked amount, and the recovery rate It was between 97.0-110%. The unmodified STX aptamer was used to determine the addition recovery rate in the range of 0.1-5.0 ng / mL STX spiked amount, and the recovery rate was between 62.5-75.8%. This shows that the STX aptamer modified by truncation and thiol modification has a higher recovery rate and better effect.

[0050] After screening and verification by the above-mentioned method, the following aptamer with the highest recovery rate and the best effect was obtained. The sequence length...

Embodiment 3

[0055] A preparation method for rapid detection of saxitoxin nucleic acid aptamer biosensor, comprising the following steps:

[0056] 1. Preparation of signal probe (cDNA-AuNPs-PV) and capture probe conjugate (AuMNPs-Apt)

[0057](1) Preparation of saxitoxin (STX) aptamer

[0058] Entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize aptamer (Aptamer, Apt) and thiolated complementary DNA (cDNA).

[0059] The specific nucleotide sequence composition of described adapter is as follows:

[0060] 5'SH-(CH 2 ) 6 - CCGTGGAAACATGTTCATTGGGCGCACTCCGCTTTCTGT-3'.

[0061] The nucleotide sequence of the thiolated cDNA is:

[0062] 5'SH-(CH 2 ) 6 -CAATGAACATGTTTCCACGG-3'.

[0063] (2) Preparation technology of signal probe (cDNA-AuNPs-PV)

[0064] First use K 2 CO 3 solution to adjust the pH value of the colloidal gold nanoparticles AuNPs to 9.0, then add the PV reagent to the solution, and incubate at room temperature for 15 min, during which the AuNPs and the free S...

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Abstract

The invention discloses a nucleic acid apt (aptamer) biosensor for rapidly detecting STX (saxitoxin) and a preparation method of the nucleic acid apt biosensor. The apt sequence of STX is 5'SH-(CH2)6-CCGTGGAAACATGTTCATTGGGCGCACTCCGCTTTCTGT-3'. According to the biosensor, STX detecting sensitivity is improved, and STX at a minimum of 0.05 ng / mL can be detected. The advantages of fastness and ultra-sensitivity of colorimetric analysis are closely combined with the characteristics of high affinity and high selectivity of the nucleic acid apt, the biosensor has the characteristics of being simpleto operate and portable of sensors, and the requirements of ultra-sensitivity, high specificity, rapid detection and on-site detection of STX in a seawater shellfish complex matrix can be met. Research results can provide technical support for research and development of rapid detection technology and equipment for shellfish toxins in seawater shellfish, and have great scientific research significance and market value.

Description

technical field [0001] The invention belongs to the technical field of biomolecules, and in particular relates to a saxitoxin rapid detection nucleic acid aptamer biosensor and a preparation method thereof. Background technique [0002] With the globalization of food trade, the convenience of processed food, and the change of people's dining habits, food safety issues have increasingly profoundly affected people's lives and received great attention from the whole society. Timely, rapid and comprehensive monitoring of food safety from all aspects requires a fast, convenient and accurate food safety rapid detection technology that can meet this requirement to ensure food safety. Seawater shellfish such as mussels and scallops are loved by people because of their delicious taste and high nutritional value. Zhejiang Province, as a province rich in marine resources, has a total aquatic product processing output exceeding 1 / 10 of the country, and is one of the most important suppo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/543G01N33/53G01N21/31
CPCC12N15/115C12N2310/16G01N21/31G01N33/5308G01N33/54326G01N33/54346
Inventor 吴益春潘道东郭海波罗海军陈翔王萍亚
Owner NINGBO UNIV
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