Adenosine determination method based on fluorescent and colorimetric dual detection system

A dual detection and fluorescence detection technology, applied in the field of analytical chemistry, can solve the problems of low sensitivity and detection limit of adenosine detection, time-consuming and labor-consuming, a large number of samples, etc., to reduce the detection concentration and detection limit, improve sensitivity, highly specific effect

Active Publication Date: 2014-11-19
UNIV OF SCI & TECH BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this separation method often requires a large amount of samples and toxic reagents and is time-consuming and labor-intensive. The sensitivity and detection limit of adenosine detection are relatively low. These shortcomings limit the application of the above methods to a certain extent.

Method used

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  • Adenosine determination method based on fluorescent and colorimetric dual detection system
  • Adenosine determination method based on fluorescent and colorimetric dual detection system
  • Adenosine determination method based on fluorescent and colorimetric dual detection system

Examples

Experimental program
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Embodiment 1

[0039] The specific operation steps are as follows:

[0040] 1), synthesis of gold nanoparticles with a diameter of 13nm;

[0041] The synthesis process is as follows: 100 mL of distilled water and 2 mL of 1% chloroauric acid were added into a 250 mL three-necked flask, and heated to boiling under vigorous stirring. Then, quickly add the freshly prepared 1% sodium citrate solution into the above boiling solution, and after reacting for 15 minutes, continue stirring to cool the solution to room temperature, and store it at 4°C.

[0042] 2), modifying sulfhydryl ssDNA onto the surface of AuNPs to form a ssDNA-AuNPs system;

[0043] Change the sequence to 5'-SH-C 6 -The DNA of CAGTCTGGACCC-3' was prepared to a concentration of 10uM, and 70ul of the above DNA solution was mixed with 200ul gold nanoparticles for 16h, and then 2M NaCl was added to make the final concentration 0.1M. After washing by centrifugation for 3 times, the obtained red substance was dissolved in 200ul Tris...

Embodiment 2

[0055] The specific operation steps are as follows:

[0056] 1), synthesis of gold nanoparticles with a diameter of 5nm;

[0057] The synthesis process is as follows: add 70mL of distilled water and 10mL of 0.1% chloroauric acid into a 250mL three-necked flask, and heat to boiling under vigorous stirring. Then, quickly add the freshly prepared mixed solution of 4mL 1% sodium citrate and 5mL 1% tannic acid to the above boiling solution, after reacting for 10min, continue stirring to cool the solution to room temperature, and store it at 4°C.

[0058] 2), modifying sulfhydryl ssDNA onto the surface of AuNPs to form a ssDNA-AuNPs system;

[0059] Change the sequence to 5'-SH-C 6 -The DNA of CTGACTGGACCC-3' was formulated to a concentration of 10uM, and 70ul of the above DNA solution was mixed with 200ul of gold nanoparticles for 16h, and then 1M NaCl was added to make the final concentration 0.1M. After washing by centrifugation for 3 times, the obtained red substance was diss...

Embodiment 3

[0070] 1), synthesis of gold nanoparticles with a diameter of 30nm;

[0071] The synthesis process is as follows: 100mL of 0.01% chloroauric acid was added into a 250mL three-necked flask, and heated to boiling under vigorous stirring. Then, quickly add 1 mL of freshly prepared 1% sodium citrate solution to the above boiling solution, and after reacting for 15 minutes, continue stirring to cool the solution to room temperature, and store at 4°C.

[0072] 2), modifying sulfhydryl ssDNA onto the surface of AuNPs to form a ssDNA-AuNPs system;

[0073] Change the sequence to 5'-SH-C 6 -The DNA of GCCACTGGACCC-3' was prepared to a concentration of 10uM, and 70ul of the above DNA solution was mixed with 200ul of gold nanoparticles for 24 hours, and then 1uL of 1M NaCl was added every 5min to make the final concentration 0.1M, and washed by centrifugation with 0.1MPBS (pH7.4) for 3 After several times, the obtained red substance was dissolved in 200ul Tris-HCl (pH7.4) buffer soluti...

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Abstract

The invention discloses an adenosine determination method based on a fluorescent and colorimetric dual detection system, and belongs to the technical field of analytical chemistry. By gold-sulfur bonds, sulfydryl DNAs are connected to gold nanoparticles, and the sulfydryl DNAs can hybridize with adenosine aptamers modified by fluorescent dye; because of fluorescence quenching in high-salt solution, the detection system is free of fluorescence or very low in fluorescence intensity and the stable gold nanoparticles keep the solution in a wine red color; addition of adenosine causes changes in the structures of the aptamers, so that the adenosine aptamers modified by the fluorescent dye are dissociated from the gold nanoparticles and the fluorescence of the fluorescent dye recovers; at the moment, the gold nanoparticles cannot exist stably, the color of the solution changes from red to blue. By the adenosine determination method, through changes in fluorescence intensity and solution color of the detection system, qualitative and quantitative adenosine determination is achieved; the adenosine determination method has the advantages of high sensitivity, good selectivity and the like.

Description

technical field [0001] The invention belongs to the technical field of analytical chemistry and relates to the construction of a biosensor for measuring adenosine based on a dual detection system of fluorescence and colorimetry. Background technique [0002] Nucleic acid aptamer (aptamer) refers to a single-stranded oligonucleotide that can bind to a target molecule with high affinity and high specificity, screened from a synthetic DNA / RNA library. It can be obtained by SELEX technology, and its general idea is to design and synthesize random oligonucleotides (10 15 -10 18 Nucleic acid molecule) after multiple rounds of screening and exponential enrichment, the target nucleic acid aptamer was finally obtained. [0003] The unique biochemical properties of nucleic acid itself make nucleic acid aptamers have the following advantages in the field of biosensing applications: 1. A wide range of target molecules. Small molecules such as ATP, amino acids, and metal ions, large b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N21/78
Inventor 温永强赵娜桂万元王文谦焦翔宇李延生
Owner UNIV OF SCI & TECH BEIJING
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