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Nucleic acid aptamer based micro-cantilever array detection method of MC-LR (microcystin LR)

A nucleic acid aptamer and microcystin technology, which is applied in measurement devices, instruments, scientific instruments, etc., to achieve the effect of combining high efficiency, easy storage, and easy operation.

Inactive Publication Date: 2018-06-19
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the field of MC-LR detection, the use of cantilever beam sensing technology in dynamic mode using its corresponding antibody has been reported, but there is no use of aptamers as probe molecules for detection on microbeam sensing platforms based on stress mode. to report

Method used

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  • Nucleic acid aptamer based micro-cantilever array detection method of MC-LR (microcystin LR)
  • Nucleic acid aptamer based micro-cantilever array detection method of MC-LR (microcystin LR)
  • Nucleic acid aptamer based micro-cantilever array detection method of MC-LR (microcystin LR)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, the modification process of microcantilever array

[0044] Experiment 1 Modification of DNA aptamers on micro-cantilever arrays

[0045] 1) Cleaning: Put the unused microbeam array (purchased from Micromotive Company, Germany) into the enzyme-labeled well plate, add Piranha Dip solution (V(H 2 o 2 ):V(H 2 SO 4 )=1:3) for several minutes, rinsed with deionized water, and dried with nitrogen;

[0046] 2) Aptamer modification: the washed micro-cantilever array was placed in an enzyme-labeled well plate, added to the DNA aptamer solution, and incubated at room temperature for 3 hours. Then use buffer (buffer containing 50mM Tris-HCl, 150mM NaCl and 2mM MgCl 2 , pH=7.5) cleaning;

[0047] 3) Blocking: the modified micro-cantilever array is placed in an enzyme-labeled well plate, 1 mM MCH is added, and incubated for 30 minutes. Then wash with buffer solution and blow dry with nitrogen gas. A modified micro-cantilever array is obtained.

[0048] Experime...

Embodiment 2

[0051] Embodiment 2, detection of MC-LR using microbeam array sensor

[0052] 1) Fixing the microbeam array: Fix the aptamer-modified microbeam array in the reaction pool of the system in Experiment 1 of Example 1 above, add buffer, and flow liquid to remove air bubbles in the reaction pool.

[0053] 2) Adjust the optical path of the system: adjust the positions of the laser, the reaction cell, and the PSD so that the eight lasers can irradiate the tip of each beam in the microbeam array and reflect to the target surface of the PSD.

[0054] 3) Sample detection: adjust the syringe pump to 1 μL s -1 The velocity of the buffer is flowing, and the deflection of each beam of the microbeam array is monitored in real time. When the detected real-time curves of each beam tend to be stable, a buffer solution containing a certain concentration of MC-LR is added to collect and record the deflection data of the microbeam array in real time.

[0055] Use a concentration of 100 μg mL pur...

Embodiment 3

[0056] Example 3. Detection of two MC isomers using a microbeam array sensor

[0057] Use a concentration of 100 μg mL purchased from Sigma-Aldrich -1 MC-LA, YR standard samples, and buffer solution were prepared to a concentration of 100 μg L -1 for each corresponding sample. Repeat the experimental process in Example 2, using the concentration of 100 μg L prepared above successively -1 Three kinds of samples MC-LR, LA, YR were tested, and the obtained data can be found in Figure 5 . From Figure 5 It can be seen that at 85 min, the concentration of the microbeam array pair modified with MC-LR aptamer was 100 μg L -1 The average deflection of the three samples MC-LR, LA, and YR is 123, 49, and 34nm in turn, and the deflection of the microbeam to MC-LR is significantly greater than that of the other two samples, and the microbeam to MC The detection results of -LR were distinguished from those of the other two samples, which to a certain extent explained that the microb...

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Abstract

The invention provides a method for detecting MC-LR (microcystin LR) in a sample solution. The method comprises the steps as follows: a nucleic acid aptamer capable of specifically binding the MC-LR is fixed on a micro-cantilever array; the micro-cantilever array fixed with the nucleic acid aptamer is contacted with the sample solution; bending deformation produced after the micro-cantilever arrayfixed with the nucleic acid aptamer is contacted with the sample solution is detected. The invention further provides the micro-cantilever array and a system for detecting the MC-LR in the sample solution.

Description

technical field [0001] The invention relates to the field of water body environmental pollution detection, in particular to a microcantilever beam array sensor based on surface stress effect and a method for detecting microcystin LR using an aptamer as a probe molecule. Background technique [0002] Microcystin (MC) is a class of biologically active cyclic heptapeptide compounds mainly produced by cyanobacteria. This compound can be produced in large quantities by cyanobacteria cells when the water body is eutrophic and causes water blooms. When the algae cells are aging, Upon death and rupture, MCs are released into bodies of water. Direct contact of human body with water containing toxins can cause allergies and acute gastroenteritis. MC is a strong hepatotoxin, which can strongly inhibit the activity of protein phosphatase, which can cause liver damage, hemorrhage and necrosis. MC is also a potential tumor promoter. Long-term drinking of water sources containing microcy...

Claims

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Application Information

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IPC IPC(8): G01N33/535G01N33/558
CPCG01N33/535G01N33/558
Inventor 张青川张广平吴尚犬李潮伍小平
Owner UNIV OF SCI & TECH OF CHINA
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