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Fluorescent biosensor for detecting ochratoxin A as well as preparation method and application thereof

A biosensor, ochratoxin technology, applied in the field of biosensors, can solve the problems of long detection period, low specificity and sensitivity, and achieve the effect of fast detection speed, easy operation and short detection cycle

Active Publication Date: 2019-03-08
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems of relatively low specificity and sensitivity and long detection period of the method for detecting ochratoxin A (OTA) in the prior art, the present invention provides a rolling circle-based method with high specificity and sensitivity and fast detection speed. Fluorescent biosensor for detection of ochratoxin A by amplification-mediated catalytic hairpin self-assembly and endonuclease feedback amplification

Method used

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  • Fluorescent biosensor for detecting ochratoxin A as well as preparation method and application thereof
  • Fluorescent biosensor for detecting ochratoxin A as well as preparation method and application thereof
  • Fluorescent biosensor for detecting ochratoxin A as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Preparation of Circular Template and Composite Probe

[0069] Prepared with 50 mM Tris-HCl, 10 mM MgCl 2 , T4 DNA Ligase Reaction Buffer with 10 mM DTT and 1 mM OTA. Formulated with 10 mM Na 2 HPO 4 , 10 mM NaH 2 PO 4 , 140 mM NaCl, 1 mM KCl, 1 mM MgCl 2 , 1mM CaCl 2 , PBS buffer solution of pH=7.4.

[0070] (1) Mix 42 μL sterilized water, 6 μL linear template (100 μM), 6 μL ligation probe (100 μM) and 6 μL 10× T4 DNA ligase buffer, denature at 95°C for 5 min, then Slowly cool down to room temperature to complete the hybridization, then add 3 μL of T4 DNA ligase (60 U / μL) to the reaction system, and react at 16°C for 20 hours; after that, the reaction system is placed in a water bath at 65°C for 15 minutes , inactivate the T4 DNA ligase in the system.

[0071] (2) Add 1 μL of exonuclease Ⅰ (20 U / μL) and 2 μL of exonuclease Ⅲ (100 U / μL) to the above reaction system and react at 37°C for 2 h; then put the reaction system at 85°C Heated in a water bath ...

Embodiment 2

[0073] Embodiment 2 Fluorescence intensity changes with endonuclease IV concentration

[0074] A method for preparing a fluorescent biosensor of the present invention, comprising the following steps:

[0075] (1) Mix 3 μL composite probe I (0.1 μM), 3 μL composite probe II (0.5 μM), 3 μL HP2 (0.5 μM), 3 μL HP3 (0.5 μM), 3 μL phi29 DNA polymerase (1 U / μL), 3 μL endonuclease IV (concentrations are 0.1 U / μL, 0.2 U / μL, 0.25 U / μL, 0.5 U / μL, 0.75 U / μL, 1 U / μL), 3 μL NMM ( 2.4 μM), 2 μL dNTP (1mM), in 3 μL buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 4 mM DTT, pH7.5) and then add OTA (100 ng / μL) respectively, and then react at a constant temperature of 37°C for 120 min after mixing;

[0076] (3) Dilute the solution obtained in step (2) with water to 100 μL, and then perform fluorescence detection; the excitation wavelength is set to 399 nm, the emission wavelength is 610 nm, and the detection range is 560 nm-640 nm, and the fluorescence signal changes are read....

Embodiment 3

[0080] Embodiment 3 Fluorescence intensity changes with the concentration of phi29 DNA polymerase

[0081] A method for preparing a fluorescent biosensor of the present invention, comprising the following steps:

[0082] (1) Mix 3 μL composite probe I (0.1 μM), 3 μL composite probe II (0.5 μM), 3 μL HP2 (0.5 μM), 3 μL HP3 (0.5 μM), 3 μL phi29 DNA polymerase (concentrations respectively 0.1 U / μL, 0.2 U / μL, 0.5 U / μL, 1 U / μL, 1.5 U / μL, 2 U / μL), 3 μL endonuclease IV (0.5 U / μL), 3 μL NMM ( 2.4 μM), 2 μL dNTP (1 mM), in 3 μL buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 4 mM DTT, pH 7.5), add OTA (100 ng / μL) respectively after mixing, and react at a constant temperature of 37°C for 120 min after mixing;

[0083] (3) Dilute the solution obtained in step (2) with water to 100 μL, and then perform fluorescence detection; the excitation wavelength is set to 399 nm, the emission wavelength is 610 nm, and the detection range is 560 nm-640 nm, and the fluorescence sign...

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Abstract

The invention relates to the technical field of biosensors, and particularly relates to a fluorescent biosensor for detecting ochratoxin A based on a rolling ring amplification mediated catalytic hairpin self-assembly and incision enzyme feedback amplification method. According to the fluorescent biosensor, as for a detection mode, a fluorescence method is adopted for detection, and a luminoscopeis utilized; before detection, a padlock probe and a connecting probe form an annular template probe; then a target object is added to a homogeneous solution of a complex probe I, a complex probe II,HP 2 and HP 3, incubating is carried out for 120 minutes at 37 DEG C, and the target object and an aptamer sequence are bound; the multiple feedback amplification process is completed under the actionof a phi29 DNA polymerase and an endonuclease IV, so that signal amplification is realized; and then, the luminoscope is used for setting the excitation wavelength to be 399 nm, the fluorescence intensity at 610 nm is detected, and the detection range is 560-640 nm. Meanwhile, the invention further provides a preparation method for the biosensor. The preparation method has the advantages of beingmild in reaction condition and easy to operate.

Description

technical field [0001] The invention relates to the technical field of biosensors, in particular to a fluorescent biosensor for detecting ochratoxin A based on rolling circle amplification-mediated catalytic hairpin self-assembly and endonuclease feedback amplification methods, and also relates to a preparation method thereof. Background technique [0002] Ochratoxins (Ochratoxins, OT) are a class of low molecular weight secondary metabolites produced by Aspergillus, Penicillium and other genera, and belong to the natural pollutants of the mycotoxin group. It can exist in the process of crop production, processing, storage, etc. Among them, ochratoxin A (Ochratoxin A, OTA) is the most toxic and widely distributed type. It was found in corn flour in South Africa in the 1960s. Find. Many studies have shown that OTA has nephrotoxicity, hepatotoxicity, embryotoxicity, teratogenicity, neurotoxicity, immunotoxicity, genotoxicity and carcinogenicity. The International Agency for ...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6402G01N21/6486
Inventor 黄加栋王敬锋王玉刘素王海旺宋晓蕾张雪赵一菡瞿晓南张儒峰
Owner UNIV OF JINAN
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