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Biosensor for detecting MicroRNA-17

A biosensor and protein technology, applied in the field of sensors, can solve the problems of complex experimental process, low sensitivity, limitations, etc., and achieve the effects of low process cost, sensitive detection, and easy operation.

Pending Publication Date: 2022-04-26
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional experimental methods such as Northern blotting, RT-PCR, etc. have limited their applications due to their complicated experimental procedures and low sensitivity.

Method used

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  • Biosensor for detecting MicroRNA-17
  • Biosensor for detecting MicroRNA-17
  • Biosensor for detecting MicroRNA-17

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Pre-trigger concentration optimization

[0035] (1) In a 1.5 mL centrifuge tube, add hairpin pre-trigger (final concentrations are 0.4 μM, 0.6 μM, 0.8 μM, 1.0 μM, 1.2 μM, 1.4 μM), H1 (10 μM), H2 (10 μM ), H3 (10 µM), H4 (10 µM) 10 µL each, add 10 µL 10×buffer2.1, shake and mix well, put in 95°C water bath for 5 min, then cool to room temperature;

[0036] (2) Add 2 µL of cas13a protein (100 nM), 2 µL of crRNA (50 nM), 1 µL of miRNA-17, and 26.7 µL of sterilized water into the solution obtained above, and place the centrifuge tube in a 37°C water bath for reaction 75 minutes;

[0037] (3) Add 3 µL of heme, 3 µL of luminol reagent, and 3.3 µL of hydrogen peroxide, and detect the peak of chemiluminescence at 420 nm with a fluorometer. The measurement range of chemiluminescence spectrum is 400 nm to 600 nm, read the change of fluorescence signal, and detect the target object;

[0038] see results figure 2 , it can be seen from the figure that the detected pea...

Embodiment 2

[0039] Example 2 H1 concentration optimization

[0040] (1) In a 1.5 mL centrifuge tube, add hairpin pre-trigger (10 µM), H1 (final concentrations are 0.4 µM, 0.6 µM, 0.8 µM, 1.0 µM, 1.2 µM, 1.4 µM), H2 (10 µM ), H3 (10 µM), H4 (10 µM) 10 µL each, add 10 µL 10×buffer2.1, shake and mix well, put in 95°C water bath for 5 min, then cool to room temperature;

[0041] (2) Add 2 µL of cas13a protein (100 nM), 2 µL of crRNA (50 nM), 1 µL of miRNA-17, and 26.7 µL of sterilized water into the solution obtained above, and place the centrifuge tube in a 37°C water bath for reaction 75 minutes;

[0042] (3) Add 3 µL of heme, 3 µL of luminol reagent, and 3.3 µL of hydrogen peroxide, and detect the peak of chemiluminescence at 420 nm with a fluorometer. The measurement range of chemiluminescence spectrum is 400 nm to 600 nm, read the change of fluorescence signal, and detect the target object;

[0043] see results image 3 , it can be seen from the figure that the detected peak chemilum...

Embodiment 3

[0044] Embodiment 3 reaction time optimization

[0045](1) In a 1.5 mL centrifuge tube, add 10 µL each of hairpin pre-trigger (10 µM), H1 (10 µM), H2 (10 µM), H3 (10 µM), H4 (10 µM), and add 10 µL10×buffer2.1, vortex and mix well, put in 95℃ water bath for 5min, then cool to room temperature;

[0046] (2) Add 2 µL of cas13a protein (100 nM), 2 µL of crRNA (50 nM), 1 µL of miRNA-17, and 26.7 µL of sterilized water into the solution obtained above, and place the centrifuge tube in a 37°C water bath for reaction 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, 120 min;

[0047] (3) Add 3 µL of heme, 3 µL of luminol reagent, and 3.3 µL of hydrogen peroxide, and detect the peak of chemiluminescence at 420 nm with a fluorometer. The measurement range of chemiluminescence spectrum is 400 nm to 600 nm, read the change of fluorescence signal, and detect the target object;

[0048] see results image 3 , it can be seen from the figure that the detected peak chemiluminescence intensi...

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Abstract

The invention belongs to the technical field of sensors, and provides a biosensor for detecting microRNA (micro Ribonucleic Acid), which comprises crRNA with nucleotide sequences as shown in SEQ ID NO: 1-6, a hairpin probe pre-trigger, H1, H2, H3, H4 and Cas13a protein, heme, a luminol reagent and H2O2. The biosensor realizes rapid, simple and sensitive detection of a target object; the process for manufacturing the biosensor is low in cost, mild in reaction condition and high in reaction speed.

Description

technical field [0001] The invention belongs to the technical field of sensors, in particular to a biosensor for detecting MicroRNA-17 by a nucleic acid aptamer. Background technique [0002] Different expression levels of microRNAs (miRNAs) in normal tissues, benign tumors, and malignant tumors make them important biomarkers for tumor diagnosis and prognosis, which highlights the urgency and importance of developing highly sensitive and specific miRNA assays. importance. However, traditional experimental methods such as Northern blotting, RT-PCR, etc. have limited their applications due to their complicated experimental procedures and low sensitivity. [0003] As the adaptive immune system of prokaryotes, CRISPR-Cas system (clustered regularly interspaced short palindromic repeats and crispr associated protein) has been widely used in gene editing of eukaryotes. Mature CRISPR RNA (crRNA), produced by the processing of precursor crRNA (pre-crRNA), is essential for the form...

Claims

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Application Information

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IPC IPC(8): G01N21/31G01N21/64
CPCG01N21/3103G01N21/6402
Inventor 王玉朱镜儒黄加栋刘素郭志强李倩茹姚玉颖李宗强李静静张清心徐婉晴朱志学
Owner UNIV OF JINAN
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