Self-calibration double-signal biosensor and application thereof in miRNA detection

A biosensor and dual-signal technology, applied in the field of biosensors, can solve problems such as poor sensitivity and specificity, complicated operation, etc., and achieve the effects of reducing false positives, improving reliability, and realizing self-calibration functions

Active Publication Date: 2020-08-25
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, commonly used miRNA detection methods include Northern blotting analysis, real-time quantitative polymerase chain reaction and microarray technology, but these methods have some technical limitations, such as semi-quantitative data, complex operation, reproducibility, poor sensitivity and specificity, etc.

Method used

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  • Self-calibration double-signal biosensor and application thereof in miRNA detection
  • Self-calibration double-signal biosensor and application thereof in miRNA detection
  • Self-calibration double-signal biosensor and application thereof in miRNA detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Preparation of self-calibrating dual-signal biosensor

[0050] (1) Clean the SiO with deionized water and isopropanol in sequence 2 oxide layer of the silicon wafer. Next, put them in piranha solution (H 2 SO 4 :H 2 o 2 =7:3) at 100°C for 8 minutes for hydroxylation.

[0051] (2) Immerse the hydroxylated silicon wafer in 3-aminopropyltrimethylsilane (APTMS) solution (ethanol:water=95:5, APTMS 2vol%) for 30 minutes, rinse and perform three times in Mill-Q water for 10 min sonication followed by N 2 Dry in medium to obtain aminated substrates. The aminated substrate was immersed in graphene oxide (GO) aqueous solution (0.1 mg / ml) for 45 min, after which the samples were rinsed and sonicated three times for 15 min in Mill-Q water and washed with N 2 Dry to obtain a bilayer (APTMS / GO) membrane. By repeating the above steps 5 times, covalently assembled (APTMS / GO) 5 film, where 5 represents the number of layers of the bilayer (APTMS / GO) film, and the thi...

Embodiment 2

[0054] Example 2: Performance investigation of self-calibrating dual-signal biosensor for detecting miRNA

[0055] 1. Linear curve and sensitivity:

[0056] (1) Prepare a self-calibrating dual-signal biosensor with Example 1, test the transfer curve of a self-calibrating dual-signal biosensor and obtain the grid voltage value D of the Dirac point 1 ;

[0057] (2) Soak the self-calibrating dual-signal biosensor in 600 μL of complementary miRNA (5'-UAA CAC UGU CUG GUA AAG AUG G-3') solution at different concentrations, and incubate at room temperature for 20 min, and then use Rinse with DEPC water to remove excess miRNA sequences, and dry at room temperature at 20°C for at least 2 seconds to test the transfer curve of the above self-calibration dual-signal biosensor and obtain the gate voltage value D of the Dirac point 2 ;

[0058] The solvent of the miRNA solution is DEPC water, and an RNase inhibitor is added during preparation, so that the final concentration of the RNase...

Embodiment 3

[0075] Example 3: Serum Sample Analysis

[0076] (1) Prepare a self-calibrating dual-signal biosensor with Example 1, test a transfer curve of a self-calibrating dual-signal biosensor and obtain the grid voltage value D of the Dirac point 1 ;

[0077] (2) Dilute fetal bovine serum 20 times with DEPC water and add 6×10 –10 M complementary miRNA (5'-UAACAC UGU CUG GUA AAG AUG G-3') to prepare miRNA test solution.

[0078] When preparing the miRNA test solution, RNase inhibitors were added so that the final concentration of RNase inhibitors was 10 μM.

[0079] (3) Soak the self-calibrating dual-signal biosensor in 600 μL of miRNA test solution and incubate for 20 minutes, then rinse with DEPC water to remove excess miRNA sequences, and dry at room temperature at 20°C for at least 2 seconds to test the performance of the above FET Transfer the curve and get the grid voltage value D at the Dirac point 2 ;

[0080] Before soaking the self-calibrating dual-signal sensor in the m...

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Abstract

The invention discloses a self-calibration double-signal biosensor and application thereof in miRNA detection, and belongs to the technical field of biosensors. Fluorescent group modified DNA is fixedon a full covalent bond graphene field effect transistor to serve as a sensitive probe for detecting miRNA. The stability of multiple subsequent processing of the device in a solution phase is realized by utilizing the characteristic of covalent bond connection between layer transfer structures; through a full covalent bond graphene field effect transistor and a fluorescence technology, simultaneous detection of double signals of miRNA is realized so as to realize a self-calibration function. Compared with the existing miRNA detection technology at present, the self-calibration biosensor fordetecting miRNA can meet the detection requirements of stability, reliability, high sensitivity, high selectivity, rapidness, simplicity and convenience, and a new thought is provided for miRNA detection.

Description

technical field [0001] The invention relates to the technical field of biosensors, in particular to a self-calibrating dual-signal biosensor and its application in miRNA detection. Background technique [0002] Micro ribonucleic acid (microRNA, miRNA) is a kind of non-coding small molecule RNA with a length of about 19 to 23 nucleotides, which inhibits the corresponding protein translation by binding to the messenger RNA (mRNA) of the target gene. Recent research results have shown that miRNA can stably exist in serum and plasma of mammals, so it can be used as a new type of disease marker for early diagnosis of disease and indication of individualized treatment. At present, commonly used miRNA detection methods include Northern blotting analysis, real-time quantitative polymerase chain reaction and microarray technology, but these methods have some technical limitations, such as semi-quantitative data, complex operation, reproducibility, poor sensitivity and specificity, et...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N27/414
CPCG01N21/6428G01N21/274G01N27/4145G01N27/4163
Inventor 张丛丛刘宏孙铭远王建
Owner UNIV OF JINAN
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