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Preparation method and application of electrochemical sensor for non-coding RNA

An electrochemical and sensor technology, applied in the field of electrochemical biosensing, can solve the problems of cumbersome sample processing, high detection cost, and high maintenance cost, and achieve the effects of good chemical stability, low detection limit, and time saving.

Active Publication Date: 2019-06-04
YUNNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the typical methods for detecting APA mainly include blot hybridization, real-time fluorescent quantitative PCR, gene chip, high-throughput sequencing technology and other methods, but there are some shortcomings in these methods, such as expensive equipment, high maintenance cost, cumbersome sample processing, Time-consuming, high detection cost, complex operation, low detection sensitivity, high false positive rate, etc., limit their application and popularization

Method used

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  • Preparation method and application of electrochemical sensor for non-coding RNA
  • Preparation method and application of electrochemical sensor for non-coding RNA
  • Preparation method and application of electrochemical sensor for non-coding RNA

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Embodiment 1: The steps of the preparation method of the electrochemical sensor of the non-coding RNA are as follows:

[0050] 1. Ferric oxide (Fe 3 o 4 ) Preparation of nanospheres

[0051] Put 1.35 g FeCl 3 .6H 2 O was added to 40 mL of ethylene glycol to form a clear solution, and sodium acetate and polyethylene glycol were added, wherein the mass ratio of sodium acetate to ferric chloride was 3:1, and the mass ratio of polyethylene glycol to ferric chloride The ratio is 7:10; after stirring for 30 minutes, put it into a hydrothermal reaction kettle and heat it to 180°C, react for 7 hours, cool to room temperature to get a black precipitate, wash the precipitate with absolute ethanol, and treat it at 60°C for 10 hours to obtain Fe 3 o 4 nanospheres;

[0052] 2. Gold nanoparticle-loaded Fe3O4 nanocomposites (Au@Fe 3 o 4 ) preparation

[0053] Step (1) Fe 3 o 4 Disperse 10 mg of nanospheres in 10 mL of ultrapure water. After ultrasonic dispersion is unifor...

Embodiment 2

[0068] Example 2: Specific detection of electrochemical sensors

[0069] In this embodiment, the electrochemical sensor in Example 1 is used to detect four kinds of mismatched sequences, 1MT-S, 2-MT-S, 1MT-L, and 2MT-L; the concentration of mismatched sequences is diluted to 10 -10 M for electrochemical detection; refer to the method of step 4 (2) of the example to prepare the complex phosphate buffer dispersion, the difference is that the target RNA is replaced by the mismatched sequence in this example, and the prepared phosphate buffer dispersion Drop on and cover the surface of the screen printing electrode, measure DPV, DPV scanning range: 0~-0.5V;

[0070] The mismatched sequences are:

[0071] 1MT-S: CCG AAA GAG CGA GAC GCG TC CAT AAT CTG GTC TCT TCT TC;

[0072] 2MT-S: CCG AAA GAG CAA GAC GCG TC CAT AAT CTG GTC TCT TCT TC;

[0073] 1MT-L: ATG ATA TAG CCA GCT GCC TT TTA AGA GGT CTT ATC TGT TC;

[0074] 2MT-L: ATG ATA TAG CAA GCT GCC TT TTA AGA GGT CTT ATC TGT TC;

...

Embodiment 3

[0076] Example 3: Application of electrochemical sensor in the detection of CCND2-S and CCND2-L in human lung cancer cell H292 and human normal lung cell Beas-2B

[0077] (1) Human normal lung cells and human lung cancer cells were selected for RNA extraction, and the extraction method was as follows:

[0078] 1) Remove the medium in the cell culture flask, add 1mL PBS to wash the cells, and aspirate and discard;

[0079] 2) Add 0.5 mL Trizol, shake by hand for 5 min to lyse the cells, and collect them into a 1.5 mL centrifuge tube;

[0080] 3) Add 0.1 mL of chloroform, shake vigorously for 15 s, and place at room temperature for 2-3 min;

[0081] 4) Centrifuge at 12000 rpm at 4°C for 15 min; (start to prepare new tubes for centrifugation; prepare RNA collection column; prepare enzyme-RDD mixed system: 10 μL DNase I stock solution and 70 μL Buffer RDD);

[0082] 5) Transfer the supernatant to a new centrifuge tube, add an equal volume of 70% ethanol, turn it upside down to m...

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Abstract

A method for preparing an electrochemical sensor for non-coding RNA, which comprises preparing a sulfonated cup [8] arene-supported reduced graphene oxide by a one-step method, synthesizing a ferroferric oxide nano material by a moist heating method, and adding gold nanoparticles to synthesize Au@SCX8-RGO and Au@Fe3O4 composite materials. After confirming successful material construction by material characterization, a target material is electrochemically detected by a screen printing electrode with toluidine blue and other materials used as electrical signal substances. The electrochemical biosensor prepared by the method can detect the expression quantity of 3'UTR transcripts of non-coding regions of different lengths produced by the selective polyadenylation (APA) phenomenon.

Description

technical field [0001] The invention belongs to the field of electrochemical biosensing, and in particular relates to an electrochemical detection method of non-coding RNA and its application in selective polyadenylation detection. Background technique [0002] Ultra-sensitive nucleic acid detection technology is widely used in functional gene detection and its biological function evaluation, disease diagnosis and other fields. Such as fluorescent quantitative PCR technology, rolling circle amplification technology and other trace nucleic acid detection technologies are based on polymerase chain reaction and fluorescent signal detection methods to achieve trace nucleic acid quantification or detection, but these detection methods have complex operation steps, expensive optical signal probes, and require Specific detection equipment and other issues. Electrochemical sensing technology has always been the most widely used biomedical and chemical analysis detection platform, w...

Claims

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Application Information

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IPC IPC(8): G01N27/48G01N27/327C12Q1/00
CPCC12Q1/00C12Q1/68G01N27/327G01N27/48
Inventor 赵卉李灿鹏张亚平刘凤
Owner YUNNAN UNIV
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