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Method for manufacturing rotary type sensor used for rapidly, sensitively and specifically detecting trace small RNAs

A sensor, rotary technology, used in the field of biosensors

Inactive Publication Date: 2011-03-30
殷勤伟 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] This research idea has not been reported at home and abroad, and it is original

Method used

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  • Method for manufacturing rotary type sensor used for rapidly, sensitively and specifically detecting trace small RNAs
  • Method for manufacturing rotary type sensor used for rapidly, sensitively and specifically detecting trace small RNAs
  • Method for manufacturing rotary type sensor used for rapidly, sensitively and specifically detecting trace small RNAs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1. Preparation, labeling and assembly of molecular motor sensors

[0028] Extraction of photosynthetic bacteria chromophore [2, 3]: Use 5g of cultured R.rubrum cells (provided by Dr. Zhang Xujia) with 50ml TSM buffer (50mM Tricine-NaOH, pH 8.0, 0.25M sucrose, 4mM MgCl) 2 ) Suspended. Use ultrasound to break cells: large probe, 25% intensity, over 5s, stop for 5s, ultrasound for 10-12min, which can prevent the denaturation and inactivation of biological macromolecules caused by the overheating reaction caused by ultrasound. Then centrifuge at 25,000g for 30min; centrifuge the supernatant in FicollPM400 (GE Healthcare, USA) at 145,000g for 90min to obtain a uniform precipitation band, that is, photosynthetic bacterial chromatophores with higher purity ( Figure 4 ). Then suspend the precipitate in 1ml TSM buffer, add an equal volume of glycerin, and store at -20°C or -80°C.

[0029] LiCl treatment of the chromophore: take 0.8ml of the newly extracted chromophore (wit...

Embodiment 2

[0034] Example 2. Biosensor feasibility test

[0035] Previous studies have shown that the addition of the proton channel permeability agent CCCP can make the membrane permeable inside and outside, thereby inhibiting the non-delta subunit F o F 1 -The rotation of ATPase [11]. In addition, F o Channel inhibitor DCCD (purchased from Sigma) is able to interact with F o The carboxyl group of aspartic acid at position 61 of the upper c subunit is covalently bound, thereby blocking the outflow of protons and causing the fluorescence to no longer increase. In the experiment, the sample was incubated with 20μM DCCD at room temperature for one hour. Due to the inhibitory effect of DCCD, the fluorescence will not increase after the temperature is increased to 37℃ ( Figure 5 ). The above results prove that the increase in fluorescence is the result of proton pumping out of the membrane, not an artifact.

[0036] Determine the light time: Before detection, the miRNA detection system is illu...

Embodiment 3

[0042] Example 3. Sensitivity of the biosensor

[0043] Previous studies have shown that mir-145 is a tumor suppressor gene in breast cancer cells, so the expression level is lower than that of normal cells [15]. Therefore, this experiment chose to detect the low content of mir-145 in human breast cancer cells MCF-7 for research. We prefer to use Trizol (purchased from invitrogen) to extract total cellular RNA to detect mir-145 in this complex environment. Figure 7 The middle curves (a, b, c, d, e, f, g) respectively represent the effects of adding different amounts of load on the changes in fluorescence of the biosensor at 37°C. The curve a represents that the control sample probe system is completely connected, and only the target miRNA is not bound. Experiments show that when adding 10 6 The slope of miR-145 curve after the total RNA of the above cells 6 The slope of the curve of total RNA of each cell is the same. Through the above results, the saturation concentration of ...

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Abstract

The invention relates to the technical field of method for manufacturing rotary type sensor used for rapidly, sensitively and specifically detecting trace small RNAs, belongs to the technical field of biosensor, in particular relates to establishment and application of a novel rotary type sensor detection method. The invention has the characteristic that sensitivity and specificity when detecting small RNA with low abundance in cell, tissue, body fluid and blood are rapidly, simply and obviously improved. The invention provides a method for manufacturing novel rotary type small RNAs sensor, used for screening, detecting and comparing expression of endogenous small RNA (such as shRNA, miRNA, piRNA, siRNA and the like) and improving the existing chromatophore purification, preparation and fluorescence labelling, design of probe and sensor assembling and preparation technique of related chip. In the invention, FoF1-ATPase has double actions of biosensor and molecular motor. In the detection process, a probe with high specificity on a molecular motor rotates like a propeller, so that centrifugal force and fluidity of peripheral liquid are improved, thus reducing time for target small RNAs to approach the probe and promoting separation of non-target RNA. Thus, the method can rapidly and simply detect small RNA molecules expressed in cell, tissue, body fluid and blood at low abundance with high sensitivity and high specificity and can be used for identifying and comparing variation of small RNA before and after cell differentiation and diagnosis and evaluation of occurrence and development of disease.

Description

Technical field [0001] The invention belongs to the technical field of biosensors, and more specifically relates to the establishment and application of a new type of rotary sensor detection method. The invention has the characteristics of rapid, simple and remarkably improving the sensitivity and specificity of detecting low-abundance small RNAs in cells, tissues, body fluids and blood. Background technique [0002] At present, the research on rapid detection technology of biological samples has become one of the most important fields of international high technology. The rapid determination technology not only requires the size of the sample particles to be small enough, but also requires rapid, sensitive and convenient determination. Therefore, the recent development of intelligent sensors with molecular assembly refers to the integration of molecular recognition, signal conversion and measurement technologies. [0003] Biosensors can be divided into static and dynamic types. ...

Claims

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Application Information

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IPC IPC(8): C12M1/34C12Q1/68
Inventor 殷勤伟乐加昌黄兵廖洁颖
Owner 殷勤伟
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