Biosensor for simultaneously detecting AFP (Alpha Fetoprotein) and miRNA-122 through resonance light scattering and preparation method of biosensor

A resonant light scattering and sensor technology, applied in biochemical equipment and methods, scattering characteristic measurement, biological material analysis, etc., can solve the problems of long detection time and high detection cost, and achieve high sensitivity, good response, and low cost effects

Inactive Publication Date: 2018-09-04
XIANGTAN UNIV
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  • Application Information

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Problems solved by technology

[0005] In order to overcome the shortcomings in the prior art and improve the problems of high detection cost and long detection time caused by relying on photochemical analysis technology in conventional tumor marker analysis, the purpose of the present invention

Method used

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  • Biosensor for simultaneously detecting AFP (Alpha Fetoprotein) and miRNA-122 through resonance light scattering and preparation method of biosensor
  • Biosensor for simultaneously detecting AFP (Alpha Fetoprotein) and miRNA-122 through resonance light scattering and preparation method of biosensor
  • Biosensor for simultaneously detecting AFP (Alpha Fetoprotein) and miRNA-122 through resonance light scattering and preparation method of biosensor

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Experimental program
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Effect test

Embodiment 1

[0037] Design of the detection probe system of the dual-function label-free sensor in the detection of tumor markers based on resonance light scattering technology:

[0038] The system includes two probes cDNA1 and cDNA2, each probe contains two parts, for the cDNA1 probe, near the 5' end is the AFP aptamer sequence, and near the 3' end is the partial complementary sequence of the miRNA-122 aptamer ; For the cDNA2 probe, it includes two parts: the partial complementary sequence of the AFP aptamer near the 5' end, and the miRNA-122 aptamer sequence near the 3' end;

Embodiment 2

[0040] Feasibility determination of dual-functional label-free sensors based on resonance light scattering technology in tumor marker detection probes:

[0041] (1) RLS determination of AFP and miRNA-122

[0042] cDNA1 and cDNA2 (1.0 nM) were added to 10 mM Tris (pH 7.4). DNA hybridization was performed at 40° C. for 40 minutes, and then 2.7 μM methyl violet (MV) was added to the solution to obtain a cDNA1cDNA2-methyl violet (MV) complex. Next, different AFP and miRNA-122 concentrations were added to the solution. The aptamer binding reaction was carried out at 40°C for 30 minutes. Finally, the RLS spectrum of the resulting reaction solution was obtained by synchronously scanning the excitation and emission monochromator (λex=λem) from 220.0 nm to 700.0 nm with an RF-5301PC fluorescence spectrophotometer (Shimadzu, Japan). Excitation and emission slit widths were kept at 3.0 nm.

[0043] (2) Circular dichroism (CD) spectrometry

[0044] Spectra were measured in the wavele...

Embodiment 3

[0051] The dual-function label-free sensor based on resonance light scattering technology is optimized in the detection method of tumor markers: the detection efficiency of this method is affected by factors such as methyl violet (MV) concentration, reaction time and working temperature, in order to obtain the best detection Performance, optimized assay detection conditions such as methyl violet (MV) concentration, reaction time and operating temperature. Through optimization, the best experimental conditions were obtained: the concentration of methyl violet (MV) was 2.7×10 -6 M; the reaction time is 30 minutes; the optimum reaction temperature is 40°C.

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Abstract

The invention discloses a biosensor for simultaneously detecting AFP (Alpha Fetoprotein) and miRNA-122 through resonance light scattering and a preparation method of the biosensor. The biosensor is composed of hybridized double chains cDNA1 and cDNA2 and methyl violet (MV), wherein the methyl violet is embedded into the hybridized double chains; cDNA1 comprises an AFP aptamer fragment; and cDNA2 comprises a miRNA-122 aptamer fragment. Under mutual actions of electrons of methyl violet (MV) and dsDNA, amplified resonance light scattering signals can be generated, a double-function label-free probe sensor has detection limits of 0.94mu g and 98mP upon AFP and miRNA-122 respectively, a relatively efficient, rapid and low tumor marker detection technique is established, and the technique has potential application in clinical screening of early-stage liver cancer.

Description

technical field [0001] The invention relates to a molecular detection probe, in particular to a dual-function label-free probe sensor for simultaneous detection of tumor markers AFP and miRNA-122 by resonance light scattering; it belongs to the field of molecular detection. Background technique [0002] Cancer has become one of the most devastating diseases in the world, causing more than 10 million new cases and 7.6 million deaths each year. Hepatocellular carcinoma (HCC) is one of the most common cancers and the third leading cause of cancer death worldwide. However, death from liver cancer is entirely avoidable through early detection and treatment. Therefore, the detection of tumor markers is important for early screening of cancer, identification of benign and malignant diseases, assessment of disease extent, monitoring of tumor treatment response and prediction of recurrence. However, the detection of a single tumor marker cannot provide a good cancer diagnosis due t...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N21/33G01N21/25G01N21/47C12Q1/6825G01N33/574
CPCC12Q1/6825G01N21/25G01N21/33G01N21/47G01N21/6486G01N33/57484G01N2800/7028
Inventor 蔡昌群韩云鹏刘易陈小明
Owner XIANGTAN UNIV
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