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SPRi biosensor for detecting exosomes of liver cancer cells as well as preparation and application of SPRi biosensor

A technology of biosensors and liver cancer cells, which is applied in the field of SPRi biosensors for detecting exosomes of liver cancer cells, can solve the problems that the sensitivity needs to be improved, and achieve the effect of enhancing the detection signal

Pending Publication Date: 2021-08-20
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method still has its inherent flaws, and the sensitivity needs to be improved when it is used to detect trace amounts of target substances in complex samples

Method used

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  • SPRi biosensor for detecting exosomes of liver cancer cells as well as preparation and application of SPRi biosensor
  • SPRi biosensor for detecting exosomes of liver cancer cells as well as preparation and application of SPRi biosensor
  • SPRi biosensor for detecting exosomes of liver cancer cells as well as preparation and application of SPRi biosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Preparation of SPRi biosensor and detection of exosomes

[0088] 1. Materials

[0089] HAuCl 4 4H 2O was purchased from Sinopharm Chem Co., Ltd (Shanghai, China). 6-Mercapto-1-hexanol (MCH) was purchased from Sigma-Aldrich (St. Louis, USA). Fetal bovine serum (FBS) and Dulbecco's Modified Eagle Medium (DMEM) were provided by Gibco (Gaithersburg, MD, USA). N-Hydroxysuccinimide (NHS) and N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC) were purchased from Alfa Aesar (Massachusetts, USA ). All HPLC purified oligonucleotides were prepared and purified by Sangon Biotech.Co., Ltd (Shanghai, China). Oligonucleotides were dissolved in Tris-EDTA (TE) buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The aqueous solution prepared by Millipore Milli-Q gradient ultrapure water system (Millipore Co., MA, USA) was used throughout the experiment.

[0090] 2. Testing instruments

[0091] The SPRi detection in the experiment was performed on a surface plasmon reso...

Embodiment 2

[0126] Validation of the feasibility of detecting SPRi biosensors

[0127] 1. Verify the combination of G4-hemin

[0128] Verify the successful combination of G4-hemin in Example 1, figure 2 The UV absorption diagram shown to verify the combination of the two is given by figure 2 It can be seen that the characteristic absorption peaks of G4 nucleic acid chain and hemin appear in the signal of the blue line at the same time, which are 260nm and 402nm respectively, indicating that the two are successfully combined.

[0129] 2. Verify the combination of gold nanoparticles and tyramide

[0130] image 3 The TEM (A) and UV characterization results (B) of the combination of gold nanoparticles and tyramide are shown.

[0131] Depend on image 3 The TEM figure shown in A shows that the gold nanoparticles synthesized in embodiment 1 have a uniform particle size and better dispersibility;

[0132] image 3 In the ultraviolet characterization diagram shown in B, the black one is...

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Abstract

The invention discloses an SPRi biosensor for detecting exosomes of liver cancer cells as well as preparation and application of the SPRi biosensor. A detection method of the SPRi biosensor comprises the following steps of: (1) extracting an exosome, specifically, extracting the exosome from liver cancer cells; (2) synthesizing a gold nanoparticle-tyramine compound, specifically, firstly synthesizing gold nanoparticles, and then combining the gold nanoparticles with tyramine to generate the gold nanoparticle-tyramine compound; and (3) performing SPRi detection, specifically, firstly modifying a chip surface with an aptamer hairpin capture probe with a G4 chain, then loading the exosome, after loading the exosome, specifically binding the aptamer with protein on the surface of the exosome, opening the hairpin, releasing a G4 sequence, forming a G4-hemin enzyme, loading the gold nanoparticle-tyramine compound, and binding the gold nanoparticles to the chip surface through tyramine signal amplification (TSA), and carrying out detection through SPR. The sensor is a label-free SPRi biosensor capable of detecting liver cancer cell-derived exosomes in real time, and is high in sensitivity and specificity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a SPRi biosensor for detecting liver cancer cell exosomes and its preparation and application. Background technique [0002] The incidence of liver cancer ranks third among malignant tumors in my country, with high risk and high mortality. The most effective way to improve the survival rate of liver cancer patients is early diagnosis and treatment. Thanks to its simple operation, non-invasiveness, ready sampling and rapid response, liquid biopsy is considered to be one of the most promising methods for early tumor screening. Among them, the selection of biomarkers is the key to determine the reliability of liquid biopsy results. Exosomes are one of the emerging tumor markers in recent years. They are double-membrane disc-shaped vesicles secreted by mammalian cells with a particle size of 30-200 nm. A large number of studies have shown that the production, content and function of e...

Claims

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Application Information

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IPC IPC(8): G01N33/574G01N33/569G01N21/552
CPCG01N33/57438G01N33/57492G01N33/56966G01N21/554
Inventor 丁世家陈文琴白惠洁
Owner CHONGQING MEDICAL UNIVERSITY