Electrochemical sensor based on short-distance hybridization of split aptamer as well as preparation method and application of electrochemical sensor

An aptamer, short-range technology, applied in the field of bioelectrochemical sensors, to achieve the effect of good linear relationship, good specificity and good selectivity

Pending Publication Date: 2020-06-26
NANJING NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the first cocaine split aptamer was reported by Stojanovic et al. in 2000, split aptamers have attracted widespread attention in the field of small molecule, protein and cell detection. However, so far, no split aptamers have been used in tumors. Reports on Exosome Detection

Method used

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  • Electrochemical sensor based on short-distance hybridization of split aptamer as well as preparation method and application of electrochemical sensor
  • Electrochemical sensor based on short-distance hybridization of split aptamer as well as preparation method and application of electrochemical sensor
  • Electrochemical sensor based on short-distance hybridization of split aptamer as well as preparation method and application of electrochemical sensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Such as figure 1 Steps shown to prepare electrochemical aptasensors.

[0044] (1) Prepare the buffer solution and DNA buffer solution required for the experimental system;

[0045] (2) Preparation of electrodes for capturing DNA modifications;

[0046] (3) Add S1, S2 and the exosome solution to be tested;

[0047] (4) Using a three-electrode system, the signal is recorded on an electrochemical workstation.

[0048] The buffer solution prepared in step (1) is a 10 mM Tris-HCl (pH=7.4) buffer solution used as a detection solution and a storage solution for DNA strands.

[0049] In step (2), the gold electrode is pretreated. 6 μL of 10 μM capture DNA, 6 μL of 10 mM tris(2-formylethyl)phosphate (TCEP) and 48 μL of 10 mM tris-hydroxymethylaminomethane (Tris-HCl) buffer were co-incubated for 2 hours to break the disulfide bonds. 10 μL of 0.1 μM thiolated capture DNA was dropped on the surface of the gold electrode (GE) and incubated at 4°C for 3 hours, and was connected ...

Embodiment 2

[0056] The aptamer sensor of the present invention analyzes and detects exosomes at different concentrations

[0057] Using the aptamer sensor prepared in Example 1 of the present invention, according to the steps and detection of the above-mentioned Example 1, different concentrations of 2.375×10 were added in step (3). 6 , 4.75×10 6 , 9.5×10 6 , 2.375×10 7 , 4.75×10 7 , 9.5×10 7 , 4.75×10 8 , 9.5×10 8 particles / mL, the exosomes secreted by HeLa cells were analyzed and detected.

[0058] The result is as image 3 As shown, the detection range of the aptasensor is 2.375×10 6 ~9.5×10 8 In particles / mL, the detection limit is 6.607×10 5 particles / mL.

[0059] image 3 As shown in A, the signal intensity of DPV increases with the concentration of exosomes from 2.375×10 6 particles / mL-9.5×10 8 particles / mL increased and enhanced. exist image 3 Within the logarithmic range of the exosome concentration shown in B, it can be observed that the logarithm of the exosome...

Embodiment 3

[0061] The aptamer sensor of the present invention analyzes and detects exosomes in complex biological samples

[0062] Using the aptamer sensor prepared in Example 1 of the present invention, according to the steps and detection of the above-mentioned Example 1, the human cervical cancer (HeLa) cell exosomes (4.75×10 8 particles / mL) were tested in PBS (pH=7.4) and PBS (pH=7.4) solutions containing 15%, 65% UC FBS (ultracentrifuged FBS). Signal intensities were compared by adding the same amount of exosomes to PBS and PBS containing (15% and 65%) UC FBS. There were no significant differences in the signal intensities obtained among the three. Therefore, the prepared aptasensor has good stability in complex biological samples, and the results are as follows: Figure 4 shown.

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Abstract

The invention discloses an electrochemical sensor based on short-distance hybridization of a split aptamer as well as a preparation method and application of the electrochemical sensor. The aptamer sensor performs quantitative analysis on the exosome by utilizing the advantages of short-distance hybridization and aptamer splitting; sulfydryl-modified captured DNA is fixed on the surface of a goldelectrode; hexammine ruthenium trichloride is taken as an electroactive substance, a target exosome and a DNA chain with an aptamer are added, the exosome is captured on a gold electrode through target-induced close-range hybridization, and self-assembled DNA can adsorb more hexammine ruthenium trichloride, so that relatively high peak current is generated. The aptamer sensor of the invention hasa good linear relation for exosome detection, the detection limit is low, meanwhile, the content of exosome surface protein secreted by various cells can be analyzed, detection of exosomes in complexbiological samples can be completed, and the aptamer sensor has a good application prospect in clinical diagnosis.

Description

technical field [0001] The invention relates to the technical field of bioelectrochemical sensors, in particular to an electrochemical sensor based on split-type aptamer short-distance hybridization and its preparation method and application. Background technique [0002] Exosomes are vesicle bodies with a lipid bilayer secreted by living cells, with a particle size of 30-150 nm. Exosomes derived from tumor cells can participate in the regulation of the tumor microenvironment and the formation of metastases, and have the characteristics of good stability and high content in body fluids. Sensitive and accurate detection of exosomes is extremely important for early diagnosis and treatment monitoring of cancer It is an important detection substance for cancer liquid biopsy. The surface of tumor exosomes has a variety of characteristic proteins derived from mother cells. The types and contents of these proteins are closely related to the type, development and treatment of tumor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/569G01N27/327
CPCG01N33/57411G01N33/57426G01N33/57492G01N33/56966G01N27/3276
Inventor 沈健张卉乔斌
Owner NANJING NORMAL UNIVERSITY
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