Electrochemical sensor for detecting telomerase activity and detection method thereof

A technology of telomerase and electrochemistry, applied in the fields of electrochemical variables of materials, scientific instruments, instruments, etc., can solve the problems of low binding efficiency and enzyme kinetics, limited recognition ability of telomerase, and unfavorable real detection of telomerase. , to avoid the steric hindrance effect, improve the sensitivity, and have a broad application prospect.

Active Publication Date: 2019-09-17
THE SECOND HOSPITAL OF NANJING +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although great progress has been made using different electrochemical methods to detect telomerase activity, telomerase has limited recognition of primers on the electrode surface, and the steric hindrance of the electrode surface makes the combination of these heterogeneous experiments difficult. Relatively low efficiency and enzyme kinetics
At the same time, because the cell lysate contains multiple enzyme proteins, it is easy to degrade the extension product of telomerase, which is not conducive to the real detection of telomerase activity.

Method used

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  • Electrochemical sensor for detecting telomerase activity and detection method thereof
  • Electrochemical sensor for detecting telomerase activity and detection method thereof
  • Electrochemical sensor for detecting telomerase activity and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1 is used to detect the preparation method of the electrochemical sensor of telomerase activity

[0079] 1. Cell culture and extraction of telomerase

[0080] (1) HeLa cell culture: 37°C, 5% CO 2 Cultured in DMEM medium containing 10% fetal bovine serum under culture conditions;

[0081] (2) Cell suspension and dispersion treatment: wash the cells in the logarithmic growth phase with PBS, digest with trypsin for 1-3min, and gently blow the cells with PBS buffer to detach them from the surface of the cell culture flask to obtain detached cells;

[0082] (3) Telomerase extraction: collect the detached cells obtained in step (2), centrifuge at 1000rpm for 5min, resuspend with 200μL of frozen 1×CHAPS lysis buffer, and put the lysate containing cells at 4°C , centrifuged at 12000rpm for 30min, collected the supernatant with an Eppendorf tube, and measured the protein concentration with a small part of the supernatant (2μL) under the absolute absorbance at 280nm...

Embodiment 2

[0092] Example 2. Detection of telomerase activity

[0093] 1. The electrochemical method used is alternating current impedance method (EIS). The electrochemical instrument is CHI660D, including a three-electrode system, Ag / AgCl as a reference electrode, and platinum wire as an auxiliary electrode and a gold electrode modified with DNA as a working electrode;

[0094] with 1M KNO 3 5mM [Fe(CN) 6 ] 3- / 4- The solution was carried out, and the EIS experimental parameters: the initial voltage was 0.224V, and the scanning frequency was 0.1Hz-100kHz.

[0095]2. The electrochemical method used is differential pulse voltammetry (DPV), and the electrochemical instrument is CHI660D, including a three-electrode system, Ag / AgCl is a reference electrode, and platinum wire is used as an auxiliary electrode and a gold electrode modified with DNA. Working electrode;

[0096] DPV is carried out in 10mM PBS buffer (PH 7.4), DPV experimental parameters: scanning voltage is -0.5V-0V, pulse a...

experiment example 1

[0098] Feasibility test of experimental example 1 telomerase activity assay

[0099] In this experimental example, differential pulse voltammetry (DPV) is used to verify the feasibility of the method for detecting telomerase activity of the present invention. Such as figure 2 As shown, the DPV current peak showed a significant enhancement in the presence of crude telomerase extract (curve e), indicating the successful synthesis of the repeat sequence (TTAGGG)n on the surface of the magnetic beads and the generation of signal amplification. However, in the absence of telomerase crude extract, the peak DPV current is negligible (curve a), indicating that in the absence of telomerase, the primer cannot be extended further and the complementary sequence has no bound sequence , Exonuclease III cannot be recognized and degraded, and the hairpin-like structure containing MB cannot be opened, nor can it be degraded by Exonuclease III, so that it cannot bind to the capture probe on t...

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Abstract

The invention provides an electrochemical sensor for detecting telomerase activity and a detection method thereof which are constructed based on functionalized nanometer magnetic beads and an exonuclease III-assisted amplification strategy, and the detection of the telomerase activity of the tumor cells is realized. In the invention, complementary probes can be hybridized with an extended telomerase primer repeated sequence, a hybrid is separated by the magnetic beads, a telomerase primer repeated sequence chain is identified and degraded by exonuclease III, a plurality of methylene blue marked hairpin DNA probes can be hybridized and opened by the released complementary probes, after the secondary degradation of the exonuclease III, the complementary probes can enter the next cycle of hybridization with an MB-marked hairpin-like structure, and the MB-marked probe is combined with a capture probe on the surface of a gold electrode, so that a peak current value which is in direct proportion to the telomerase activity is generated. According to the electrochemical sensor and the detection method thereof, the telomerase activity can be detected at a single-cell level.

Description

technical field [0001] The invention relates to the field of electrochemical detection of telomerase, in particular to an electrochemical sensor for detecting telomerase activity and a detection method thereof. Background technique [0002] Telomerase, or telomerase for short, was originally discovered in single-celled organisms (Tetrahymena). grain end. Nearly a decade after its discovery, telomerase is considered a universal biomarker of human cancer. It is well known that telomeres gradually shorten with age, and through a series of changes such as activation or upregulation of telomerase, have short Cells with telomeres escape senescence and become immortal cells. Most human tumor cells (85%–90%) highly express telomerase, whereas in most normal tissues or cells telomerase activity is absent or highly inhibited, making telomerase an attractive candidate for cancer therapy. A valuable tumor marker protein for early diagnosis of tumors. Studies have shown that the abno...

Claims

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Application Information

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IPC IPC(8): G01N27/327G01N27/38G01N27/48
CPCG01N27/3276G01N27/38G01N27/48
Inventor 李金龙杨永峰张永臣许传军胡凯
Owner THE SECOND HOSPITAL OF NANJING
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