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NDM-1 locked nucleic acid probe modified electrode as well as preparation method and application thereof

A technology of locking nucleic acid probes and modifying electrodes, which is applied in the field of electrochemical detection, can solve the problems of high detection cost, time-consuming and cumbersome diagnostic methods, low sensitivity and specificity, and achieve the effect of specific detection tools

Active Publication Date: 2014-12-24
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these diagnostic methods have the disadvantages of time-consuming and cumbersome, low sensitivity and specificity, and high detection cost.

Method used

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  • NDM-1 locked nucleic acid probe modified electrode as well as preparation method and application thereof
  • NDM-1 locked nucleic acid probe modified electrode as well as preparation method and application thereof
  • NDM-1 locked nucleic acid probe modified electrode as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The preparation method of NDM-1 locking nucleic acid probe modified electrode comprises the following steps:

[0029] a. Cleaning of the gold electrode: take a gold electrode with a diameter of 3 mm, and use 0.3 μm and 0.05 μm Al respectively 2 o 3 The powder was ground and polished to a mirror surface, rinsed with ultra-pure water after each grinding, and then ultrasonically washed in nitric acid, acetone, and ultra-pure water for 5 minutes respectively, and dried to obtain a cleaned gold electrode for future use.

[0030] b. Immobilization of LNA probes: Take 20 μL of NDM-1 LNA probe solution with a concentration of 1.5 μmol / L modified thiol at the 5’ end and drop it on the surface of the cleaned gold electrode, and then keep the temperature in an incubator at 37°C for 1 Hours, the locked nucleic acid is modified to the surface of the electrode through the chemical bonding of the Au-S bond, and then the electrode is immersed in a mercaptohexanol solution with a conce...

Embodiment 2

[0032] Embodiment 2, build the electrochemical biosensor that detects NDM-1

[0033] The NDM-1-locked nucleic acid probe-modified electrode was used as the working electrode, the saturated calomel electrode was used as the reference electrode, and the platinum electrode was used as the counter electrode to form an electrochemical biosensor for detecting NDM-1. Connect the prepared electrochemical sensor to the electrochemical workstation, and use pH 7.4 containing 2mmol / L K 3 Fe(CN) 6 -K 4 Fe(CN) 6 , 5mmol KCl in PBS was used as the test base solution, and then the scanning measurement was carried out by cyclic voltammetry at room temperature, the working potential was -0.3V~0.7V, and the scanning speed was 50mV / s. The detection principle of the electrochemical biosensor for detecting NDM-1 is as follows: after the hybridization reaction occurs on the surface of the electrode modified by NDM-1 locked nucleic acid probe, the hybrid complex formed hinders the electron transfe...

Embodiment 3

[0034] Example 3, Detection of Electrochemical Properties of NDM-1 Locked Nucleic Acid Probe Modified Electrode

[0035] Use cyclic voltammetry to characterize the electrochemical characteristics of the electrode during the modification and detection process. The specific detection method is: after the electrodes at different stages are constructed as an electrochemical biosensor according to the method in Example 2, the electrodes are soaked in 150 μL NDM-1 In a solution (pH7.40.1 mol / L phosphate solution) with a final concentration of the target sequence of 100 μg / L, hybridize in a 37° C. incubator for 45 minutes. During the hybridization process, the sensor uses a three-electrode system to measure the sensor current, and the obtained cyclic voltammetry curve is as follows: figure 1 shown.

[0036] Among them, curve a is that the bare gold electrode contains 2mmol / L K at pH7.4 3 [Fe(CN) 6 ] / K 4 [Fe(CN) 6 ] and the cyclic voltammetry curve in PBS solution of 5mmol / l KCl,...

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Abstract

The invention discloses an NDM-1 locked nucleic acid probe modified electrode as well as a preparation method and application thereof. Specifically, a locked nucleic acid modified probe is designed, which immobilized on the surface of an electrode to serve as a specific probe, wherein the 5' end of the locked nucleic acid probe is modified with a sulfydryl and then the sulfydryl can be immobilized on the surface of the electrode by virtue of chemical bonding of an Au-S bond; the locked nucleic acid probe is mutually complementary with partial sequence of a target gene. The NDM-1 locked nucleic acid probe modified electrode provides a brand new thought and a detection method for the detection of multiple drug-resistant bacteria.

Description

technical field [0001] The invention belongs to the field of electrochemical detection, and in particular relates to an NDM-1 locked nucleic acid (Locked nucleic acid, LNA) probe modified electrode, and also relates to a preparation method and application of the electrode. Background technique [0002] Most of the sensors reported in the current research are immobilized on the surface of DNA probes, which have many shortcomings. The length of the probe is generally about 20 bases. When the DNA probe hybridizes to a longer target sequence, the binding force is not high, and the base mismatch recognition ability is low. In addition, since the single-stranded DNA cannot directly combine with the untwisted double-stranded target sequence, the specimen to be detected needs to be amplified by PCR before it can react with it. At the same time, the reaction of the NDM-1 probe is affected by the ionic strength. The amount of DNA immobilized is the largest in acidic or alkaline envir...

Claims

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Application Information

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IPC IPC(8): G01N27/327
Inventor 张立群王云霞府伟灵
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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