Nuclease amplified high-sensitivity electrochemical immunoassay method

An analytical method and electrochemical technology, applied in the field of highly sensitive electrochemical immunoassay, can solve the problems of high cost and long time required, and achieve the effects of simple preparation, good versatility and improved sensitivity

Active Publication Date: 2015-02-04
NANJING UNIV +1
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  • Description
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  • Application Information

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Problems solved by technology

However, this method basically uses PCR technology to detect the gen

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  • Nuclease amplified high-sensitivity electrochemical immunoassay method
  • Nuclease amplified high-sensitivity electrochemical immunoassay method

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Embodiment 1

[0025] Embodiment 1: in combination with figure 1 , Preparation of highly sensitive and general-purpose nucleic acid electrochemical immunosensing interface

[0026] Grind and clean the gold electrode with a diameter of 2 mm, add 6 μL of 0.5 μM hairpin DNA labeled with sulfhydryl (SH) and methylene blue (MB) on the treated gold electrode dropwise, react at room temperature for 2 hours in the dark, and use 0.1 The electrodes were washed with 0.01M phosphate buffer solution of M NaCl and dried with nitrogen gas. Then, 6 μL of 1 mM mercaptohexanol was added dropwise on the surface of the electrode, reacted at room temperature for 40 minutes, washed with phosphate buffer solution and dried with nitrogen gas. The nucleic acid electrochemical immunosensing interface was prepared and stored at 4°C until use.

Embodiment 2

[0027] Embodiment 2: in combination with figure 1 , taking carcinoembryonic antigen (CEA) as an example to illustrate the application of this nuclease-amplified high-sensitivity electrochemical immunoassay method.

[0028] (1) Preparation of sample incubation solution: mix CEA standard solution (or sample solution containing CEA) and DNA1-antibody 1, DNA2-antibody 2, DNA3-antibody 3 in 30mM MgCl 2 0.01M phosphate buffer, in which the concentrations of DNA1-antibody 1, DNA2-antibody 2, and DNA3-antibody 3 were all 50 nM.

[0029] (2) Add 6 μL of sample incubation solution dropwise to the electrochemical immunosensing interface, incubate at room temperature for 40 minutes, and then rinse with washing solution.

[0030] (3) Insert the rinsed electrode into the detection electrolyte, use this electrode as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire as the counter electrode, and perform differential pulse voltammetry in the potent...

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Abstract

The invention relates to a nuclease amplified high-sensitivity electrochemical immunoassay method. The method comprises the steps of carrying out self-assembly to methylene blue (MB) marked hairpin DNA on the surface of a gold electrode to construct an electrochemical immunosensing interface under the Au-S bonding effect; enabling antibody 1, antibody 2 and antibody 3 to recognize target proteins simultaneously to form immune complex under the existence of the target proteins by taking the mixed solution of Mg<2+>, DNA1-antibody1, DNA2-antibody2 and DNA3-antibody3 as detection liquid, and enabling DNA1, DNA2 and DNA3 to be close with one another to perform ortho-position hybridization to form Mg<2+>-dependent nuclease, so as to perform catalytic cracking to the hairpin DNA on the sensing interface, so that the MB is separated from the surface of the electrode and the oxidized current is reduced; determining the concentration of the target protein by detecting the change of the current of the MB. The immunoassay method utilizes three DNA-antibody conjugates to form a nuclease amplified detection signal in an ortho-position manner, can improve the detection sensitivity and the selectivity, can detect the target proteins rapidly in a one-step manner, and has good clinical application value.

Description

1. Technical field [0001] The invention is a highly sensitive electrochemical immunoassay method for nuclease amplification. The hairpin DNA labeled electrochemically active molecule (MB) was immobilized on the electrode surface through Au-S bonds to construct an electrochemical immunosensing interface. Induction of proximity effects and formation of Mg by immune recognition of target proteins 2+ Nuclease-dependent nuclease, thereby catalyzing the cleavage of hairpin DNA, detaching MB molecules from the sensor surface, and realizing the simple, rapid and highly sensitive determination of target proteins by detecting the reduction of MB oxidation current. 2. Background technology [0002] As a highly selective and sensitive analytical method, immunoassay has been widely used in environmental monitoring, clinical diagnosis, food safety and other fields. Immunoassays can be divided into heterogeneous immunoassays and homogeneous immunoassays. The latter is widely used in clin...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/44
CPCC12Q1/6804C12Q2525/301C12Q2565/607C12Q2563/137
Inventor 鞠熀先严枫任克维吴洁
Owner NANJING UNIV
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