Dinucleotide-labelled ratio electrochemical immunosensor

An immunosensing and electrochemical technology, applied in the field of ratiometric electrochemical immunosensors, can solve the problem that the detection signal is easily affected by the environment, and achieve the effects of broadening the detection linear range, improving biosensing efficiency, and simplifying the analysis process

Inactive Publication Date: 2014-05-14
NANJING UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Traditional electrochemical immunoassay methods use single-signal detection, and the detection signal is easily affected by the environment

Method used

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  • Dinucleotide-labelled ratio electrochemical immunosensor
  • Dinucleotide-labelled ratio electrochemical immunosensor
  • Dinucleotide-labelled ratio electrochemical immunosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: in combination with image 3 , Preparation of dual nucleic acid-labeled ratiometric electrochemical immunosensor

[0023] Grind and clean a gold electrode with a diameter of 2 mm, add 6 μL of 0.5 μM capture probe DNA3 labeled with sulfhydryl (SH) and ferrocene (Fc) at both ends, and react at room temperature for 2 hours in the dark. Clean the electrode with 0.01M phosphate washing solution containing 0.5M NaCl, and dry it 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 washing solution, and dried with nitrogen gas. Continue to drop 6 μL of 1 μM MB-labeled DNA1-antibody 1 on the electrode surface, react overnight at room temperature, wash with phosphate washing solution and blow dry with nitrogen to make a ratiometric electrochemical immunosensor for double nucleic acid labeling, and store it at 4°C stand-by.

Embodiment 2

[0024] Embodiment 2: in combination with figure 1 , taking prostate specific antigen (PSA) as an example to illustrate the application of this ratiometric electrochemical immunosensor.

[0025] (1) Preparation of sample incubation solution: mix the test solution containing the target protein (prostate specific antigen) and DNA2-antibody 2 in 0.01M phosphate buffer containing 0.5M NaCl, wherein the concentration of DNA2-antibody 2 is 50nM.

[0026] (2) Add 6 μL of sample incubation solution dropwise to the ratiometric electrochemical immunosensing interface, incubate at room temperature for 40 minutes, and then rinse with washing solution.

[0027] (3) Insert the rinsed electrode into the detection solution, 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 AC voltammetry in the potential range of -0.4V to +0.60V, The pulse amplitude was 25mV, the pulse frequency was 25Hz...

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Abstract

The invention relates to a dinucleotide-labelled ratio electrochemical immunosensor. The dinucleotide-labelled ratio electrochemical immunosensor is characterized in that DNA3 of sulfydryl and ferrocene are respectively labelled at the two assembled ends of the surface of a gold electrode and are hybridized with methylene-blue-labelled complementary DNA1-antibody1 so as to form a ratio electrochemical immunosensor interface. When target protein exists, a DNA2-antibody2, the target protein and the DNA1-antibody1 form sandwiched immune complex to generate a vicinal effect, and DNA2 is hybridized with DNA1, so that the DNA1-antibody1 is separated from a sensing interface, and freed DNA3 forms a hairpin structure on the surface. In the process, methylene blue and the ferrocene with electrochemical activity are separated from and close to the surface of the electrode respectively, and the oxidized electric currents of the methylene blue and the ferrocene are reduced and increased respectively. By detecting the ratio of the two currents, the concentration of the target protein in the solution is detected. The immunosensor is high in sensitivity, wide in the detection range, realizes fast and one-step detection of protein, and has clinical application value.

Description

1. Technical field [0001] The invention is a ratiometric electrochemical immunosensor based on dual nucleic acid labels. Two kinds of nucleic acid chains labeled with electrochemically active molecules are used to construct a dual electrochemical signal immunosensing interface, in which one chain (capture probe) is fixed on the electrode with Au-S bonds, and the other chain is labeled with an antibody, and passed through Complementary base pairing binds to the capture probe, and leaves the sensor surface under the action of the proximity effect induced by the immune reaction, making the two electroactive molecules away from and close to the electrode surface respectively. By detecting the ratio of the oxidation current of the two electroactive molecules, Realize the simple, rapid and highly sensitive determination of the target protein. 2. Background technology [0002] As an analytical method with high selectivity and high sensitivity, immunoassay has been widely used in e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N27/26
CPCG01N27/3276G01N33/6803
Inventor 鞠熀先严枫任克维吴洁
Owner NANJING UNIV
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