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Gold-labeled secondary antibody signal amplified SRP immunosensing method for detection of atrazine

A technology of atrazine and sensors, applied in measuring devices, material analysis through optical means, instruments, etc., can solve the problems of solvent consumption and time consumption

Active Publication Date: 2015-04-15
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are time-consuming and solvent-intensive

Method used

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  • Gold-labeled secondary antibody signal amplified SRP immunosensing method for detection of atrazine
  • Gold-labeled secondary antibody signal amplified SRP immunosensing method for detection of atrazine
  • Gold-labeled secondary antibody signal amplified SRP immunosensing method for detection of atrazine

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Experimental program
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Effect test

Embodiment approach 1

[0022] Embodiment 1: Construction of SPR detection platform:

[0023] (1) Immerse the clean SPR chip in the 1.0mmol / L undecyl mercaptoalkanoic acid solution, overnight at room temperature, rinse the SPR chip with carboxyl group with ethanol, deionized water successively and blow dry with nitrogen gas, then pack into the SPR sensor ( Autolab ESPRIT, Netherlands).

[0024] (2) Using pH 4.5 acetic acid-sodium acetate buffer as the coupling buffer, add 50 μL of 0.4mol / LEDC and 0.1mol / L NHS (1 / 1, v / v) mixture to the SPR sensor reaction cell to activate the chip Carboxyl groups on the surface. Add 50 μL of complete antigen atrazine-OVA solution diluted in coupling buffer, then add 1.0mol / L ethanolamine solution with pH8.5 as blocking solution, choose 0.1mol / L hydrochloric acid as regeneration solution, and coupling buffer as reference solution ( figure 2 ).

Embodiment approach 2

[0025] Embodiment 2: Using nano-gold-labeled secondary antibodies as SPR sensor amplification components for signal amplification

[0026] (1) Use phosphate buffer solution PBST buffer (10 mmol / LPBS (pH7.0) + 0.9% NaCl + 0.005% (v / v) Tween-20) to stabilize the chip surface until the baseline is stable.

[0027] (2) Take 50 μL of atrazine monoclonal antibody, nano-gold-labeled secondary antibody and atrazine small molecule mixture for 10 minutes of immunoreaction on the surface of the chip, desorb with carrier liquid for 5 minutes to remove weak non-specific binding, Measure the concentration of atrazine SPR sensory response value. Finally, use 0.1mol / L hydrochloric acid + 0.1% SDS as the regeneration solution, and regenerate twice in 120s to remove the AT-mAb combined with AT-OVA, so that AT-OVA can be used for the next detection.

[0028] (3) The standard curve of the detection method was drawn by using the measured signal and the corresponding atrazine concentration, the mi...

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Abstract

The invention discloses a gold-labeled secondary antibody signal amplified SRP (surface plasmon resonance) immunosensing method for detection of atrazine. The method adopts antigen-antibody immunoreaction as the basic principle, an antigen or antibody is taken as a sensor sensitive recognition element, an indirect competitive technique and an SPR sensing technology are combined to detect the herbicide atrazine, an atrazine complete antigen (AT-OVA) is fixed on the surface of an SPR chip through a chemical bond, competitive binding with the antibody is carried out on the atrazine small molecules in a to-be-detected solution, a nanogold-labeled secondary antibody is added to perform signal amplification, the detection sensitivity is enhanced, the detection range is improved, and the signal amplified indirect competitive inhibition method is established. The lowest detection limit of the method is 0.72ng / mL, IC50 is 15.70ng / mL, and the detection range is 2.18-629.06ng / mL.

Description

technical field [0001] The invention relates to a signal amplification surface plasmon resonance (surface plasmon resonance, SPR) immunosensing method, which is used for rapid detection of atrazine with high specificity and high sensitivity, in particular to the SPR signal amplification of nano-gold-labeled secondary antibodies method. Background technique [0002] Atrazine (AT) is a triazine herbicide that kills weeds by inhibiting the electron transfer of photosynthesis. weeds. Most of the AT sprayed on farmland diffuses into the air or soil, and then seeps into the ground with precipitation and irrigation water, causing groundwater pollution. Studies have shown that AT is an estrogen disruptor with teratogenic potential, and even very low doses of exposure can increase health risks. Moreover, it can still maintain activity in the environment for several years, causing damage to the ecological environment. Therefore, in 2004, the European Union listed it as a banned pe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/552G01N33/577G01N33/532
Inventor 彭媛高志贤宁保安白家磊孙思明柳明
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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