A biosensing assay method for detecting estrogen-binding activity in water samples

A technology combining activity and estrogen, which is applied in the direction of material stimulation analysis, material analysis, biological testing, etc., can solve the problems of increasing detection complexity and detection cost, inability to fully characterize estrogen binding activity, etc., and achieve easy online and in situ The effect of monitoring, reducing detection costs, and stabilizing regeneration

Active Publication Date: 2020-03-10
TSINGHUA UNIV
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Problems solved by technology

Compared with the traditional biological method for the detection of estrogen compounds, this method is more efficient and easier to operate, but it needs to be combined with LC-MS, NMR and other techniques to quantitatively analyze the concentration of estrogen pollutants. On the one hand This greatly increases the complexity and cost of detection, and on the other hand, it cannot fully characterize the estrogen-binding activity of water samples

Method used

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  • A biosensing assay method for detecting estrogen-binding activity in water samples
  • A biosensing assay method for detecting estrogen-binding activity in water samples
  • A biosensing assay method for detecting estrogen-binding activity in water samples

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Embodiment 1

[0031] 1) Make a receptor protein affinity column

[0032] Resuscitate the Escherichia coli transformed with the His-ER expression plasmid, smear it on the LB plate containing 50 mg / L kanamycin, pick a single clone into the LB medium containing kanamycin after 16 hours, and culture with shaking at 37°C At 20 hours, absorb 15mL to 100mL LB medium containing 15% sucrose, culture at 37°C for 3h with shaking, then cool down gradually, and the temperature drops to 15°C within 3h, and add IPTG with a final concentration of 0.5mM for low temperature induction for 16h. Centrifuge the bacterial liquid to obtain the bacterial cells, add 10 mL of lysis buffer to resuspend the bacterial cells, crush on an ultrasonic breaker, and centrifuge at a high speed and low temperature to obtain the supernatant. Add the supernatant to a 200 μL nickel column agarose gel column, shake and incubate at 4°C for 2 hours, centrifuge to discard the supernatant, wash the impurity protein with purification bu...

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Abstract

The invention discloses a biosensing analysis method for detecting the binding activity of estrogen in a water sample. According to the method, an estrogen receptor is used as a bio-recognition molecule; a signal probe and a sensor chip are placed in a to-be-detected water sample, and the sensor chip captures the signal probe and excites fluorescence-labeled molecules in the signal probe; the fluorescence intensity of the excited fluorescence-labeled molecules is proportional to the content of estrogen in the to-be-detected water sample, so the content of estrogen in the water sample is calculated based on the fluorescence intensity of the excited fluorescence-labeled molecules; and the signal probe is a fluorescently labeled estradiol-streptavidin conjugate. The method of the invention can realize the quantitative analysis of the binding activity of estrogen in the water sample; a biosensing interface can be stably regenerated, so detection cost is greatly reduced; and the method hasthe advantages of economic performance, simplicity and rapidity, and is easy to realize online and in-situ monitoring.

Description

technical field [0001] The invention relates to a biosensing analysis method for detecting water sample estrogen binding activity, belonging to the field of environmental monitoring analysis. Background technique [0002] Complex water pollution includes toxic and harmful chemical pollutants with different structures and complex types, and the single concentration of water complex pollution and its risks has great limitations. With the increasing emphasis on environmental pollution effect-oriented analysis, biosensing analysis methods using nuclear receptors as recognition elements have been rapidly developed. Estrogen receptor is a nuclear receptor protein molecule that can specifically bind to estrogen in the steroid hormone receptor protein superfamily. When different estrogen pollutants bind to estrogen receptors, they will induce structural changes in receptor proteins, form or inhibit the formation of dimers, and then produce different biological effects, showing estr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/74G01N21/64
CPCG01N21/6428G01N33/74
Inventor 周小红刘兰华施汉昌陆韻
Owner TSINGHUA UNIV
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