| Estrogen is one of the most important hormones in human body and animal body,with extremely extensive and important physiological functions.Cardiovascular system,nervous system,bone and other target organs(or tissues)of estrogen in the body.But estrogen is a double-edged sword for people,too high or too high can lead to disease.In addition,there are also some exogenous estrogen with estrogen class functions such as a variety of plant estrogen,synthetic estrogen compounds etc widely exists in the usual diet and food packaging,this kind of natural and synthetic estrogen compounds of misuse or illegal use of pose great threat to human health,is likely to lead to the human body endocrine disorders,It can even cause cancer.Therefore,how to accurately evaluate the function of natural estrogen or exogenous estrogen with estrogen effect(food functional components or toxins)is particularly important.G-protein coupled estrogen receptor(GPER)is one of the seven transmembrane estrogen receptors,which is expected to be developed as a platform for determining or evaluating the biological(or cellular)functions of estrogen compounds.However,if the ligand compounds of estrogen receptor are analyzed by G protein coupling and functional evaluation,it is necessary to understand the interaction and signaling mechanism between GPER and its ligand compounds more precisely.To explore a quantitative method to determine or evaluate the physiological activity of estrogen compounds.Studies have shown that G protein-coupled estrogen receptors play physiological functions such as cardio-cerebral and vascular function regulation and metabolism,reproductive function regulation and metabolism and balance,and energy metabolism and balance through mediating non-genomic pathways.The GPER-mediated non-genomic pathway is often achieved within a short period of time,i.e.,minutes or even seconds,during which GPER ligand compounds(estrogens or estrogen analogues)interact with GPER,resulting in the dissociation of G proteins into G?and G??,followed by activation of their intracellular downstream signals.Although there are more and more researches on GPER at present,no report has been found on establishing GPER sensor to measure estrogen and its interaction dynamics with different ligands.In this study,an electrochemical GPER receptor sensor was successfully constructed through the GPER receptor protein synthesized in vitro and the signal amplification system created by our laboratory,and the quantitative kinetics of 10 representative GPER ligand compounds(estrogen or estrogen analogues)were determined.The interaction between these ligands and GPER was discussed in detail.In this study,the GPER receptor electrochemical biosensor modified by gold nanoparticles was prepared by self-assembly method,which was successively assembled on the surface of glassy carbon electrode in the order of chitosan-gold nano-sol-gper-thiansy chitosan-gold nano-horseradish peroxidase-GPER-BSA.The interaction kinetics of estrogen or estrogen analogues with GPER was studied by current-time assay.The results showed that the linkage allosteric constants(Ka value)of estradiol(E2)were 8.80×10-17mol/L,the linkage allosteric constant(Ka value)of G-1 is 7.85×10-15mol/L,the linkage allosteric constant(Ka value)of G-15 is 4.97×10-15mol/L,the linkage allosteric constant(Ka value)of bisphenol A(BPA)was 6.53×10-16mol/L,the linkage allosteric constant(Ka value)of resveratrol was 8.35×10-16mol/L,the linkage allosteric constant(Ka value)of epicatechin was 6.47×10-15mol/L,the linkage allosteric constant(Ka value)of daidzein was4.28×10-16mol/L,the linkage allosteric constant(Ka value)of genistein was 4.48×10-16mol/L,the linkage allosteric constant(Ka value)of nonylphenol is 3.89×10-16mol/L,the linkage allosteric constant(Ka value)of coustrostrol was 1.02×10-15mol/L.By using the same GPER electrochemical receptor sensor to measure estradiol solution in 10-6mol/L for10 times continuously,the calculated relative standard deviation(RSD)of current change rate was 5.81%.By using 5 different GPER electrochemical receptor sensors to measure estradiol concentration with the same concentration,the calculated RSD is:1.82%;The assembled GPER electrochemical receptor sensor was stored in a refrigerator at 4?for reserve and measured regularly.The results showed that the response current value on the10th day was 79.65%of that on the first day.It is proved that the GPER receptor sensor can be successfully used to quantify the dynamics of interaction between GPER and estrogen or estrogen analogues,and has good repeatability,reproducibility and stability.The results also showed that the sensitivity of the receptor sensor to 10 ligand compounds was up to10-17mol/L,far superior to other detection methods,and it has the advantages of high sensitivity,simple operation,fast determination and good stability.This study by using electrochemical signal amplification system simulation cell receptor signaling cascade amplifier system,to help direct research GPER interaction and its ligands and explore its sensor dynamics,from the perspective of receptors for food ingredients or toxins to quantitatively function evaluation and the law of interaction of research provides a new method and means. |
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