Study On Novel BLMs Electrochemical Immunosensor For Detection Of Several Type Environmental Estrogens

Biomembranes are fundamental to life.The structure and functions of biomembranes arecomplicated. BLMs has served as one of the most useful models for biological membranes.Thelipid bilayer, after suitable modification, acts as a conduit for ion transport, as a framework forantigen–antibody binding, as a bipolar electrode for redox reactions, and as a reactor for energyconversion. Furthermore, a modified lipid bilayer performs as a transducer for signaltransduction （i.e. sensing）, and numerous other functions as well. Environmental estrogens（EEs）are a kind of chemical compounds which exist in the environment. It has been proved that EEshave estrogenic activity similar to the in vivo estrogens. More and more evidences show lots ofchemical contaminants had similar function of estrogens and were being released in theenvironment.With the growing concern on EEs, people are more foused on the detection orscreening methods for EEs.It is very important to monitor the concentration of estrogen andpredict the pollution of environmental estrogen.Foused on immobilized method for developing of novel biosensor，we have constructednovel BLM electrochemical immunosensors with EEs antibody estrogen or membranereceptor（mER） as a functional element. The biosensor was fabricated to directly detect17β-estradiol, diethylstilbestrol and bisphenol A, and the properties of the modified electrodeswere characterized by Linear sweep voltammetry.1. Study on Solid supported BLMs systemLecithin and cholesterol were used as the main component of the membrance formingsolution.The preparation method and the stability of the BLMs were studied using Patch clampelectrode or agar as supporting material.The effects of lecithin and cholesterol concentrationsand ratios on the stability of BLMs was studied. The stable BLMs was obtained by agarsupported electrode.2. Study on preparation of arginine-riched polypeptide modified antibodyDifferent kinds of hydrophobic groups was screened.The coupling of8-arginine-containingcell penetrating peptide （R8） and anti-17β-estradiol antibody was realized by carbodiimide method.The reaction conditions were optimized. The molar ratio of EDC and R8was10:1andthe activation time for the reaction was about20min.Under optimized conditions, the couplingratio of R8and anti-17β-estradiol antibody was about5:1. Surface plasmon resonancetechnology was used to study the affinity constant of antibody. Affinity constants of Ab-E2was3.8509×10^-5and that of R8-Ab-E2was2.3233×10^-6.The results suggested the modification ofR8to antibody does not affect the binding site of E2with estradiol antibody. The response of ofR8-Ab-E2was high than that of Ab-E2in the same molar concentration.The resluts furtherconfirmed the modification of R8to antibodies. Peptide mass fingerprinting（PMF） of R8-Ab-E2and Ab-E2was Compared.Trypin was used as the enzymatic reagents.The PMF was changedwhen the Ab-E2was modified by R8. The peptide fingerprint peaks of Ab-E2at774.2623Da，1102.4167Da，1496.6604Da，1624.7266Da，2012.9243Da was disappear when it was modifiedby R8.However new peptide fingerprint peaks was appeared in low mass charge ratio area. A newMatrix [CHC][NH3+-Si-SBA-15-Si-NH3+][CHC] was synthesised in order to remove thesignal interference of matrix.3. Study on novel BLMs electrochemical immunosensor modified by R8coupling antibodyA novel arginine-riched polypeptide modified BLM electrochemical biosensor wasproposed for determination of17β-Estradiol and diethylstilbestrol. Themodification ofantibodies on BLM was performed at the same time with the formation of BLMs.With sameratio of lecithin and cholesterol, higher concentration of lecithin was more easy to formBLMs.TheBLMs was very stable with20%lecithin when the ratio of lecithin and cholesterolwas4:1to4.5:1. A two-step self-assembly method was used to prepared estradiol antibodymodified BLMs.The AFM was used to test the blank BLMs-forming solution and the BLMSforming solution after self-assembly.The results show it was very smooth for blankBLMs-forming soulution.The patches of projections were observed on BLMS forming solutionafter self-assembly.The current was lower when R8-Ab-E2was modified on BLMS. Theself-assembly time for antibody incorporated on BLMs was optimized and30minutes wasselected for the fixed process. The response on17β-estradiol to the biosensor was studied byLinear sweep voltammetry. Competitive assay experiments demonstrated the feasibility of thebiosensor with an response concentration of17β-estradiol ranging from0.01to0.20ng/mL anda detection limit of0.01ng/mL. Diethylstilbestrol antibody was further used to validate thefeasibility of the system. The response concentration of diethylstilbestrol ranging from0.1to0.2 ng/mL and a detection limit of0.10ng/mL.The biosensor showed good has been used fordetection of EEs in running water.4. Study on novel BLMs electrochemical immunosensor modified by mERA novel mER modified BLMs electrochemical biosensor was proposed for determinationof environmental estrogens. mER modified BLMs forming solution was achieved withliquid-liquid extraction method.The rough projections was observed when mER modified BLMsforming solution was scan by the atomic force scanning electron microscope.The conditons onliquid-liquid extraction method was optimized. One part mER solution and one part blankmembrance forming solution was mixed about5min.The mER modified BLMs showed goodstability. Compared with blank solution, the membrane currents of mER modified BLM wasincreased slightly, which further confirmed modification of mER on the BLM. Linear sweepvoltammetry method was used to detect the response of mER modified BLM to17β-estradiol.The detection limit of the system to17β-estradiol was1×10^-10mol/L.The responsewas saturated when the concentration of17β-estradiol was high than1×10^-8mol/L.The systemcan produce response to1×10^-5mol/L bisphenol A. The response of the sensor was increasedwhen1×10^-10mol/L17β-estradiol and1×10^-5mol/L bisphenol A was coexist.This research established two kinds of new immobilized methods for active materialincorportaed into BLMs. A self-assembled method was developed for incorporatingwater-soluble antibody into BLM. A peptide composed of R8was used as an intramolecularanchor that allows the incorporation of modified antibody into BLM. Competitive assayexperiments demonstrated the feasibility of the biosensor for determination of17β-estradiol anddiethylstilbestrol. The results suggested that arginine-rich peptides can be applied inelectrochemical sensors for the immunoassay and investigation of the interactions of antibodywith other molecules on BLM. mER modified BLM biosensor show good response to17β-estradiol and Bisphenol A. Related research is further expanding and improving the rangeof BLMs applications.It was a useful complement to other technology with better theoreticalvalue and application prospect. Further studies involving look for suitalbe electroactivesubstance to improve the sensibility of the syetem.