Construction Of Aptamer-functionalized Graphite Nanoparticle Fluorescent Probe And Specific Detection Of 17?-estradiol

17?-estradiol(E2)is a natural steroidal estrogen and also one of endocrine disrupting chemicals,which can interfere with the normal endocrine system.Even at lower concentration it will cause toxic effects on the organism and result in endocrine disorder.Therefore,it is essential to develop a fast,high efficiency and specificity method for E2 detection.As single stranded oligonucleotides,aptamers obtained by SELEX have many unique characteristics,including short screening time,small molecule,easy synthesis,high specificity and selective detection of various analytes(like metal ions,small organic molecules,amino peptides,proteins and the whole cells,etc.).Besides,the conformation of aptamer would change after capturing analyte,therefore,it is usually acted as molecular recognition tool for biosensor construction.Graphite nanoparticle(GN)as a spherical carbon nanomaterial,consisting of several layers of graphene sheets,has the advantages of simple synthesis,low cost,uniform size,easy modification,large specific surface area and good quenching characteristics for fluorescent dye(FAM).In this paper,we employed GN as an excellent nanoquencher and short aptamer as recognition element,based on the mechanism of fluorescence resonance energy transfer(FRET)to establish a novel method by physical adsorption or covalent crossing method for specific detection of E2 based on aptamer-functionalized graphite nanoparticle as fluorescent probe.And then the mechanism of E2 detection and effect of various factors including GN particle size,the length of aptamer,and experimental conditions on sensitivity of E2 detection were studied.In addition,the performance and specificity of E2 detection and its applicability in real environmental water detection were also demonstrated The conclusions were obtained:(1)Using GN as good nanoquencher and short aptamer as recognition element,we have successfully constructed a novel method for specific detection of E2 based on aptamer-functionalized graphite nanoparticle as fluorescent probe.The probe obtained the highest fluorescence signal for E2 detection at p H 8.0 and temperature 25 °C;The change of fluorescence signal by GN/aptamer probe prepared with shorter length aptamer was 3.5 times higher than that of using the conventional longer length aptamer;And the GN/aptamer probe with 5 nm GN presented 2.24 times fluorescence signal than that with 100 nm GN.(2)The relative fluorescence intensity was linearly correlated to E2 concentrations ranging from 0 to 20 ng/mL and the detection limit was 1.02 ng/L;Moreover,it presented no cross reaction with E2 analogs and was successfully utilized for the real environmental water monitoring.(3)Under the optimal condition(1 ?M of aptamer used for the probe preparation and 4 ?g/mL GN/Pyr/aptamer probe used to detect E2),the relative fluorescence intensity of the GN/Pyr/aptamer probe was linearly correlated to E2 concentrations ranging from 50 to 800 ng/mL and the detection limit was 34.5 ng/mL.(4)The GN/aptamer probe constructed by physisorption is much more stable and higher sensitivity(34 times higher than GN/Pyr/aptamer probe),while still retaining similar selectivity.