Upconversion Fluorescence-based Test Strip And Biosensor For Rapid Detection Of Estradiol In Milk

Estrogen abuse has now gradually become a key factor to food security.Estradiol(E2),a typical estrogen,is considered the strongest hormones in the human body.Excessive intake of exogenous estradiol can cause serious harm to human health,such as sexual prematurity and even cancer.It is imperative to establish a fast,easy and efficiency framework for estradiol detection because of short estradiol detection methods in milk.ObjectivesThis paper took estradiol detection as research object,based on the principle of competitive binding reaction,fluorescent nanomaterials and biosensor technology to devise two simple,efficient and sensitive methods for E2 detection in milk,including immune chromatography and biosensor method.Methods1.The upconversion nanoparticles(UCNPs)doped with rare earth were synthesized by classical solvothermal approach,then UCNPs were modified and characterized with different techniques.2.UCNPs-based immune chromatography method for estradiol detection.The UCNPs modified with amino groups were conjugated to the estradiol monoclonal antibody(E2-mAb)by glutaraldehyde method and fixed on the conjugate pad.The estradiol coating(E2-BSA)was immobilized on the test line(T),the goat anti-mouse antibody was immobilized on the control line(C).E2 was detected based on the principle of competitive immunoassay.Then drew standard curve and calculated he limits of detection.3.UCNPs-based fluorescence resonance energy system(FRET)for estradiol detection.The UCNPs modified with amino groups were conjugated to the streptavidin by glutaraldehyde method,and streptavidin was linked to the biotinylated E2 aptamer by non-covalent,then aptamer hybridized with complementary strand with fluorescent group.The fluorescent group and the UCNPs could undergo FRET which led to fluorescence quenching.Once the E2 was added,the structural aptamers changed and the complementary strand separated from aptamers led to fluorescence recovery.The Hybridization buffer and the concentration of streptavidin were optimized,and then drew standard curve calculated the limits of detection.Results1.The characterization results of UCNPs.The UCNPs are approximately 100 nm and shell thickness size were approximately 10 nm,which indicated that the particles were successfully coated with the silicon shell.The modified UCNPs showed good redispersibility in polar solvent such as water.2.The optimization results of immune chromatography method.The sample pad and binding pad were 1.6 cm and 0.9 cm long respectively;the concentration of test line and control line were 1.25 mg/mL and 0.8 mg/mL respectively.The results showed a good linear relationship under the optimal conditions.The detection range of the method for E2 was from 5 ng/mL to 2000 ng/mL,and the limit of detection was 2.75 ng/mL.3.The optimization results of biosensor method.The concentration of streptavidin was 0.033 mg/mL.The hybridisation buffer was composed of 0.1 mol/L NaCl and 0.01 mol/L phosphate buffer(PBS)(pH 7.6),and the concentration of aptamer and complementary chain both were 5 ?mol/L.Under the optimal conditions,the linear ranged from 0.8 ng/mL to 180 ng/mL,and the limit of detection for E2 was down to 0.4 ng/mL.ConclusionsOne the basis of immunochromatography test strip and biosensor techniques,this study develops two high specific,sensitive and precise methods for the E2 determination which takes the UCNPs as fluorescence label.The methods are efficient,easy,rapid,and shows great potential in the field of food safety analysis.