Phosphorescent Sensor And Lateral Flow Assay For Estradiol

Estradiol is a naturally occurring steroid estrogen that can be enriched in the human body through the food chain.Excessive intake of exogenous estradiol will disturb the endocrine system and endanger human health.Most of the existing estradiol quantitative analysis methods require expensive separation equipment and complicated sample pretreatment.Almost all the estradiol detection methods based on the test strip use antibodies as recognition units,and cannot overcome the drawbacks of the antibody itself such as poor stability.Therefore,the establishment of stable,simple,rapid and sensitive estradiol determination method based on the aptamer recognition technology,is of great significance for ensuring food safety.The contents of this thesis are as follows:Firstly,based on the principle of phosphorescence resonance energy transfer,a room temperature phosphorescent sensor for estradiol was established and applied to the determination of estradiol in milk samples:persistent luminescence nanoparticles(PLNPs)doped with rare earth element were prepared by hydrothermal synthesis and modified with aptamers.The structure and properties were characterized.A phosphorescence resonance energy transfer sensor was constructed using PLNPs as an energy donor and molybdenum disulfide nanosheets as an energy acceptor.Under optimized experimental conditions,the phosphorescence intensity at 710 nm and estradiol concentration in the range of 0.5-1200ng/mL were at a good linear relationship(R~2=0.982)and the limit of detection was 0.3ng/mL.The recoveries in spiked milk samples was 93.6%-102.4%,indicating the method can be applied to the determination of estradiol in milk samples.The phosphorescence sensing method can effectively avoid the fluorescence interference from proteins and coexisting substances in milk so as to improve accuracy of the method.Secondly,a rapid detection method for estradiol based on aptamer recognition-test zone responsive allosteric-sandwich-like lateral flow assay was established and applied to the determination of estradiol in milk samples:a 35 mer estradiol aptamer sequence was used as the annular part of the molecular beacon on test zone.Estradiol in the sample solution could bind to this part forcing the detachment of the two neck ends at the molecular beacon,and one of the end is complementary to the nanogold-modified DNA single strand to allow the test zone to develop color.Under the optimized experimental conditions,the ratio of signal intensity between test zone and control zone has a good linear relationship with the concentration of estradiol in the range of 27.2-27200?g/mL(R~2=0.920),and the naked eye detection limit is 27.2 ng/mL.The recoveries in spiked milk samples is 89%-103%,and it takes about 15 minutes for a single test,indicating the method can be used for rapid detection of estradiol in milk samples.The determination of estradiol by the method is faster and simpler than the above mentioned room temperature phosphorescence sensing method,but the detection limit is higher.Finally,to decrease the detection limit of the lateral flow assay,a test-zone pre-enrichment method was established:miR-210 mimic was adopted as a model target;conjugate pads were inserted between the sample pads and the nitrocellulose membranes after enriching the model target in the test-zone pre-enrichment method.The visual detection limit is reduced by 1 to 3 orders of magnitude and the specificity is not significantly different compared with direct loading method in both the sandwich and competitive formats.In a 1:1diluted serum sample the pre-enrichment method yields the same detection limit as in the buffer solution.The method can provide new insight for improving the detection limit of lateral flow assay for estradiol.