Development of Electrochemical Aptamer-Based Biosensors for the Detection of Hormonal Contaminants in Water

Endocrine disrupters (EDs) are compounds that specifically interfere with the normal functions of the body’s endocrine system (system of glands for hormonal secretion) by causing adverse effects in both human and wildlife. Therefore, routine, sensitive, reliable and cost effective detection methods for EDs are highly demanded. In this work we present the selection and characterization of ssDNA aptamers that exhibit high affinity, specificity and sensitivity to progesterone (P4), 17b-estradiol (E2) and, norethisterone (NET). The aptamer selection was conducted by in vitro selections by incubating a highly diverse ssDNA library of about  random 60mer sequences with the target analyte. After 15 cycles of selections, the enriched aptamers pool was cloned and sequenced. The dissociation constants (K_D) of the selected aptamers determined by fluorometry and by electrochemical impedance spectroscopy (EIS) methods are in the subnanomolar order. Cross-reactivity tests for each aptamer demonstrated the high specificity to their target analyte. In addition, circular dichroism (CD) spectroscopy measurements of the selected aptamers for progesterone showed a marked conformational change upon binding the target analyte. This change in the conformation was exploited to design a biosensing platform to generate a measurable signal after binding the specific analyte by following the redox process of a couple composed for [Fe(CN)₆]^3-/4-. For first time, an impedimetric aptasensor for progesterone detection was developed with limit of detection of 0.90 ng/mL. We believe, based in our finding, that the continuous selection of high affinity aptamers for other EDs and their integration in a biosensing platform will facilitate the routine monitoring of EDs in environment as well as clinical and medical diagnosis purposes.