Aptamer-Based Electrochemical Biosensing Platform for Detection of Hormonal Pollutants 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, the 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 huge ssDNA library of about 1.80x〖10〗^15 random sequences with the target analyte which has been previously immobilized on agarose beads. After certain cycles of selections, the enriched aptamers pool was cloned and sequenced. The dissociation constants (KD) of the selected aptamers were determined by fluorometry and by electrochemical impedance spectroscopy (EIS) methods. The KD values are in the low nanomolar range. Cross-reactivity tests for each aptamer demonstrated the high specificity to their target analyte. Additionally, circular dichroism (CD) spectroscopy measurements of the selected aptamers showed a remarkable conformational change upon binding the target analyte. The change in the conformation was exploited to design a biosensing platform to get 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 to detect progesterone was developed by yielding a limit of detection of 0.90ng/mL; for other analytes, the data will be presented and discussed in the programmed session of the conference.
We believe 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.
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 huge ssDNA library of about 1.80x〖10〗^15 random sequences with the target analyte which has been previously immobilized on agarose beads. After certain cycles of selections, the enriched aptamers pool was cloned and sequenced. The dissociation constants (KD) of the selected aptamers were determined by fluorometry and by electrochemical impedance spectroscopy (EIS) methods. The KD values are in the low nanomolar range. Cross-reactivity tests for each aptamer demonstrated the high specificity to their target analyte. Additionally, circular dichroism (CD) spectroscopy measurements of the selected aptamers showed a remarkable conformational change upon binding the target analyte. The change in the conformation was exploited to design a biosensing platform to get 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 to detect progesterone was developed by yielding a limit of detection of 0.90ng/mL; for other analytes, the data will be presented and discussed in the programmed session of the conference.
We believe 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.
