Aptamer-Based Label-Free Impedimetric Biosensor for Detection of Progesterone

Abstract

Rising progesterone (P4) levels in humans due to its overconsumption through hormonal therapy, food products, or drinking water can lead to many negative health effects. Thus, the simple and accurate assessment of P4 in both environmental and clinical samples is highly important to protect public health. In this work, we present the selection, identification, and characterization of ssDNA aptamers with high binding affinity to P4. The aptamers were selected in vitro from a single-stranded DNA library of 1.8 × 10(15) oligonucleotides showing dissociation constants (KD) in the low nanomolar range. The dissociation constant of the best aptamer, designated as P4G13, was estimated to be 17 nM by electrochemical impedance spectroscopy (EIS) as well as fluorometric assay. Moreover, the aptamer P4G13 did not show cross-reactivity to analogues similar to progesterone such as 17β-estradiol (E2) and norethisterone (NET). An impedimetric aptasensor for progesterone was then fabricated based on the conformational change of P4G13 aptamer, immobilized on the gold electrode by self-assembly, upon binding to P4, which results in an increase in electron transfer resistance. Aptamer-complementary DNA (cDNA) oligonucleotides were tested to maximize the signal gain of the aptasensor after binding with progesterone. Significant signal enhancement was observed when the aptamer hybridized with a short complementary sequence at specific site was used instead of pure aptamer. This signal gain is likely due to the more significant conformational change of the aptamer-cDNA than the pure aptamer upon binding with P4, as confirmed by circular dichroism (CD) spectroscopy. The developed aptasensor exhibited a linear range for concentrations of P4 from 10 to 60 ng/mL with a detection limit of 0.90 ng/mL. Moreover, the aptasensor was applied in spiked tap water samples and showed good recovery percentages. The new selected progesterone aptamers can be exploited in further biosensing applications for environmental, clinical, and medical diagnostic purposes.

A label-free colorimetric progesterone aptasensor based on the aggregation of gold nanoparticles

“…The ultrapure water used in all experiments and aqueous solution preparation was purified by a Milli-Q Advantage A10 System (Millipore Inc., Bedford, MA, USA). The sequence of P4 aptamer is 5′-GCATCACACACCGATAC TCACCCGCCTGATTAACATTAGCCCACCGCCCACCC-CCGCTGC-3′ (60-mer) [4], synthesized by Sangon Biotechnology Inc. (Shanghai, China; www.sangon.com). The aptamer was dissolved in ultrapure water and stored at 4°C before use.…”

“…The interaction between aptamers and AuNPs can keep AuNPs coated with aptamers and be isolated. When NaCl was added afterwards and interacted with AuNPs, liquid chromatography-electrospray tandem mass spectrometry 5 × 10 −5 [36] TIRF-based biosensor 2 × 10 −4 [8] Label-free impedimetric aptasensor 0.9 [4] Label-free colorimetric aptasensor 0.8 This work…”

“…Yet, excessive consumption of P4 may produce side effects like breast tenderness, tiredness, headache et al to women. As for men, high amounts of P4 may affect the secretion of gonadotropin releasing hormone (GnRH), which is likely to generate a decrease of testosterone secretion [4]. Nowadays, surface waters are polluted by natural hormones and endocrine-disrupting chemicals (EDCs) from wastewater treatment plants and intensive stock-rearing operations [5].…”

“…The first is immunoassay method (radioimmunoassay, enzyme-linked immunosorbent assay, electrochemical immunoassay et al), which is sensitive to detect P4 [4,[7][8][9]. The other is chromatography method (high performance liquid chromatography, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, etc) [4,6,7]. Although they are sensitive, these methods are not suitable for field tests because of exorbitant price, long detecting time and requirement for professional operation.…”

“…needs. In 2015, Indian researchers selected the aptamer of P4 successfully and developed an electrochemical aptasensor with a detection limit of 0.90 ng·mL −1 , which created a new way based on aptamers for rapid and sensitive detection of P4 [4].…”