A fluorescent amplification strategy for high-sensitive detection of 17 β-estradiol based on EXPAR and HCR

Abstract

Environmental endocrine disruptors in the environment and food, especially 17 β-estradiol (E2), are important factors affecting the growth and development of organisms. In this research, we constructed a fluorescence strategy for two-step amplification that combined two currently popular methods, exponential amplification reaction (EXPAR) and hybridization chain reaction (HCR). E2 competed with the complementary DNA (cDNA) to bind the aptamer modified on the magnetic beads. The free complementary strand in the supernatant was used as a trigger sequence to activate EXPAR, producing a large amount of short single-stranded DNA (ssDNA). The amplified ssDNA can trigger the second HCR amplification, producing many long double-stranded DNA (dsDNA) analogues. According to the principle of fluorescence resonance energy transfer, the carboxyfluorescein (FAM) signals in H1 and H2 hairpins were quenched by black hole quencher (BHQ-1). After the addition of E2 and initiation of amplification, the initially quenched fluorescent signal would be restored. This strategy with a detection limit of 0.37 pg mL^-1 (S/N = 3) showed a good linear relationship in the range of 0.4-800 pg mL^-1. In addition, the recovery rates of the method for milk and water samples were 98.55%-116.95% and 92.32%-107.00%, respectively. This is the first report of the combined detection of EXPAR and HCR, providing a reference for rapid and highly sensitive detection using multiple isothermal amplification methods.

Keywords: 17 β-estradiol; Exponential amplification reaction (EXPAR); Hybridization chain reaction (HCR); Sensitively fluorescent detection; Two-step amplification.