An ultrasensitive fluorescent strategy for 17β-estradiol based on aptamer and isothermal exponential amplification reaction

Abstract

The abuse of 17β-estradiol (E2) in breeding and the excretion into the environment by human beings result in exposure of E2 to humans. Excessive levels of E2 can cause an adverse effect on the human endocrine systems. Therefore, an efficient and sensitive method for the analysis of E2 is required. Herein, based on the specific capture of aptamer and the digestion od Exonuclease III, efficient amplification of isothermal exponential amplification reaction (EXPAR), a highly sensitive and selective strategy is established for E2 analysis. E2 binds to the aptamer due to their high affinity, preventing the complementary DNA (cDNA) of the aptamer from hybridizing with it. Thus, the cDNA remains unbound and freely available in the solution. Then, Exonuclease III digests the cDNA-aptamer complex, leaving the free cDNA intact. cDNA serves as primers to trigger EXPAR with the help of Bst 2.0 WarmStart DNA polymerase and Nt.BstNBⅠ nicking enzyme, achieving sensitive detection of E2. Without E2, cDNA hybridizes with the aptamer to form double-stranded DNA, which is degraded by Exonuclease III. As a result, the amplification reaction fails to initiate. The approach shows a linear range of 100 aM-10 pM, spanning 5 orders of magnitude, with the limit of detection as low as 36 aM. In addition, the selectivity and reproducibility are great, and the recoveries in tap water and milk ranged from 99.4% to 117.3%, suggesting good practicability. This method may also provide a new possibility for highly sensitive detection of other small-molecule targets via using the corresponding aptamers.