Ultrasensitive Detection of 17β-Estradiol (E2) Based on Multistep Isothermal Amplification

Abstract

17β-Estradiol (E2) can cause an adverse effect on the human endocrine system even at the nanomolar level. Measurements of very low levels of E2 remain a critical challenge due to insufficient sensitivity. In this study, a multistep isothermal amplification fluorescence strategy was constructed, which could realize the exponential amplification of target E2. Specifically, strand displacement reaction (SDA), rolling circle amplification (RCA), and multiprimed rolling circle amplification (MRCA) were combined in a series to quantify trace complementary strand of E2 (cDNA). The E2 aptamer and cDNA were hybridized and modified on the magnetic beads. E2 could bind to its aptamer and cause the release of the cDNA. Then, cDNA would combine with the template DNA, initiating the SDA−RCA−MRCA. The molecular beacons, possessing low background signal, whose fluorescence was quenched in the state of chain folding, could be specifically recognized by the long single-stranded DNA (L-ssDNA) generated by the multistep isothermal amplification triggered by cDNA, and then the fluorescence of the molecular beacons could be restored. Therefore, the E2 could be quantitatively detected by the recovery fluorescence intensity. The fluorescence value showed a good linear relationship with the concentration of E2 in the range of 0.001836−183.6 nM, and the limit of detection (LOD) was as low as 63.09 fM. In addition, the recovery rates of this method spiked in milk and water were 80.8−107.0%, respectively. This method has the advantage of multistep isothermal amplification to obtain abundant fluorescence signals, which may provide a new possibility for highly sensitive detection of other small-molecule targets.

Capture-SELEX of DNA Aptamers for Estradiol Specifically and Estrogenic Compounds Collectively

“…The first E2 aptamer was reported by Gu’s group in 2007 . This 76-mer aptamer ( K d 130 nM) has been used in many sensors in its full length, , and little effort was made to truncate it . In 2014, Hodgkiss’s group reported a 75-mer DNA aptamer .…”

Programmable design of isothermal nucleic acid diagnostic assays through abstraction-based models

“…The design of functionalities into an isothermal amplification system requires significant expertise 16 and is often an iterative approach. Our reaction graph abstraction method provides the means to enable the design of the sequences necessary for an assay, based on relationships between different sequences of building blocks, enabling us to automate the compilation of the functional motifs for hairpin-mediated isothermal amplification.…”

Ultrasensitive Automatic Detection of Small Molecules by Membrane Imaging of Single Molecule Assays

“…The optimization results of our previous experiment were used for defining the sequences of PLP, P, and MB and the number of reagents added . The DNA sequences are listed in Table S1.…”