Highly Sensitive Fluorescence Assay for miRNA Detection:
Investigation of the DNA Spacer Effect on the DSN Enzyme Activity
toward Magnetic-Bead-Tethered Probes
posted on 2022-01-07, 16:33authored byKhouloud Djebbi, Biao Shi, Ting Weng, Mohamed Bahri, Mohamed Amin Elaguech, Jin Liu, Chaker Tlili, Deqiang Wang
Researchers have
recently designed various biosensors combining
magnetic beads (MBs) and duplex-specific nuclease (DSN) enzyme to
detect miRNAs. Yet, the interfacial mechanisms for surface-based hybridization
and DSN-assisted target recycling are relatively not well understood.
Thus, herein, we developed a highly sensitive and selective fluorescent
biosensor to study the phenomenon that occurs on the local microenvironment
surrounding the MB-tethered DNA probe via detecting microRNA-21 as
a model. Using the above strategy, we investigated the influence of
different DNA spacers, base-pair orientations, and surface densities
on DSN-assisted target recycling. As a result, we were able to detect
as low as 170 aM of miR-21 under the optimized conditions. Moreover,
this approach exhibits a high selectivity in a fully matched target
compared to a single-base mismatch, allowing the detection of miRNAs
in serum with improved recovery. These results are attributed to the
synergetic effect between the DSN enzyme activity and the neutral
DNA spacer (triethylene glycol: TEG) to improve the miRNA detection’s
sensitivity. Finally, our strategy could create new paths for detecting
microRNAs since it obliterates the enzyme-mediated cascade reaction
used in previous studies, which is more expensive, more time-consuming,
less sensitive, and requires double catalytic reactions.