A rapid, inexpensive, and sensitive DNA assay is essential for point-of-care molecular diagnosis. The ultrasensitive detection of DNA requires time-consuming, expensive, complicated enzyme-based DNA amplification. Recently, toehold-mediated strand displacement (TMSD) cascade has been employed to detect low abundance nucleic acid without enzymatic amplification, resulting in the fM-to-pM range of detection limit. However, relatively slow amplification compared to enzymatic amplification requires 2-3 hours to reach high sensitivity. Herein, we present a target DNA-catalyzed self-assembly of QD-DNA hydrogel as a new enzyme-free nanosensor for a femtomolar detection limit of a short DNA in one hour. We employ a TMSD cascade, where a single target DNA catalyzes the multiplication of Y-shaped DNA (Y-DNA) attached with a fluorescence quencher. Single-stranded DNA (ssDNA)-functionalized QDs are prepared by the aqueous synthesis of CdTe/CdS core-shell QDs with phosphorothioated ssDNA. Y-DNAs are self-assembled with DNA-functionalized QDs to form a hydrogel, which leads to efficient Förster resonance energy transfer (FRET) from QD to the Y-DNA quencher. The QD-DNA hydrogel-based assay exhibits the detection limit of ~ 6 fM, five orders of magnitude lower than the conventional sandwich-type FRET assay, without any additional enzymatic amplification. The new method can also quickly discriminate against a single-base mismatch in the target sequence.

Catalytic self-assembly of DNA hydrogel for enzyme-free femtomolar DNA assay

ACS Spring Meeting 2021

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