Cascade Amplification-Mediated In Situ Hot-Spot Assembly for MicroRNA Detection and Molecular Logic Gate Operations

Abstract

MicroRNAs (miRNAs) play important roles in many biological processes and are associated with various diseases, especially cancers. Combination of technological developments such as nanomaterials, functional enzyme-mediated reactions, and DNA nanotechnology holds great potential for high-performance detection of miRNAs in molecular diagnostic systems. In this work, we have fabricated a cascade signal amplification platform through integrating duplex-specific nuclease (DSN)-assisted target recycling with catalytic hairpin assembly (CHA) reaction for the detection of microRNA-141 (miR-141). The target recycling process driven by DSN results in highly amplified translation of target miRNA to single-stranded connector DNA fragments. The CHA reaction is further initiated by connector DNAs using hairpin-modified gold nanoparticles (HP-AuNPs) as the sensing unit, leading to the formation of AuNP network architecture on the electrode for electrochemical and photoelectrochemical detection of miR-141 in signal-on and signal-off modes, respectively. The developed electrochemical biosensor exhibits a detection limit down to 25.1 aM miR-141 (60 copies in 4 μL sample) and excellent selectivity to discriminate a single base-mismatched sequence and other miRNAs. This assay is also applied to the determination of miR-141 in total RNAs extracted from human breast cancer cells (MDA-MB-231), confirming the applicability of this method for absolute quantification of specific miRNAs in real-world samples. Furthermore, two-input AND and INHIBIT (INH) logic gates are constructed to detect miRNAs. In particular, the AND gate achieves cell-specific gate activation based on expression profiles of miR-141 and microRNA-21 (miR-21). Therefore, our proposed cascade amplification platform has great potential applications in miRNA-related clinical diagnostics and biochemical research.

Applications of Gold Nanoparticles in Non-Optical Biosensors

“…This strategy achieved a wide detection linear range of 10 −17 to 10 −11 M, with limit of detection as low as 6.8 aM (Figure 6) [111]. Yu et al developed AuNPs hot-spots self-assemble structure by catalytic hairpin assembly (CHA) reaction to improve the absorption amount of [Ru(NH 3 ) 6 ] 3+ , obtaining a detection limit as low as 25.1 aM for miRNA-141 [112]. It suggests that AuNPs combined with bio-amplification technologies such as bio-barcode HCR, CHA lead to a detection limit as low as aM level, which is of great prospect to enhance the sensitivity of electrochemical biosensors.…”

Applications of Catalytic Hairpin Assembly Reaction in Biosensing

“…Electrochemical signals (current, differential pulse voltammetry) of reactants possess electrocatalytic capability for H 2 O 2 or electron transfer. Electrochemical biosensors based on CHA are classified into four types: 1) hairpins synthesized with electroactive indicators (toluidine blue, methylene blue, streptavidin‐alkaline phosphatase); 2) horseradish peroxidases or peroxidase mimics (G‐quadruplex/hemin complex, PdNPs@Fe‐metal organic frameworks microcrystals) formed in the resultant products that generate signals; 3) modified nanoparticles (Au nanoparticles, Fe 3 O 4 /CeO 2 @Au magnetic nanoparticles, Ag nanoclusters, MoS 2 nanoflowers, Au@Pt nanospheres) covalently bound to CHA products to magnify currents; and 4) electrochemical indicators (Pb nanoparticles, methylene blue) intercalated into the backbone of CHA products to produce signal changes …”

Nanomaterials for molecular signal amplification in electrochemical nucleic acid biosensing: recent advances and future prospects for point-of-care diagnostics

“…In this manner, a 32-fold enhancement in detection limits has been reported when compared to a single gold nanoparticle label. 120,121 On the other hand, a unique approach for miRNA detection was recently described by Gooding and co-workers, as illustrated in Fig. 8.…”