Search

A rapid lateral flow assay (LFA) is developed for the colorimetric and surface-enhanced Raman scattering (SERS) dual-mode detection of microRNA (miRNA) based on the robust immunoassay-like (immuno-like) recognition mechanism of S9.6 antibody to DNA/miRNA duplexes. Different from the traditional target-mediated sandwich-type hybridization-based LFA methods, the formation of S9.6 antibody/miRNA/DNA complexes is more rapid and stable, achieving 40 times higher sensitivity with only 10 min assaying time. Furthermore, taking benefit of the versatility of the immuno-like recognition mode, the multiplexed detection of miRNAs can be realized with the SERS signal readout, providing a versatile LFA design towards sensitive, specific, and multiplexed miRNA analysis. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: Most conventional digital bioassays rely on the use of fully‐sealed microchambers as independent units to compartmentalize the target molecules and the signal generation reaction, which require specialized equipment or proprietary reagents/consumables. Herein, we report a microchamber‐free and spherical nucleic acid (SNA)‐amplified digital flow cytometric bead assay (dFBA) for ultrasensitive protein and exosome analysis with simple workflows, easily accessible instruments/reagents, and high discriminating ability towards the fluorescence‐positive and fluorescence‐negative beads. In this dFBA, microbeads are employed as independent carriers to anchor the single target molecule‐initiated signal amplification reaction, avoiding the use of sealed droplets or microwell microchambers. Meanwhile, antibody‐functionalized SNAs (FSNAs) with a high density of DNA probes act as a bridge for efficiently amplified target‐to‐DNA signal conversion, which allows the use of DNA‐based rolling circle amplification (RCA) as the fluorescence signal amplification technique to quantify non‐nucleic acid targets. Even a single target‐induced on‐bead RCA and fluorescence enriching are sufficient to make the target‐loaded bead bright enough to be clearly discriminated from the negative ones just by use of a most common flow cytometer (FCM). This dFBA has successfully realized the digital analysis of ultralow levels of protein and exosome biomarkers, enlarging the toolbox of digital bioassays for clinical applications. [ABSTRACT FROM AUTHOR]

Plant microRNAs (miRNAs) are naturally 2′‐O‐methylated at the 3′‐terminal; as a consequence, they cannot be efficiently detected by traditional target‐triggered polymerization reactions. Here, a simple but robust enzyme‐free sensing strategy was developed for plant miRNA analysis by using dynamic light scattering (DLS) to monitor the crosslinking of gold nanoparticles (AuNPs) amplified by click chemical ligation. Combining the enzyme‐free cycling chemical‐ligation‐mediated signal amplification, and the intrinsic outstanding ability of DLS for discriminating the extremely low level of particle aggregation in a large pool of monodisperse AuNPs, high sensitivity was achieved and as low as 78.6 fm plant miRNA could be easily detected. A simple but robust enzyme‐free sensing strategy was developed for plant microRNA analysis by using dynamic light scattering (DLS) to monitor the crosslinking of gold nanoparticles (AuNPs) amplified by click chemical ligation. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: Most conventional digital bioassays rely on the use of fully‐sealed microchambers as independent units to compartmentalize the target molecules and the signal generation reaction, which require specialized equipment or proprietary reagents/consumables. Herein, we report a microchamber‐free and spherical nucleic acid (SNA)‐amplified digital flow cytometric bead assay (dFBA) for ultrasensitive protein and exosome analysis with simple workflows, easily accessible instruments/reagents, and high discriminating ability towards the fluorescence‐positive and fluorescence‐negative beads. In this dFBA, microbeads are employed as independent carriers to anchor the single target molecule‐initiated signal amplification reaction, avoiding the use of sealed droplets or microwell microchambers. Meanwhile, antibody‐functionalized SNAs (FSNAs) with a high density of DNA probes act as a bridge for efficiently amplified target‐to‐DNA signal conversion, which allows the use of DNA‐based rolling circle amplification (RCA) as the fluorescence signal amplification technique to quantify non‐nucleic acid targets. Even a single target‐induced on‐bead RCA and fluorescence enriching are sufficient to make the target‐loaded bead bright enough to be clearly discriminated from the negative ones just by use of a most common flow cytometer (FCM). This dFBA has successfully realized the digital analysis of ultralow levels of protein and exosome biomarkers, enlarging the toolbox of digital bioassays for clinical applications. [ABSTRACT FROM AUTHOR]

The high-sensitive and rapid detection of critical biomarkers, e.g., disease-related nucleic acids and proteins, is always desired. Compared with the routine homogenous detection strategies, the on-bead flow cytometry (FCM)-based assays have drawn a lot of interests owing to their unique advantages. On one hand, microbeads (MBs) are employed for the enrichment of fluorescent signals, allowing the size encoding for multiplexed detection of biomarkers. On the other hand, FCM enables the fast read-out of the total fluorescent signals enriched on the MBs and the decoding of MBs' size information. For an improved sensitivity and versatile application scenarios, the signal amplification on MBs is required. However, the enzyme-catalyzed on-bead reactions remain challenging owing to the critical reaction conditions on the MBs/solution interface. Toward the high-sensitive detection of target biomolecules in real-samples, a series of on-bead enzyme-catalyzed signal amplification strategies have been developed. After careful optimization of the reaction conditions, the proposed sensors are proven to have ultra-high sensitivities to fulfill the requirement of real-sample detection., (© 2020 Elsevier Inc. All rights reserved.)