Tethered Catalytic Hairpin Assembly with Plasmon-Enhanced Fluorescence Readout for Single Molecule Detection
Status Publisher Jazyk angličtina Země Německo Médium print-electronic
Typ dokumentu časopisecké články
Grantová podpora
DIPLAB I 5119
Austrian Science Fund
LS20-014
Gesellschaft für Forschungsförderung Niederösterreich
22-30456J
Grantová Agentura České Republiky
SVV-2023-260716
Univerzita Karlova v Praze
CZ.02.01.01/00/22_008/0004596
Ministerstvo Školství, Mládeže a Tělovýchovy
PubMed
40207774
DOI
10.1002/smtd.202500037
Knihovny.cz E-zdroje
- Klíčová slova
- catalytic hairpin assembly, flexible DNA linker, plasmon‐enhanced fluorescence, sandwich immunoassay, single molecule detection,
- Publikační typ
- časopisecké články MeSH
Here a novel digital bioassay readout concept is reported that does not rely on enzymatic amplification nor compartmenting of an analyzed liquid sample. Rather, it is based on counting individual affinity-captured target biomolecules via the use of a tethered catalytic hairpin assembly (tCHA) deployed on a solid sensor surface with spatial confinement utilized by a flexible polymer linker (FPL). Wide-field plasmon-enhanced fluorescence (PEF) imaging is employed for optical real-time probing of the reaction kinetics, where affinity-captured target molecules are manifested as spatially distinct bright fluorescent spots. The effect of the length of the FPLs is investigated, and the analytical performance of the dual amplification tCHA-PEF concept is tested by using a model short single-stranded DNA analyte. When applied in a sandwich immunoassay, the detection of target proteins at sub-femtomolar concentrations is demonstrated. The reported experiments are supported by diffusion-limited mass transfer models and document the potential of tCHA-PEF as a new class of generic enzyme-free bioanalytical tools enabling the ultrasensitive analysis of trace amounts of protein and nucleic acid analytes, making it attractive for future molecular diagnostics and research applications.
Faculty of Mathematics and Physics Charles University Prague 121 16 Czech Republic
FZU Institute of Physics Czech Academy of Sciences Na Slovance 2 Prague 182 21 Czech Republic
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