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.

• Klíčová slova
• catalytic hairpin assembly, flexible DNA linker, plasmon‐enhanced fluorescence, sandwich immunoassay, single molecule detection,
• MeSH
• biosenzitivní techniky * metody   MeSH
• fluorescence MeSH
• imunoanalýza metody   MeSH
• jednovláknová DNA chemie   analýza   MeSH
• katalýza MeSH
• povrchová plasmonová rezonance * metody   MeSH
• zobrazení jednotlivé molekuly * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• jednovláknová DNA MeSH

There has recently been a growing use of surface bound nanorods within electrochemical and optical sensing applications. Predictions of the microfluidic rate of analyte transport to such nanorods (either individual or to an array) remain important for sensor design and data analysis; however, such predictions are difficult, as nanorod aspect ratios can vary by several orders of magnitude. In this study, through the use of numerical simulation, we propose an explicit analytical approach to predict the steady-state diffusion-limited rate of mass transport to (individual) surface bound nanorods of variable aspect ratio. We show that, when compared to simulation, this approach provides accurate estimations across a wide range of Péclet numbers.

• Klíčová slova
• biosensors, electrochemistry, microfluidics, nanoparticles, surface plasmon resonance,
• Publikační typ
• časopisecké články MeSH