Bringing Attomolar Detection to the Point‐of‐Care with Nanopatterned DNA Origami Nanoantennas

Creating increasingly sensitive and cost‐effective nucleic acid detection methods is critical for enhancing point‐of‐care (POC) applications. This requires highly specific capture of biomarkers and efficient transduction of capture events. However, the signal from biomarkers present at extremely low amounts often falls below the detection limit of typical fluorescence‐based methods, necessitating molecular amplification. Here, we present single‐molecule detection of a non‐amplified, 151‐nucleotide sequence specific to antibiotics‐resistant Klebsiella pneumoniae down to attomolar concentrations, using Trident NanoAntennas with Cleared HOtSpots (NACHOS). This NACHOS‐diagnostics assay leverages a compact microscope with a large field‐of‐view, including microfluidic flow to enhance capturing efficiency. Fluorescence enhancement is provided by NanoAntennas, arranged using a combination of nanosphere lithography and site‐specific DNA origami placement. This method can detect 200 ± 50 out of 600 molecules in a 100 µL sample volume within an hour. This represents a typical number of pathogens in clinical samples commonly detected by Polymerase Chain Reaction. We achieve similar sensitivity in untreated human plasma, enhancing the practical applicability of the system. This platform can be adapted to detect shorter nucleic acid fragments that are not compatible with traditional amplification‐based technologies. This provides a robust and scalable solution for sensitive nucleic acid detection in diverse clinical settings.