Biosensing with plasmonic signal amplification
Signal amplification strategies are essential for improving sensitivity, speed, and robustness of sensing assays for point-of-care diagnostic applications. One strategy to achieve this relies on physical fluorescence signal amplification by plasmonic nanostructures that act as optical nanoantennas concentrating the incident excitation light into zeptoliter volumes and enhancing the radiative decay rate of fluorescent molecules. In our lab, we exploit the unique positioning precision of DNA origami to directly place the diagnostic elements in the plasmonic hotspots of silver and gold dimer nanoantennas (Figure 1). Using fluorescence-based diagnostic assays in the hotspots of such nanoantennas we can achieve the signal amplification reaching several hundred-fold.
Our recent progress in the development of DNA nanoantenanas for diagnostics includes the development of addressable NAnoantennas with Cleared plasmonic HOtspotS (NACHOS). NACHOS enabled us to incorporate the diagnostic assay specific to DNA of antibiotic resistant bacteria directly into the hotspot of dimer DNA nanoantenna as well as demonstrate the detection of single molecules on a cheap and low-tech diagnostic platform such as a portable smartphone microscope (Figure 2).
Molecular voltage sensors
The electrical potential of the cell membrane is important for many signaling pathways, e.g. neuronal activity or apoptosis. Using DNA nanotechnology, we are developing a novel family of voltage sensitive probes with fluorescent read-out where we can design the different sensor elements individually and modularly to combine them into a nanometer-sized device. The sensor is based on FRET and employs a new principle using a hydrophobic ATTO647N dye that anchors the sensor unit in the membrane (Figure 3a). A donor dye is placed on negatively charged DNA linkers that are attached to the DNA origami. Depending on the potential, the flexible element with the donor changes its position relative to the FRET acceptor in the membrane yielding a FRET change that is read out on single FRET pairs. Besides transmembrane potentials, a similar principle can also be employed to study surface potentials induced by the composition of lipids with different charges in large unilamellar vesicles (Figure 3b).
In addition to the single-molecule sensing approaches described above we are also exploring the possibility of using DNA origami method to build a general platform for the development of tunable fluorescence sensors. We aim at tuning and controlling the dynamic range of the single-molecule sensors, introducing signal amplification mechanisms and expanding the utility of the biosensors beyond the limits dictated by the bio-recognition interaction itself.
In: Anal Chem, 2022, ISSN: 1520-6882.
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