PT  - JOURNAL ARTICLE
AU  - Oliver Vanderpoorten
AU  - Ali Nawaz Babar
AU  - Georg Krainer
AU  - Raphaёl P.B. Jacquat
AU  - Pavan K. Challa
AU  - Quentin Peter
AU  - Zenon Toprakcioglu
AU  - Catherine K. Xu
AU  - Ulrich F. Keyser
AU  - Jeremy Baumberg
AU  - Clemens F. Kaminski
AU  - Tuomas P. J. Knowles
TI  - 2-photon-fabricated nano-fluidic traps for extended detection of single macromolecules and colloids in solution
AID  - 10.1101/2021.11.17.468989
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.11.17.468989
4099  - http://biorxiv.org/content/early/2021/11/19/2021.11.17.468989.short
4100  - http://biorxiv.org/content/early/2021/11/19/2021.11.17.468989.full
AB  - The analysis of nanoscopic species, such as proteins and colloidal assemblies, at the single-molecule level has become vital in many areas of fundamental and applied research. Approaches to increase the detection timescales for single molecules in solution without immobilising them onto a substrate surface and applying external fields are much sought after. Here we present an easy-to-implement and versatile nanofluidics-based approach that enables increased observational-timescale analysis of single biomacromolecules and nanoscale colloids in solution. We use two-photon-based hybrid lithography in conjunction with soft lithography to fabricate nanofluidic devices with nano-trapping geometries down to 100 nm in height. We provide a rigorous description and characterisation of the fabrication route that enables the writing of nanoscopic 3D structures directly in photoresist and allows for the integration of nano-trapping and nano-channel geometries within micro-channel devices. Using confocal fluorescence burst detection, we validated the functionality of particle confinement in our nano-trap geometries through measurement of particle residence times. All species under study, including nanoscale colloids, α-synuclein oligomers, and double-stranded DNA, showed a three to five-fold increase in average residence time in the detection volume of nano-traps, due to the additional local steric confinement, in comparison to free space diffusion in a nearby micro-channel. Our approach thus opens-up the possibility for single-molecule studies at prolonged observational timescales to analyse and detect nanoparticles and protein assemblies in solution without the need for surface immobilisation.Competing Interest StatementThe authors have declared no competing interest.