Babar, A. N. | bioRxiv Science

bioRxiv2021

Single-molecule sizing through nano-cavity confinement

An approach relying on nano-cavity confinement is developed in this paper for the sizing of nanoscale particles and single biomolecules in solution. The approach, termed nano-cavity diffusional sizing (NDS), measures particle residence times within fluidic nano-cavities to determine their hydrodynamic radii. Using theoretical modeling and simulation, we show that the residence time of particles within nano-cavities above a critical timescale depends on the diffusion coefficient of the particle, which allows estimation of the particles size. We demonstrate this approach experimentally through measurement of particle residence times within nano-fluidic cavities using single-molecule confocal microscopy. Our data show that the residence times scale linearly with the sizes of nanoscale colloids, protein aggregates and single DNA oligonucleotides. NDS thus constitutes a new single molecule optofluidic approach that allows rapid and quantitative sizing of nanoscale particles for potential application in nanobiotechnology, biophysics, and clinical diagnostics. Table of content graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/472369v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@15abf7eorg.highwire.dtl.DTLVardef@d2180dorg.highwire.dtl.DTLVardef@1290f57org.highwire.dtl.DTLVardef@ae2087_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

2-photon-fabricated nano-fluidic traps for extended detection of single macromolecules and colloids in solution

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, -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.

biophysics↗