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Introduction: The study of polymers in nanofluidic systems such as nanopores and nanochannels is an important avenue of research in the physical and life sciences today. Complex nanofluidic devices containing varying topography are ideal for quantifying the behaviour of polymers under confinement. This study investigates the Marko-Siggia force-extension relationship under confinement. We measure the transverse fluctuations of deoxyribonucleic acid (DNA) confined between two pits in a nanofluidic slit to measure the potential breakdown of this model. Methods: We took images of fluorescently tagged single DNA molecules in the nanopit array with video-fluorescence microscopy and analyzed the standard deviation of the peak position along the direction of the molecular extension. Results: We were able to measure the parabolic relation between position and the strength of transverse fluctuations. By examining the peak variance as a function of slit height, our results indicate that the two dimensional version of the force-fluctuation relationship may be appropriate in the limit of strong confinement. However, we could not consistently measure the absolute length of the DNA stretched between two pits, which prevented us from fully exploring the force-extension and force-fluctuation relationships of confined DNA. Conclusions: These experiments further demonstrate that nanofluidic confinement serves as a useful tool for testing the extreme properties of polymers, and our results suggest further investigation into the breakdown of the Marko-Siggia force law. [ABSTRACT FROM AUTHOR]

We use molecular dynamics (MD) simulation and nanofluidic experiments to probe the non-equilibrium transient physics of two nanochannel-confined polymers driven against a permeable barrier in a flow field. For chains with a persistence length P smaller than the channel diameter D, both simulation and experiment with dsDNA reveal nonuniform mixing of the two chains, with one chain dominating locally in what we term "aggregates." Aggregates undergo stochastic dynamics, persisting for a limited time, then disappearing and reforming. Whereas aggregate-prone mixing occurs immediately at sufficiently high flow speeds, chains stay segregated at intermediate flow for some time, often attempting to mix multiple times, before suddenly successfully mixing. Observation of successful mixing nucleation events in nanofluidic experiments reveal that they arise through a peculiar "back-propagation" mechanism whereby the upstream chain, closest to the barrier, penetrates and passes through the downstream chain (farthest from the barrier) moving against the flow direction. Simulations suggest that the observed back-propagation nucleation mechanism is favored at intermediate flow speeds and arises from a special configuration where the upstream chain exhibits one or more folds facing the downstream chain, while the downstream chain has an unfolded chain end facing upstream.

Extracellular vesicles (EVs) are continually released from cancer cells into biofluids, carrying actionable molecular fingerprints of the underlying disease with considerable diagnostic and therapeutic potential. The scarcity, heterogeneity and intrinsic complexity of tumor EVs present a major technological challenge in real-time monitoring of complex cancers such as glioblastoma (GBM). Surface-enhanced Raman spectroscopy (SERS) outputs a label-free spectroscopic fingerprint for EV molecular profiling. However, it has not been exploited to detect known biomarkers at the single EV level. We developed a multiplex fluidic device with embedded arrayed nanocavity microchips (MoSERS microchip) that achieves 97% confinement of single EVs in a minute amount of fluid (<10 μL) and enables molecular profiling of single EVs with SERS. The nanocavity arrays combine two featuring characteristics: (1) An embedded MoS 2 monolayer that enables label-free isolation and nanoconfinement of single EVs due to physical interaction (Coulomb and van der Waals) between the MoS 2 edge sites and the lipid bilayer; and (2) A layered plasmonic cavity that enables sufficient electromagnetic field enhancement inside the cavities to obtain a single EV level signal resolution for stratifying the molecular alterations. We used the GBM paradigm to demonstrate the diagnostic potential of the SERS single EV molecular profiling approach. The MoSERS multiplexing fluidic achieves parallel signal acquisition of glioma molecular variants (EGFRvIII oncogenic mutation and MGMT expression) in GBM cells. The detection limit of 1.23% was found for stratifying these key molecular variants in the wild-type population. When interfaced with a convolutional neural network (CNN), MoSERS improved diagnostic accuracy (87%) with which GBM mutations were detected in 12 patient blood samples, on par with clinical pathology tests. Thus, MoSERS demonstrates the potential for molecular stratification of cancer patients using circulating EVs.