Mixing and demixing arising from compression of two semiflexible polymer chains in nanochannels.

We use molecular dynamics simulation to probe the non-equilibrium physics of two nanochannel-confined semiflexible polymers in a homogeneous flow field. We find that for sufficiently stiff chains the internal organization of the two chains takes the form of interwoven folds and circular coils. This organization can lead to mixing or demixing depending on chain stiffness and flow speed. At low and intermediate flow, the two chains adopt a folded configuration, which favours mixing. At high flow, the two chains adopt a predominantly coiled configuration. The coiled configuration results in demixing when the chains are compressed from an initially demixed condition and mixing when the chains are compressed from an initially mixed condition. We find that the mixing/demixing behaviour is governed by the ratio of the number of folded segments of one chain relative to the other at low flow and by the degree of coiling in both chains at high flow. For decreasing stiffness, the chains start to aggregate locally instead of mixing smoothly at low and intermediate flow. In the limit of completely flexible chains, the two chains either completely segregate at low flow, or adopt a locally demixed configuration consisting of large aggregates of one chain relative to the other that undergo complex stochastic dynamics, diffusing, disintegrating, and reforming at intermediate flow. The transition from complete segregation to the aggregate-dominated configuration occurs when the linear intra-chain ordering breaks down.

High Osmotic Power Generation via Nanopore Arrays in Hybrid Hexagonal Boron Nitride/Silicon Nitride Membranes.

Nanopores embedded in two-dimensional (2D) nanomaterials are a promising emerging technology for osmotic power generation. Here, coupling our new AFM-based pore fabrication approach, tip-controlled local breakdown (TCLB), with a hybrid membrane formed by coating silicon nitride (SiN) with hexagonal boron nitride (hBN), we show that high osmotic power density can be obtained in systems that do not possess the thinness of atomic monolayers. In our approach, the high osmotic performance arises from charge separation induced by the highly charged hBN surface rather than charge on the inner pore wall. Moreover, exploiting TCLB's capability of producing sub 10 nm pore arrays, we investigate the effects of pore-pore interaction on the overall power density. We find that an optimum pore-to-pore spacing of ∼500 nm is required to maintain an efficient selective transport mechanism.

Transient physics in the compression and mixing dynamics of two nanochannel-confined polymer chains.

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.

MoS2-Plasmonic Nanocavities for Raman Spectra of Single Extracellular Vesicles Reveal Molecular Progression in Glioblastoma.

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 MoS2 monolayer that enables label-free isolation and nanoconfinement of single EVs due to physical interaction (Coulomb and van der Waals) between the MoS2 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.

Organized states arising from compression of single semiflexible polymer chains in nanochannels.

We use molecular dynamics simulation to probe the nonequilibrium physics of single nanochannel-confined semiflexible polymers in a homogeneous flow field. The flow field compresses the polymer against the end of the nanochannel, simulating an experiment of a nanochannel confined chain compressed against a slit barrier. The flow-based compression gives rise to a packing of the chain against the channel end that possesses a striking organization, consisting of interweaving of folds and circular coils. For stiff chains at low flow, we find that the organization is dominated by repeated hairpin folds. For stiff chains at higher flow, we observe that circular coils arise along with the folds, with folding and coiling domains becoming interwoven at the highest flow speeds. Chain organization is retained even when the chain persistence length is on order of the channel width. We show that the global polymer organization, consisting of a number of defined folds and coiled loops, arises from the minimization of the total chain free energy.

Electronic Mapping of a Bacterial Genome with Dual Solid-State Nanopores and Active Single-Molecule Control.

We present an electronic mapping of a bacterial genome using solid-state nanopore technology. A dual-nanopore architecture and active control logic are used to produce single-molecule data that enables estimation of distances between physical tags installed at sequence motifs within double-stranded DNA. Previously developed "DNA flossing" control logic generates multiple scans of each captured DNA. We extended this logic in two ways: first, to automate "zooming out" on each molecule to progressively increase the number of tags scanned during flossing, and second, to automate recapture of a molecule that exited flossing to enable interrogation of the same and/or different regions of the molecule. Custom analysis methods were developed to produce consensus alignments from each multiscan event. The combined multiscanning and multicapture method was applied to the challenge of mapping from a heterogeneous mixture of single-molecule fragments that make up the Escherichia coli (E. coli) chromosome. Coverage of 3.1× across 2355 resolvable sites of the E. coli genome was achieved after 5.6 h of recording time. The recapture method showed a 38% increase in the merged-event alignment length compared to single-scan alignments. The observed intertag resolution was 150 bp in engineered DNA molecules and 166 bp natively within fragments of E. coli DNA, with detection of 133 intersite intervals shorter than 200 bp in the E. coli reference map. We present results on estimating distances in repetitive regions of the E. coli genome. With an appropriately designed array, higher throughput implementations could enable human-sized genome and epigenome mapping applications.

Collapse of DNA in ac electric fields.

We report that double-stranded DNA collapses in the presence of ac electric fields at frequencies of a few hundred Hertz, and does not stretch as commonly assumed. In particular, we show that confinement-stretched DNA can collapse to about one quarter of its equilibrium length. We propose that this effect is based on finite relaxation times of the counterion cloud, and the subsequent partitioning of the molecule into mutually attractive units. We discuss alternative models of those attractive units.

Transverse dielectrophoretic-based DNA nanoscale confinement.

Confinement of single molecules within nanoscale environments is crucial in a range of fields, including biomedicine, genomics, and biophysics. Here, we present a method that can concentrate, confine, and linearly stretch DNA molecules within a single optical field of view using dielectrophoretic (DEP) force. The method can convert an open surface into one confining DNA molecules without a requirement for bonding, hydrodynamic or mechanical components. We use a transverse DEP field between a top coverslip and a bottom substrate, both of which are coated with a transparent conductive material. Both layers are attached using double-sided tape, defining the chamber. The nanofeatures lie at the "floor" and do not require any bonding. With the application of an alternating (AC) electric field (2 Vp-p) between the top and bottom electrodes, a DEP field gradient is established and used to concentrate, confine and linearly extend DNA in nanogrooves as small as 100-nm in width. We also demonstrate reversible loading/unloading of DNA molecules into nanogrooves and nanopits by switching frequency (between 10 kHz to 100 kHz). The technology presented in this paper provides a new method for single-molecule trapping and analysis.

Waves of DNA: Propagating excitations in extended nanoconfined polymers.

We use a nanofluidic system to investigate the emergence of thermally driven collective phenomena along a single polymer chain. In our approach, a single DNA molecule is confined in a nanofluidic slit etched with arrays of embedded nanocavities; the cavity lattice is designed so that a single chain occupies multiple cavities. Fluorescent video-microscopy data shows fluctuations in intensity between cavities, including waves of excess fluorescence that propagate across the cavity-straddling molecule, corresponding to propagating fluctuations of contour overdensity in the cavities. The transfer of DNA between neighboring pits is quantified by examining the correlation in intensity fluctuations between neighboring cavities. Correlations grow from an anticorrelated minimum to a correlated maximum before decaying, corresponding to a transfer of contour between neighboring cavities at a fixed transfer time scale. The observed dynamics can be modeled using Langevin dynamics simulations and a minimal lattice model of coupled diffusion. This study shows how confinement-based sculpting of the polymer equilibrium configuration, by renormalizing the physical system into a series of discrete cavity states, can lead to new types of dynamic collective phenomena.

Denaturation mapping of Saccharomyces cerevisiae.

Optical mapping of DNA provides large-scale genomic information that can be used to assemble contigs from next-generation sequencing, and to detect rearrangements between single cells. A recent optical mapping technique called denaturation mapping has the advantage of using physical principles rather than the action of enzymes to probe genomic structure. Denaturation mapping uses fluorescence microscopy to image the pattern of partial melting along a DNA molecule extended in a channel of cross-section 120 nm at the heart of a nanofluidic device. We used denaturation mapping to locate single DNA molecules on the yeast genome (12.1 Mbp) by comparing images to a computationally predicted map for the entire genome sequence. By locating 84 molecules we assembled an optical map of the yeast genome with > 50% coverage.

From the Cover: The dynamics of genomic-length DNA molecules in 100-nm channels.

We show that genomic-length DNA molecules imaged in nanochannels have an extension along the channel that scales linearly with the contour length of the polymer, in agreement with the scaling arguments developed by de Gennes for self-avoiding confined polymers. This fundamental relationship allows us to measure directly the contour length of single DNA molecules confined in the channels, and the statistical analysis of the dynamics of the polymer in the nanochannel allows us to compute the SD of the mean of the extension. This statistical analysis allows us to measure the extension of lambda DNA multimers with a 130-nm SD in 1 min.