Tunable nanofluidic device for digital nucleic acid analysis.

Nano/microfluidic-based nucleic acid tests have been proposed as a rapid and reliable diagnostic technology. Two key steps for many of these tests are target nucleic acid (NA) immobilization followed by an enzymatic reaction on the captured NAs to detect the presence of a disease-associated sequence. NA capture within a geometrically confined volume is an attractive alternative to NA surface immobilization that eliminates the need for sample pre-treatment (e.g. label-based methods such as lateral flow assays) or use of external actuators (e.g. dielectrophoresis) that are required for most nano/microfluidic-based NA tests. However, geometrically confined spaces hinder sample loading while making it challenging to capture, subsequently, retain and simultaneously expose target NAs to required enzymes. Here, using a nanofluidic device that features real-time confinement control via pneumatic actuation of a thin membrane lid, we demonstrate the loading of digital nanocavities by target NAs and exposure of target NAs to required enzymes/co-factors while the NAs are retained. In particular, as proof of principle, we amplified single-stranded DNAs (M13mp18 plasmid vector) in an array of nanocavities via two isothermal amplification approaches (loop-mediated isothermal amplification and rolling circle amplification).

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.

Dynamics and quantitative contribution of the aminoglycoside 6'-N-acetyltransferase type Ib to amikacin resistance.

Aminoglycosides are essential components in the available armamentarium to treat bacterial infections. The surge and rapid dissemination of resistance genes strongly reduce their efficiency, compromising public health. Among the multitude of modifying enzymes that confer resistance to aminoglycosides, the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] is the most prevalent and relevant in the clinical setting as it can inactivate numerous aminoglycosides, such as amikacin. Although the mechanism of action, structure, and biochemical properties of the AAC(6')-Ib protein have been extensively studied, the contribution of the intracellular milieu to its activity remains unclear. In this work, we used a fluorescent-based system to quantify the number of AAC(6')-Ib per cell in Escherichia coli, and we modulated this copy number with the CRISPR interference method. These tools were then used to correlate enzyme concentrations with amikacin resistance levels. Our results show that resistance to amikacin increases linearly with a higher concentration of AAC(6')-Ib until it reaches a plateau at a specific protein concentration. In vivo imaging of this protein shows that it diffuses freely within the cytoplasm of the cell, but it tends to form inclusion bodies at higher concentrations in rich culture media. Addition of a chelating agent completely dissolves these aggregates and partially prevents the plateau in the resistance level, suggesting that AAC(6')-Ib aggregation lowers resistance to amikacin. These results provide the first step in understanding the cellular impact of each AAC(6')-Ib molecule on aminoglycoside resistance. They also highlight the importance of studying its dynamic behavior within the cell.IMPORTANCEAntibiotic resistance is a growing threat to human health. Understanding antibiotic resistance mechanisms can serve as foundation for developing innovative treatment strategies to counter this threat. While numerous studies clarified the genetics and dissemination of resistance genes and explored biochemical and structural features of resistance enzymes, their molecular dynamics and individual contribution to resistance within the cellular context remain unknown. Here, we examined this relationship modulating expression levels of aminoglycoside 6'-N-acetyltransferase type Ib, an enzyme of clinical relevance. We show a linear correlation between copy number of the enzyme per cell and amikacin resistance levels up to a threshold where resistance plateaus. We propose that at concentrations below the threshold, the enzyme diffuses freely in the cytoplasm but aggregates at the cell poles at concentrations over the threshold. This research opens promising avenues for studying enzyme solubility's impact on resistance, creating opportunities for future approaches to counter resistance.

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.

Characterizing interaction of multiple nanocavity confined plasmids in presence of large DNA model nucleoid.

Bacteria have numerous large dsDNA molecules that freely interact within the cell, including multiple plasmids, primary and secondary chromosomes. The cell membrane maintains a micron-scale confinement, ensuring that the dsDNA species are proximal at all times and interact strongly in a manner influenced by the cell morphology (e.g. whether cell geometry is spherical or anisotropic). These interactions lead to non-uniform spatial organization and complex dynamics, including segregation of plasmid DNA to polar and membrane proximal regions. However, exactly how this organization arises, how it depends on cell morphology and number of interacting dsDNA species are under debate. Here, using an in vitro nanofluidic model, featuring a cavity that can be opened and closed in situ, we address how plasmid copy number and confinement geometry alter plasmid spatial distribution and dynamics. We find that increasing the plasmid number alters the plasmid spatial distribution and shortens the plasmid polar dwell time; sharper cavity end curvature leads to longer plasmid dwell times.

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.

Dynamics and quantitative contribution of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] to amikacin resistance.

Aminoglycosides are essential components in the available armamentarium to treat bacterial infections. The surge and rapid dissemination of resistance genes strongly reduce their efficiency, compromising public health. Among the multitude of modifying enzymes that confer resistance to aminoglycosides, the aminoglycoside acetyltransferase AAC(6')-Ib is the most prevalent and relevant in the clinical setting as it can inactivate numerous aminoglycosides, such as amikacin. Although the mechanism of action, structure, and biochemical properties of the AAC(6')-Ib protein have been extensively studied, the contribution of the intracellular milieu to its activity remains unclear. In this work, we used a fluorescent-based system to quantify the number of AAC(6')-Ib per cell in Escherichia coli, and we modulated this copy number with the CRISPR interference method. These tools were then used to correlate enzyme concentrations with amikacin resistance levels. Our results show that resistance to amikacin increases linearly with a higher concentration of AAC(6')-Ib until it reaches a plateau at a specific protein concentration. In vivo imaging of this protein shows that it diffuses freely within the cytoplasm of the cell, but it tends to form inclusion bodies at higher concentrations in rich culture media. Addition of a chelating agent completely dissolves these aggregates and partially prevents the plateau in the resistance level, suggesting that AAC(6')-Ib aggregation lowers resistance to amikacin. These results provide the first step in understanding the cellular impact of each AAC(6')-Ib molecule on aminoglycoside resistance. They also highlight the importance of studying its dynamic behavior within the cell.

Discriminating protein tags on a dsDNA construct using a Dual Nanopore Device.

We report Brownian dynamics simulation results with the specific goal to identify key parameters controlling the experimentally measurable characteristics of protein tags on a dsDNA construct translocating through a double nanopore setup. First, we validate the simulation scheme in silico by reproducing and explaining the physical origin of the asymmetric experimental dwell time distributions of the oligonucleotide flap markers on a 48 kbp long dsDNA at the left and the right pore. We study the effect of the electric field inside and beyond the pores, critical to discriminate the protein tags based on their effective charges and masses revealed through a generic power-law dependence of the average dwell time at each pore. The simulation protocols monitor piecewise dynamics at a sub-nanometer length scale and explain the disparate velocity using the concepts of nonequilibrium tension propagation theory. We further justify the model and the chosen simulation parameters by calculating the Péclet number which is in close agreement with the experiment. We demonstrate that our carefully chosen simulation strategies can serve as a powerful tool to discriminate different types of neutral and charged tags of different origins on a dsDNA construct in terms of their physical characteristics and can provide insights to increase both the efficiency and accuracy of an experimental dual-nanopore setup.

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.

Confinement anisotropy drives polar organization of two DNA molecules interacting in a nanoscale cavity.

There is growing appreciation for the role phase transition based phenomena play in biological systems. In particular, self-avoiding polymer chains are predicted to undergo a unique confinement dependent demixing transition as the anisotropy of the confined space is increased. This phenomenon may be relevant for understanding how interactions between multiple dsDNA molecules can induce self-organized structure in prokaryotes. While recent in vivo experiments and Monte Carlo simulations have delivered essential insights into this phenomenon and its relation to bacteria, there are fundamental questions remaining concerning how segregated polymer states arise, the role of confinement anisotropy and the nature of the dynamics in the segregated states. To address these questions, we introduce an artificial nanofluidic model to quantify the interactions of multiple dsDNA molecules in cavities with controlled anisotropy. We find that two dsDNA molecules of equal size confined in an elliptical cavity will spontaneously demix and orient along the cavity poles as cavity eccentricity is increased; the two chains will then swap pole positions with a frequency that decreases with increasing cavity eccentricity. In addition, we explore a system consisting of a large dsDNA molecule and a plasmid molecule. We find that the plasmid is excluded from the larger molecule and will exhibit a preference for the ellipse poles, giving rise to a non-uniform spatial distribution in the cavity that may help explain the non-uniform plasmid distribution observed during in vivo imaging of high-copy number plasmids in bacteria.

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.

Nanofluidics for Simultaneous Size and Charge Profiling of Extracellular Vesicles.

Extracellular vesicles (EVs) are cell-derived membrane structures that circulate in body fluids and show considerable potential for noninvasive diagnosis. EVs possess surface chemistries and encapsulated molecular cargo that reflect the physiological state of cells from which they originate, including the presence of disease. In order to fully harness the diagnostic potential of EVs, there is a critical need for technologies that can profile large EV populations without sacrificing single EV level detail by averaging over multiple EVs. Here we use a nanofluidic device with tunable confinement to trap EVs in a free-energy landscape that modulates vesicle dynamics in a manner dependent on EV size and charge. As proof-of-principle, we perform size and charge profiling of a population of EVs extracted from human glioblastoma astrocytoma (U373) and normal human astrocytoma (NHA) cell lines.

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.

Time-dependent knotting of agitated chains.

Agitated strings serve as macroscale models of spontaneous knotting, providing valuable insight into knotting dynamics at the microscale while allowing explicit analysis of the resulting knot topologies. We present an experimental setup for confined macroscale knot formation via tumbling along with a software interface to process complex knot data. Our setup allows characterization of knotting probability, knot complexity, and knot formation dynamics for knots with as many as 50 crossings. We find that the probability of knotting saturates below 80% within 100 s of the initiation of tumbling and that this saturation probability does not increase for chains above a critical length, an indication of nonequilibrium knot-formation conditions in our experiment. Despite the saturation in knot formation, we show that longer chains, while being more confined, will always tend to form knots of higher complexity since the free end can access a greater number of loops during tumbling.

Flossing DNA in a Dual Nanopore Device.

Solid-state nanopores are a single-molecule technique that can provide access to biomolecular information that is otherwise masked by ensemble averaging. A promising application uses pores and barcoding chemistries to map molecular motifs along single DNA molecules. Despite recent research breakthroughs, however, it remains challenging to overcome molecular noise to fully exploit single-molecule data. Here, an active control technique termed "flossing" that uses a dual nanopore device is presented to trap a proteintagged DNA molecule and up to 100's of back-and-forth electrical scans of the molecule are performed in a few seconds. The protein motifs bound to 48.5 kb λ-DNA are used as detectable features for active triggering of the bidirectional control. Molecular noise is suppressed by averaging the multiscan data to produce averaged intertag distance estimates that are comparable to their known values. Since nanopore feature-mapping applications require DNA linearization when passing through the pore, a key advantage of flossing is that trans-pore linearization is increased to >98% by the second scan, compared to 35% for single nanopore passage of the same set of molecules. In concert with barcoding methods, the dual-pore flossing technique could enable genome mapping and structural variation applications, or mapping loci of epigenetic relevance.

Correction: Probing the organization and dynamics of two DNA chains trapped in a nanofluidic cavity.

Correction for 'Probing the organization and dynamics of two DNA chains trapped in a nanofluidic cavity' by Xavier Capaldi et al., Soft Matter, 2018, 14, 8455-8465.

Controlling DNA Tug-of-War in a Dual Nanopore Device.

Methods for reducing and directly controlling the speed of DNA through a nanopore are needed to enhance sensing performance for direct strand sequencing and detection/mapping of sequence-specific features. A method is created for reducing and controlling the speed of DNA that uses two independently controllable nanopores operated with an active control logic. The pores are positioned sufficiently close to permit cocapture of a single DNA by both pores. Once cocapture occurs, control logic turns on constant competing voltages at the pores leading to a "tug-of-war" whereby opposing forces are applied to regions of the molecules threading through the pores. These forces exert both conformational and speed control over the cocaptured molecule, removing folds and reducing the translocation rate. When the voltages are tuned so that the electrophoretic force applied to both pores comes into balance, the life time of the tug-of-war state is limited purely by diffusive sliding of the DNA between the pores. A tug-of-war state is produced on 76.8% of molecules that are captured with a maximum two-order of magnitude increase in average pore translocation time relative to the average time for single-pore translocation. Moreover, the translocation slow-down is quantified as a function of voltage tuning and it is shown that the slow-down is well described by a first passage analysis for a 1D subdiffusive process. The ionic current of each nanopore provides an independent sensor that synchronously measures a different region of the same molecule, enabling sequential detection of physical labels, such as monostreptavidin tags. With advances in devices and control logic, future dual-pore applications include genome mapping and enzyme-free sequencing.

Single Molecule DNA Resensing Using a Two-Pore Device.

A nanofluidic device is presented that, enables independent sensing and resensing of a single DNA molecule translocating through two nanopores with sub-micrometer spacing. The device concept is based upon integrating a thin nitride membrane with microchannels etched in borosilicate glass. Pores, coupled to each microchannel, are connected via a fluid-filled half-space on the device backside, enabling translocation of molecules across each pore in sequence. Critically, this approach allows for independent application of control voltage and measurement of trans-pore ionic current at each of the two pores, leading to 1) controlled assessment of molecular time of flight, 2) voltage-tuned selective molecule recapture, and 3) ability to acquire two correlated translocation signatures for each molecule analyzed. Finally, the rare cocapture of a single chain threading simultaneously through each of the two pores is reported.

Probing the organization and dynamics of two DNA chains trapped in a nanofluidic cavity.

Here we present a pneumatically-actuated nanofluidic platform that has the capability of dynamically controlling the confinement environment of macromolecules in solution. Using a principle familiar from classic devices based on soft-lithography, the system uses pneumatic pressure to deflect a thin nitride lid into a nanoslit, confining molecules in an array of cavities embedded in the slit. We use this system to quantify the interactions of multiple confined DNA chains, a key problem in polymer physics with important implications for nanofluidic device performance and DNA partitioning/organization in bacteria and the eukaryotes. In particular, we focus on the problem of two-chain confinement, using differential staining of the chains to independently assess the chain conformation, determine the degree of partitioning/mixing in the cavities and assess coupled diffusion of the chain center-of-mass positions. We find that confinement of more than one chain in the cavity can have a drastic impact on the polymer dynamics and conformation.

A nanofluidic knot factory based on compression of single DNA in nanochannels.

Knots form when polymers self-entangle, a process enhanced by compaction with important implications in biological and artificial systems involving chain confinement. In particular, new experimental tools are needed to assess the impact of multiple variables influencing knotting probability. Here, we introduce a nanofluidic knot factory for efficient knot formation and detection. Knots are produced during hydrodynamic compression of single DNA molecules against barriers in a nanochannel; subsequent extension of the chain enables direct assessment of the number of independently evolving knots. Knotting probability increases with chain compression as well as with waiting time in the compressed state. Using a free energy derived from scaling arguments, we develop a knot-formation model that can quantify the effect of interactions and the breakdown of Poisson statistics at high compression. Our model suggests that highly compressed knotted states are stabilized by a decreased free energy as knotted contour contributes a lower self-exclusion derived free energy.