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Complex light intensity patterns formed by closely-spaced multiple apertures in a metal film can be faithfully mapped with sub-wavelength resolution, from near-field to far-field, by interferometric lithography.

The goal of PrecisionTox is to overcome conceptual barriers to replacing traditional mammalian chemical safety testing by accelerating the discovery of evolutionarily conserved toxicity pathways that are shared by descent among humans and more distantly related animals. An international consortium is systematically testing the toxicological effects of a diverse set of chemicals on a suite of five model species comprising fruit flies, nematodes, water fleas, and embryos of clawed frogs and zebrafish along with human cell lines. Multiple forms of omics and comparative toxicology data are integrated to map the evolutionary origins of biomolecular interactions that are predictive of adverse health effects, to major branches of the animal phylogeny. These conserved elements of adverse outcome pathways (AOPs) and their biomarkers are expected to provide mechanistic insight useful for regulating groups of chemicals based on their shared modes of action. PrecisionTox also aims to quantify risk variation within populations by recognizing susceptibility as a heritable trait that varies with genetic diversity. This initiative incorporates legal experts and collaborates with risk managers to address specific needs within European chemicals legislation, including the uptake of new approach methodologies (NAMs) for setting precise regulatory limits on toxic chemicals.

Virus assembly and disassembly are critical steps in the virus lifecycle; however, virus disassembly is much less well understood than assembly. For hepatitis B virus (HBV) capsids, disassembly of the virus capsid in the presence of guanidine hydrochloride (GuHCl) exhibits strong hysteresis that requires additional chemical energy to initiate disassembly and disrupt the capsid structure. To study disassembly of HBV capsids, we mixed T = 4 HBV capsids with 1.0 to 3.0 M GuHCl, monitored the reaction over time by randomly selecting particles, and measured their size with resistive-pulse sensing. Particles were cycled forward and backward multiple times to increase the observation time and likelihood of observing a disassembly event. The four-pore device used for resistive-pulse sensing produces four current pulses for each particle during translocation that improves tracking and identification of single particles and increases the precision of the particle-size measurements when the pulses are averaged. We studied disassembly at GuHCl concentrations below and above denaturing conditions of the dimer, the fundamental unit of HBV capsid assembly. As expected, capsids showed little disassembly at low GuHCl concentrations (e.g., 1.0 M GuHCl), whereas at higher GuHCl concentrations (≥ 1.5 M), capsids exhibited disassembly, sometimes as a complex series of events. In all cases, disassembly was an accelerating process, where capsids catastrophically disassembled within a few 100 ms of reaching critical stability; disassembly rates reached tens of dimers per second just before capsids fell apart. Some disassembly events exhibited metastable intermediates that appeared to lose one or more trimers of dimers in a stepwise fashion.

Structural isomers of sialylated N-glycans contribute to the diversity of the N-glycome and to a range of biological functions. Sialyl linkage isomers can be readily distinguished by mass spectrometry with mass differences between α2,3- and α2,6-linkages generated by a two-step sialic acid linkage-specific alkylamidation. To improve the identification of N-glycans from complex mixtures, we added a delactonization step after the first alkylamidation step, which regenerates negatively charged carboxylic acids on α2,3-sialic acids. N-glycan isomers with α2,3-sialic acids are then fractionated by ion-exchange chromatography prior to the second alkylamidation step. With this modified alkylamidation method, sialylated N-glycans were enriched and stabilized for structural characterization by capillary electrophoresis-mass spectrometry and tandem mass spectrometry. We identified 52 sialylated N-glycan structures, including 107 linkage isomers, in human serum and confirmed the presence of positional isomers of specific sialyl linkage isomers. Due to the reduced sample complexity after ion-exchange fractionation and CE separation, substructural features of N-glycans were rapidly evaluated and included core- and antenna-fucosylation and poly-lactosamine.

Resistive-pulse sensing with solid-state nanopores is a sensitive, label-free technique for analyzing single molecules in solution. To add functionality to resistive-pulse measurements, direct coupling of the nanopores to other pores and nanoscale fluidic elements, e.g., reactors, separators, and filters, in the same device is an important next step. One approach is monolithic fabrication of the fluidic elements in the plane of the substrate, but methods to generate pores with circular cross sections are needed to improve sensing performance with in-plane devices. Here, we report a fabrication method that directly patterns nanopores with circular cross sections in series and in plane with the substrate. A focused ion beam (FIB) instrument is used to mill a lamella in a nanochannel and, subsequently, bore a nanopore through the lamella. The diameter and geometry of the nanopore are controlled by the current and dose of the ion beam and by the tilt angle and thickness of the lamella. We fabricated devices with vertical and tilted lamellae and nanopores with diameters from 40 to 90 nm in cylindrical and conical geometries. To test device performance, we conducted resistive-pulse measurements of hepatitis B virus (HBV) capsids. Current pulses from T = 3 capsid (~31 nm diameter) and T = 4 capsid (~35 nm diameter) were well resolved and exhibited relative pulse amplitudes (Δi/i) up to 5 times higher than data obtained on nanopores with rectangular cross sections. For smaller pore diameters (≤ 45 nm), which approach the diameters of the capsids, a dramatic increase in the pulse amplitude was observed for both T = 3 and T = 4 capsids. Two and three pores fabricated in series further improved the resolution between the relative pulse amplitude distributions for the T = 3 and T = 4 capsids by up to 2-fold.

Significance All organisms sense and respond to their environments. One way bacteria interact with their surroundings is by dynamically extending and retracting filamentous appendages from their surface called pili. While pili are critical for many functions, such as attachment, motility, and DNA uptake, the factors that regulate their dynamic activity are poorly understood. Here, we describe how an environmental signal induces a signaling pathway to promote the retraction of mannose-sensitive hemagglutinin pili in Vibrio cholerae . The retraction of these pili promotes the detachment of V. cholerae from a surface and may provide a means by which V. cholerae can respond to changes in its environment.

Bacteria use surface appendages called type IV pili to perform diverse activities including DNA uptake, twitching motility, and attachment to surfaces. The dynamic extension and retraction of pili are often required for these activities, but the stimuli that regulate these dynamics remain poorly characterized. To address this question, we study the bacterial pathogen

During growth, bacteria increase in size and divide. Division is initiated by the formation of the Z-ring, an intense ring-like cytoskeletal structure formed by treadmilling protofilaments of the tubulin homolog FtsZ. FtsZ localization is thought to be controlled by the Min and Noc systems, and here, we explore why cell division fails at high temperature when the Min and Noc systems are simultaneously mutated. Microfluidic analysis of a minD noc double mutant indicated that FtsZ formed proto-Z-rings at periodic inter-chromosome locations but that the rings failed to mature and become functional. Extragenic suppressor analysis indicated that a variety of mutations restored high temperature growth to the minD noc double mutant, and while many were likely pleiotropic, others implicated the proteolysis of the transcription factor Spx. Further analysis indicated that a Spx-dependent pathway activated the expression of ZapA, a protein that primarily compensates for the absence of Noc. Additionally, an Spx-independent pathway increased the activity of the divisome to reduce the length of the cytokinetic period. Finally, we provide evidence of an as-yet-unidentified protein that is activated by Spx and governs the frequency of polar division and minicell formation.IMPORTANCEBacteria must properly position the location of the cell division machinery in order to grow, divide, and ensure each daughter cell receives one copy of the chromosome. In B. subtilis, cell division site selection is thought to depend on two systems called Min and Noc, and while neither is individually essential, cells fail to grow at high temperature when both are mutated. Here, we show that cell division fails in the absence of Min and Noc, not due to a defect in FtsZ localization, but rather a failure in the maturation of the cell division machinery. To understand what happens when the division machinery fails to mature, suppressor mutations that bypass the need for Min, Noc, or both were selected. Some of the mutants activated the Spx stress response pathway while others appeared to directly enhance divisome activity.

The study of exosomes has become increasingly popular due to their potentially important biological roles. Urine can be used as an effective source of exosomes for noninvasive investigations into the pathophysiological states of the genitourinary tract, but first, detailed characterization of exosomal components in healthy individuals is essential. Here, we significantly extend the number of N-glycan compositions, including sulfated species, identified from urinary exosomes and determine the sialic acid linkages for many of those compositions. Capillary electrophoresis-mass spectrometry (CE-MS), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to identify N-glycan and sulfated N-glycan compositions. Second, because the alteration of sialylation patterns has been previously implicated in various disease states, ion-exchange chromatography, microfluidic capillary electrophoresis (CE), and MALDI-MS were adopted to resolve positional isomers of sialic acids. Structures of the sialyl-linkage isomers were assigned indirectly through α2–3 sialidase treatment and sialic acid linkage-specific alkylamidation (SALSA). In total, we have identified 219 N-glycan structures that include 175 compositions, 64 sialic acid linkage isomers, 26 structural isomers, and 27 sulfated glycans.

The self-assembly of virus capsids is a potential target for antivirals due to its importance in the virus lifecycle. Here, we investigate the effect of phenylpropenamide derivatives B-21 and AT-130 on the assembly of hepatitis B virus (HBV) core protein. Phenylpropenamides are widely believed to yield assembly of spherical particles resembling native, empty HBV capsids. Because the details of assembly can be overlooked with ensemble measurements, we performed resistive-pulse measurements on nanofluidic devices with four pores in series to characterize the size distributions of the products in real time. With its single particle sensitivity and compatibility with typical assembly buffers, resistive-pulse sensing is well suited for analyzing virus assembly in vitro. We observed that assembly with B-21 and AT-130 produced a large fraction of partially complete virus particles that may be on-path, off-path, or trapped. For both B-21 and AT-130, capsid assembly was more sensitive to disruption under conditions where the inter-protein association energy was low at lower salt concentrations. Dilution of the reaction solutions led to the rearrangement of the incomplete particles and demonstrated that these large intermediates may be on-path, but are labile, and exist in a frustrated dynamic equilibrium. During capsid assembly, phenylpropenamide molecules modestly increase the association energy of dimers, prevent intermediates from dissociating, and lead to kinetic trapping where the formation of too many capsids has been initiated leading to both empty and incomplete particles.

During growth, bacteria increase in size and divide. Division is initiated by the formation of the Z-ring, a ring-like cytoskeletal structure formed by treadmilling protofilaments of the tubulin homolog FtsZ. FtsZ localization is thought to be controlled by the Min and Noc systems, and here we explore why cell division fails at high temperature when the Min and Noc systems are simultaneously mutated. Microfluidic analysis of a

Bacteria use surface appendages called type IV pili to perform diverse activities including DNA uptake, twitching motility, and attachment to surfaces. Dynamic extension and retraction of pili is often required for these activities, but the stimuli that regulate these dynamics remain poorly characterized. To study this question, we use the bacterial pathogen Vibrio cholerae, which uses mannose-sensitive hemagglutinin (MSHA) pili to attach to surfaces in aquatic environments as the first step in biofilm formation. Here, we find that V. cholerae cells retract MSHA pili and detach from a surface in microaerobic conditions. This response is dependent on the phosphodiesterase CdpA, which decreases intracellular levels of cyclic-di-GMP (c-di-GMP) under microaerobic conditions to induce MSHA pilus retraction. CdpA contains a putative NO-sensing NosP domain, and we demonstrate that nitric oxide (NO) is necessary and sufficient to stimulate CdpA-dependent detachment. Thus, we hypothesize that microaerobic conditions result in endogenous production of NO (or an NO-like molecule) in V. cholerae. Together, these results describe a regulatory pathway that allows V. cholerae to rapidly abort biofilm formation. More broadly, these results show how environmental cues can be integrated into the complex regulatory pathways that control pilus dynamic activity and attachment in bacterial species.

Symmetrical protein complexes are ubiquitous in biology. Many have been re-engineered for chemical and medical applications. Viral capsids and their assembly are frequent platforms for these investigations. A means to create asymmetric capsids may expand applications. Here, starting with homodimeric Hepatitis B Virus capsid protein, we develop a heterodimer, design a hierarchical assembly pathway, and produce asymmetric capsids. In the heterodimer, the two halves have different growth potentials and assemble into hexamers. These preformed hexamers can nucleate co-assembly with other dimers, leading to Janus-like capsids with a small discrete hexamer patch. We can remove the patch specifically and observe asymmetric holey capsids by cryo-EM reconstruction. The resulting hole in the surface can be refilled with fluorescently labeled dimers to regenerate an intact capsid. In this study, we show how an asymmetric subunit can be used to generate an asymmetric particle, creating the potential for a capsid with different surface chemistries., Viruses have been re-engineered for many applications but the ability to build asymmetric capsids in vitro has been lacking. Here, the authors report the design of asymmetric subunits with different growth rates leading to capsids with discrete patches and potential for distinct surface chemistries.

Exosomes represent a class of secreted biological vesicles, which have recently gained attention due to their function as intertissue and interorganism transporters of genetic materials, small molecules, lipids, and proteins. Although the protein constituents of these exosomes are often glycosylated, a large-scale characterization of the glycoproteome has not yet been completed. This study identified 3144 unique glycosylation events belonging to 378 glycoproteins and 604 unique protein sites of glycosylation. With these data, we investigated the level of glycan microheterogeneity within the urinary exosomes, finding on average 5.9 glycans per site. The glycan family abundance on individual proteins showed subtle differences, providing an additional level of molecular characterization compared to the unmodified proteome. Finally, we show protein site-specific changes in regard to the common urinary glycoprotein, uromodulin. While uromodulin is an individual case, these same site-specific analyses provide a way forward for developing diagnostic glycoprotein biomarkers with urine as a noninvasive biological fluid. This study represents an important first step in understanding the functional urinary glycoproteome.

Bidirectional cell-cell communication involving exosome-borne cargo such as miRNA, has emerged as a critical mechanism for wound healing. Unlike other shedding vesicles, exosomes selectively package miRNA by SUMOylation of heterogeneous nuclear ribonucleoproteinA2B1 (hnRNPA2B1). In this work, we elucidate the significance of exosome in keratinocyte-macrophage crosstalk following injury. Keratinocyte-derived exosomes were genetically labeled with GFP reporter (Exo(κ-GFP)) using tissue nanotransfection and were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exo(κ-GFP) were also characterized. Unlike skin exosome, wound-edge Exo(κ-GFP) demonstrated characteristic N-glycan ions with abundance of low base pair RNA and were selectively engulfed by wound-macrophages (ωmϕ) in granulation tissue. In vitro addition of wound-edge Exo(κ-GFP) to proinflammatory ωmϕ resulted in conversion to a proresolution phenotype. To selectively inhibit miRNA packaging within Exo(κ-GFP) in vivo, pH-responsive keratinocyte-targeted siRNA-hnRNPA2B1 functionalized lipid nanoparticles (TLNP(κ)) were designed with 94.3% encapsulation efficiency. Application of TLNP(κ/si-hnRNPA2B1) to murine dorsal wound-edge significantly inhibited expression of hnRNPA2B1 by 80% in epidermis compared to TLNP(κ/si-control) group. Although no significant difference in wound closure or re-epithelialization was observed, TLNP(κ/si-hnRNPA2B1) treated group showed significant increase in ωmϕ displaying proinflammatory markers in the granulation tissue at day 10 post-wounding compared to TLNP(κ/si-control) group. Furthermore, TLNP(κ/si-hnRNPA2B1) treated mice showed impaired barrier function with diminished expression of epithelial junctional proteins, lending credence to the notion that unresolved inflammation results in leaky skin. This work provides insight wherein Exo(κ-GFP) are recognized as a major contributor that regulates macrophage trafficking and epithelial barrier properties post-injury.

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Through direct coupling of capillary electrophoresis (CE) to mass spectrometry (MS) with a sheathless interface, we have identified 77 potential N-glycan structures derived from human serum. We confirmed the presence of N-glycans previously identified by indirect methods, e.g., electrophoretic mobility standards, obtained 31 new N-glycan structures not identified in our prior work, differentiated co-migrating structures, and determined specific linkages on isomers featuring sialic acids. Serum N-glycans were cleaved from proteins, neutralized via methylamidation, and labeled with the fluorescent tag 8-aminopyrene-1,3,6-trisulfonic acid, which renders the glycan fluorescent and provides a -3 charge for electrophoresis and negative-mode MS detection. The neutralization reaction also stabilizes the labile sialic acids. In addition to methylamidation, native charges from sialic acids were neutralized through reaction with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium to amidate α2,6-linked sialic acids in the presence of ammonium chloride and form lactones with α2,3-linked sialic acids. This neutralization effectively labels each type of sialic acid with a unique mass to determine specific linkages on sialylated N-glycans. For both neutralization schemes, we compared the results from microchip electrophoresis and CE.

Hepatitis B virus (HBV) core protein is a model system for studying assembly and disassembly of icosahedral structures. Controlling disassembly will allow re-engineering the 120 subunit HBV capsid, making it a molecular breadboard. We examined removal of subunits from partially crosslinked capsids to form stable incomplete particles. To characterize incomplete capsids, we used two single molecule techniques, resistive-pulse sensing and charge detection mass spectrometry. We expected to find a binomial distribution of capsid fragments. Instead, we found a preponderance of 3 MDa complexes (90 subunits) and no fragments smaller than 3 MDa. We also found 90-mers in the disassembly of uncrosslinked HBV capsids. 90-mers seem to be a common pause point in disassembly reactions. Partly explaining this result, graph theory simulations have showed a threshold for capsid stability between 80 and 90 subunits. To test a molecular breadboard concept, we showed that missing subunits could be refilled resulting in chimeric, 120 subunit particles. This result may be a means of assembling unique capsids with functional decorations. This article is protected by copyright. All rights reserved.

To improve the precision of resistive-pulse measurements, we have used a focused ion beam instrument to mill nanofluidic devices with 2, 4, and 8 pores in series and compared their performance. The in-plane design facilitates the fabrication of multiple pores in series, which, in turn, permits averaging of the series of pulses generated from each translocation event. The standard deviations (σ) of the pulse amplitude distributions decrease by 2.7-fold when the average amplitudes of eight pulses are compared to the amplitudes of single pulses. Similarly, standard deviations of the pore-to-pore time distributions decrease by 3.2-fold when the averages of the seven measurements from 8-pore devices are contrasted to single measurements from 2-pore devices. With signal averaging, the inherent uncertainty in the measurements decreases; consequently, the resolution (mean/σ) improves by a factor equal to the square root of the number of measurements. We took advantage of the improved size resolution of the 8-pore devices to analyze in real time the assembly of Hepatitis B Virus (HBV) capsids below the pseudo-critical concentration. We observe that abundances of assembly intermediates change over time. During the first hour of the reaction, the abundance of smaller intermediates decreased, whereas the abundance of larger intermediates with sizes closer to a T = 4 capsid remained constant.

Colorectal cancer is the fourth most prevalent cancer in the United States, yet there are no reliable non-invasive early screening methods available. Serum-based glycomic profiling has the necessary sensitivity and specificity to distinguish disease states and provide diagnostic potential for this deadly form of cancer. We applied microchip electrophoresis and MALDI-TOF-MS-based glycomic procedures to 20 control serum samples and 42 samples provided by patients diagnosed with colorectal cancer. Within the identified glycans, the position of fucose units was located to quantitate possible changes of fucosyl isomeric species associated with the pathological condition. MALDI-MS data revealed several fucosylated tri- and tetra-antennary glycans which were significantly elevated in their abundance levels in the cancer samples and distinguished the control samples from the colorectal cancer cohort in the comprehensive profiles. When compared to other cancers studied previously, some unique changes appear to be associated with colorectal cancer, being primarily associated with fucosyl isomers. Through MS and microchip electrophoresis-based glycomic methods, several potential biomarkers were identified to aid in the diagnosis and differentiation of colorectal cancer. With its unique capability to resolve isomers, microchip electrophoresis can yield complementary analytical information to MS-based profiling.

We describe a microfluidic device with an integrated nanochannel array to trap individual bacteria and monitor growth and reproduction of lineages over multiple generations. Our poly(dimethylsiloxane) device comprises a pneumatically actuated nanochannel array that includes 1280 channels with widths from 600 to 1000 nm to actively trap diverse bacteria. Integrated pumps and valves perform on-chip fluid and cell manipulations that provide dynamic control of cell loading and nutrient flow, permitting chemostatic growth for extended periods of time (typically 12 to 20 h). Nanochannels confine bacterial growth to a single dimension, facilitating high-resolution, time-lapse imaging and tracking of individual cells. We use the device to monitor the growth of single bacterial cells that undergo symmetric (Bacillus subtilis) and asymmetric (Caulobacter crescentus) division and reconstruct their lineages to correlate growth measurements through time and among related cells. Furthermore, we monitor the motility state of single B. subtilis cells across multiple generations by the expression of a fluorescent reporter protein and observe that the state of the epigenetic switch is correlated over five generations. Our device allows imaging of cellular lineages with high spatiotemporal resolution to facilitate the analysis of biological processes spanning multiple generations.

Disruption of virus capsid assembly has compelling antiviral potential that has been applied to hepatitis B virus (HBV). HBV core protein assembly can be modulated by heteroaryldihydropyrimidines (HAPs), and such molecules are collectively termed core protein allosteric modulators (CpAMs). Although the antiviral effects of CpAMs are acknowledged, the mechanism of action remains an open question. Challenging aspects of characterizing misdirected assembly are the large size and nonuniform nature of the final particles. In this study of HBV assembly, we observed a competition between normative and CpAM-induced aberrant assembly with electron microscopy and resistive-pulse sensing on nanofluidic devices. This competition was a function of the strength of the association energy between individual core proteins, which is proportional to ionic strength. At strong association energy, assembly reactions primarily yielded morphologically normal HBV capsids, despite the presence of HAP-TAMRA. At weak association energy, HAP-TAMRA led to increased assembly product size and disrupted morphology. The smallest particles were T = 4 icosahedra, whereas the larger particles were defective spheres, ellipsoids, and bacilliform cylinders, with regions of T = 4 geometry interspersed with flat regions. Deviation from spherical geometry progressively increased with particle size, which is consistent with the interpretation of a competition between two alternative assembly pathways.

Virus self-assembly is a critical step in the virus lifecycle. Understanding how viruses assemble and disassemble provides needed insight into developing antiviral pharmaceuticals. Few tools offer sufficient resolution to study assembly intermediates that differ in size by a few dimers. Our goal is to improve resistive-pulse sensing on nanofluidic devices to offer better particle-size and temporal resolution to study intermediates and capsids generated along the assembly pathway. To increase the particle-size resolution of the resistive-pulse technique, we measured the same, single virus particles up to a thousand times, cycling them back and forth across a series of nanopores by switching the polarity of the applied potential, i.e., virus ping-pong. Multiple pores in series provide a unique multi-pulse signature during each cycle that improves particle tracking and, therefore, identification of a single particle, and reduces the number of cycles needed to make the requisite number of measurements. With T = 3 and T = 4 Hepatitis B Virus (HBV) capsids, we showed the standard deviation of the particle size distribution decreased with the square root of the number of measurements and approached discriminating particles differing in size by single dimers. We then studied in vitro assembly of HBV capsids and observed that the ensemble of intermediates shift to larger sizes over two days of annealing. On the contrary, assembly reactions diluted to lower dimer concentrations an hour after initiation had fewer intermediates that persisted after the two-day incubation and had a higher ratio of T = 4 to T = 3 capsids. These reactions indicate that labile T = 4 intermediates are formed rapidly, and dependent on conditions, intermediates may be trapped as metastable species or progress to yield complete capsids.

In the environment, most bacteria form surface-attached cell communities called biofilms. The attachment of single cells to surfaces involves an initial reversible stage typically mediated by surface structures such as flagella and pili, followed by a permanent adhesion stage usually mediated by polysaccharide adhesives. Here, we determine the absolute and relative timescales and frequencies of reversible and irreversible adhesion of single cells of the bacterium Caulobacter crescentus to a glass surface in a microfluidic device. We used fluorescence microscopy of C. crescentus expressing green fluorescent protein to track the swimming behavior of individual cells prior to adhesion, monitor the cell at the surface, and determine whether the cell reversibly or irreversibly adhered to the surface. A fluorescently labeled lectin that binds specifically to polar polysaccharides, termed holdfast, discriminated irreversible adhesion events from reversible adhesion events where no holdfast formed. In wild-type cells, the holdfast production time for irreversible adhesion events initiated by surface contact (23 s) was 30-times faster than the holdfast production time that occurs through developmental regulation (13 min). Irreversible adhesion events in wild-type cells (3.3 events/min) are 15-times more frequent than in pilus-minus mutant cells (0.2 events/min), indicating the pili are critical structures in the transition from reversible to irreversible surface-stimulated adhesion. In reversible adhesion events, the dwell time of cells at the surface before departing was the same for wild-type cells (12 s) and pilus-minus mutant cells (13 s), suggesting the pili do not play a significant role in reversible adhesion. Moreover, reversible adhesion events in wild-type cells (6.8 events/min) occur twice as frequently as irreversible adhesion events (3.3 events/min), demonstrating that most cells contact the surface multiple times before transitioning from reversible to irreversible adhesion.

This review covers conductivity detection in fabricated nanochannels and nanopores. Improvements in nanoscale sensing are a direct result of advances in fabrication techniques, which produce devices with channels and pores with reproducible dimensions and in a variety of materials. Analytes of interest are detected by measuring changes in conductance as the analyte accumulates in the channel or passes transiently through the pore. These detection methods take advantage of phenomena enhanced at the nanoscale, such as ion current rectification, surface conductance, and dimensions comparable to the analytes of interest. The end result is the development of sensing technologies for a broad range of analytes, e.g., ions, small molecules, proteins, nucleic acids, and particles.

Simian virus 40 capsid protein (VP1) is a unique system for studying substrate-dependent assembly of a nanoparticle. Here, we investigate a simplest case of this system where 12 VP1 pentamers and a single polyanion, e.g., RNA, form a T = 1 particle. To test the roles of polyanion substrate length and structure during assembly, we characterized the assembly products with size exclusion chromatography, transmission electron microscopy, and single-particle resistive-pulse sensing. We found that 500 and 600 nt RNAs had the optimal length and structure for assembly of uniform T = 1 particles. Longer 800 nt RNA, shorter 300 nt RNA, and a linear 600 unit poly(styrene sulfonate) (PSS) polyelectrolyte produced heterogeneous populations of products. This result was surprising as the 600mer PSS and 500-600 nt RNA have similar mass and charge. Like ssRNA, PSS also has a short 4 nm persistence length, but unlike RNA, PSS lacks a compact tertiary structure. These data indicate that even for flexible substrates, shape as well as size affect assembly and are consistent with the hypothesis that work, derived from protein-protein and protein-substrate interactions, is used to compact the substrate.

Electrophoretic mobilities and particle sizes of individual Hepatitis B Virus (HBV) capsids were measured in nanofluidic channels with two nanopores in series. The channels and pores had three-dimensional topography and were milled directly in glass substrates with a focused ion beam instrument assisted by an electron flood gun. The nanochannel between the two pores was 300 nm wide, 100 nm deep, and 2.5 μm long, and the nanopores at each end had dimensions 45 nm wide, 45 nm deep, and 400 nm long. With resistive-pulse sensing, the nanopores fully resolved pulse amplitude distributions of T = 3 HBV capsids (32 nm outer diameter) and T = 4 HBV capsids (35 nm outer diameter) and had sufficient peak capacity to discriminate intermediate species from the T = 3 and T = 4 capsid distributions in an assembly reaction. Because the T = 3 and T = 4 capsids have a wiffle-ball geometry with a hollow core, the observed change in current due to the capsid transiting the nanopore is proportional to the volume of electrolyte displaced by the volume of capsid protein, not the volume of the entire capsid. Both the signal-to-noise ratio of the pulse amplitude and resolution between the T = 3 and T = 4 distributions of the pulse amplitudes increase as the electric field strength is increased. At low field strengths, transport of the larger T = 4 capsid through the nanopores is hindered relative to the smaller T = 3 capsid due to interaction with the pores, but at sufficiently high field strengths, the T = 3 and T = 4 capsids had the same electrophoretic mobilities (7.4 × 10(-5) cm(2) V(-1) s(-1)) in the nanopores and in the nanochannel with the larger cross-sectional area.

We report the measurement of electroosmotic mobilities in nanofluidic channels with rectangular cross sections and compare our results with theory. Nanofluidic channels were milled directly into borosilicate glass between two closely spaced microchannels with a focused ion beam instrument, and the nanochannels had half-depths (h) of 27, 54, and 108 nm and the same half-width of 265 nm. We measured electroosmotic mobilities in NaCl solutions from 0.1 to 500 mM that have Debye lengths (κ–1) from 30 to 0.4 nm, respectively. The experimental electroosmotic mobilities compare quantitatively to mobilities calculated from a nonlinear solution of the Poisson–Boltzmann equation for channels with a parallel-plate geometry. For the calculations, ζ-potentials measured in a microchannel with a half-depth of 2.5 μm are used and range from −6 to −73 mV for 500 to 0.1 mM NaCl, respectively. For κh > 50, the Smoluchowski equation accurately predicts electroosmotic mobilities in the nanochannels. However, for κh < 10, the electrical double layer extends into the nanochannels, and due to confinement within the channels, the average electroosmotic mobilities decrease. At κh ≈ 4, the electroosmotic mobilities in the 27, 54, and 108 nm channels exhibit maxima, and at 0.1 mM NaCl, the electroosmotic mobility in the 27 nm channel (κh = 1) is 5-fold lower than the electroosmotic mobility in the 2.5 μm channel (κh = 100).

Ion, particle, and fluid transport in nanofluidic devices has received considerable attention over the past two decades due to unique transport properties exhibited at the nanoscale.1,2 Phenomena such as double layer overlap, high surface-to-volume ratios, surface charge, ion-current rectification, and entropic barriers can influence transport in and around nanofluidic structures because the length scales of these forces and the critical dimensions of the device are similar. Advances in micro- and nanofabrication techniques provide the ability to design a variety of well-defined nanofluidic geometries to study these phenomena and their effects on ion and fluid transport. Integration of micro- and nanofluidic structures into lab-on-a-chip devices permits increased functionality that is useful for a range of analytical applications.3,4 This Review focuses on recent advances in nanofabrication techniques as well as studies of fundamental transport in nanofluidic devices. Nanopores, nanochannels, and nanopipets are three common nanofluidic structures that have been influential in studying nanofluidic transport. Because of space limitations, we have limited the scope of this Review to studies with these three structures, and we focus our attention primarily on work published between January 2011 and August 2014. We do not discuss work with carbon nanotubes,5 nanomeshes,6 or nanowires.7 Figure ​Figure11 shows examples of the three nanofluidic geometries discussed here. Nanopores are typically formed perpendicular to the plane of a substrate and are characterized by a critical limiting dimension, which is measured by scanning electron microscopy (SEM), transmission electron microscopy (TEM), or conductance measurements. Pores are fabricated in a variety of materials, e.g., poly(carbonate), poly(ethylene terephthalate), or silicon nitride, and can have an asymmetric (Figure ​(Figure1a)1a) or symmetric (Figure ​(Figure1b)1b) shape, depending on the fabrication technique. Symmetric pores are either cylindrically shaped with a constant critical dimension determined by electron microscopy or hourglass-shaped with a critical dimension at the center of the pore. Although electron microscopy is capable of measuring exterior pore dimensions, the exact inner geometry is often unknown and may contain an asymmetry between two symmetric features, e.g., cigar-shaped pores. Asymmetric nanopores typically have a narrow tip and a wide base with a funnel-shaped geometry along the pore axis. Tip and base dimensions are measured by SEM, but the exact pore geometry is often unknown. Nanochannels often refer to in-plane structures with either symmetric (Figure ​(Figure1c)1c) or asymmetric (Figure ​(Figure1d)1d) geometries. Channels may be confined to the nanoscale in depth, width, or both, depending on the fabrication method. Nanochannels are commonly fabricated in glass and polymer substrates and characterized by SEM and atomic force microscopy (AFM). The in-plane nature of these channels allows the integration of well-defined features into more complex geometries, and any two-dimensional (2D) channel architecture can be designed. Nanopipets are specialized nanopores fabricated from pulled glass or fused-silica capillaries (Figure ​(Figure1e,f). The1e,f). The geometry of a nanopipet is conically shaped with a critical tip diameter of tens to hundreds of nanometers, which can be measured by electron microscopy. Unlike nanopores and nanochannels, nanopipets can be easily coupled with position control, which allows the tip of the nanopipets to be positioned in specific locations or used in scanned probe microscopies. Figure 1 Nanopores, nanochannels, and nanopipets are three common nanofluidic platforms. Nanopores are typically out-of-plane structures and have either an asymmetric or symmetric geometry. Conical nanopores have a wide base as shown in panel a that tapers to ...

We report free-solution microchip electrophoresis performed at elevated temperatures and high separation field strengths. We used microfluidic devices with 11 cm long separation channels to conduct separations at temperatures between 22 (ambient) and 45°C and field strengths from 100 to 1000 V/cm. To evaluate separation performance, N-glycans were used as a model system and labeled with 8-aminopyrene-1,3,6-trisulfonic acid to impart charge for electrophoresis and render them fluorescent. Typically, increased diffusivity at higher temperatures leads to increased axial dispersion and poor separation performance; however, we demonstrate that sufficiently high separation field strengths offset the impact of increased diffusivity in order to maintain separation efficiency. Efficiencies for these free-solution separations are the same at temperatures of 25, 35, and 45°C with separation field strengths ≥ 500 V/cm.

Current lab-on-a-chip (LoC) devices are assay-specific and are custom-built for each single experiment. Performing an experiment requires scientists or engineers to go through the time-consuming process of designing, fabricating, and testing a chip before conducting the actual experiment. This prolonged cycle can take months to complete, increasing effort and cost and reducing productivity. Similarly, minor modifications to an assay protocol re-incur the overheads of the design cycle. In this paper, we develop a multi-purpose, software-programmableLab-on-a-Chip (SPLoC), where the user simply writes or downloads a program for each experiment. We describe the components necessary to realize the SPLoC, which include a high-level programming language, an abstract instruction set, a runtime and control system, and a microfluidic device. We describe two key features of our high-level language compiler, and describe a novel variable-volume variable-ratio mixer. Finally, we demonstrate our SPLoC on four diverse, real-world assays.

The dimorphic alphaproteobacterium Prosthecomicrobium hirschii has both short-stalked and long-stalked morphotypes. Notably, these morphologies do not arise from transitions in a cell cycle. Instead, the maternal cell morphology is typically reproduced in daughter cells, which results in microcolonies of a single cell type. In this work, we further characterized the short-stalked cells and found that these cells have a Caulobacter -like life cycle in which cell division leads to the generation of two morphologically distinct daughter cells. Using a microfluidic device and total internal reflection fluorescence (TIRF) microscopy, we observed that motile short-stalked cells attach to a surface by means of a polar adhesin. Cells attached at their poles elongate and ultimately release motile daughter cells. Robust biofilm growth occurs in the microfluidic device, enabling the collection of synchronous motile cells and downstream analysis of cell growth and attachment. Analysis of a draft P. hirschii genome sequence indicates the presence of CtrA-dependent cell cycle regulation. This characterization of P. hirschii will enable future studies on the mechanisms underlying complex morphologies and polymorphic cell cycles. IMPORTANCE Bacterial cell shape plays a critical role in regulating important behaviors, such as attachment to surfaces, motility, predation, and cellular differentiation; however, most studies on these behaviors focus on bacteria with relatively simple morphologies, such as rods and spheres. Notably, complex morphologies abound throughout the bacteria, with striking examples, such as P. hirschii , found within the stalked Alphaproteobacteria . P. hirschii is an outstanding candidate for studies of complex morphology generation and polymorphic cell cycles. Here, the cell cycle and genome of P. hirschii are characterized. This work sets the stage for future studies of the impact of complex cell shapes on bacterial behaviors.

We report the development of an automated microfluidic "baby machine" to synchronize the bacterium Caulobacter crescentus on-chip and to move the synchronized populations downstream for analysis. The microfluidic device is fabricated from three layers of poly(dimethylsiloxane) and has integrated pumps and valves to control the movement of cells and media. This synchronization method decreases incubation time and media consumption and improves synchrony quality compared to the conventional plate-release technique. Synchronized populations are collected from the device at intervals as short as 10 min and at any time over four days. Flow cytometry and fluorescence cell tracking are used to determine synchrony quality, and cell populations synchronized in minimal growth medium with 0.2% glucose (M2G) and peptone yeast extract (PYE) medium contain >70% and >80% swarmer cells, respectively. Our on-chip method overcomes limitations with conventional physical separation methods that consume large volumes of media, require manual manipulations, have lengthy incubation times, are limited to one collection, and lack precise temporal control of collection times.

We report analysis of N-glycans derived from disease-free individuals and patients with Barrett's esophagus, high-grade dysplasia, and esophageal adenocarcinoma by microchip electrophoresis with laser-induced fluorescence detection. Serum samples in 10-μL aliquots are enzymatically treated to cleave the N-glycans that are subsequently reacted with 8-aminopyrene-1,3,6-trisulfonic acid to add charge and a fluorescent label. Separations at 1250 V/cm and over 22 cm yielded efficiencies up to 700,000 plates for the N-glycans and analysis times under 100 s. Principal component analysis (PCA) and analysis of variance (ANOVA) tests of the peak areas and migration times are used to evaluate N-glycan profiles from native and desialylated samples and to determine differences among the four sample groups. With microchip electrophoresis, we are able to distinguish the three patient groups from each other and from disease-free individuals.

In-plane nanofluidic channels with 3D topography are fabricated. Nanochannel masters are written by electron beam lithography in SU-8 resist and shaped by electron-beam-induced etching (EBIE) with water as the precursor gas. Nanofunnel replicas cast from unmodified and EBIE-modified masters show that the funnel tip dimensions decrease from a 150-nm depth and 80-nm width to a 70-nm depth and 40-nm width.

We study ionic current rectification observed in a nanofluidic device with a nanofunnel positioned between two straight nanochannels. Ion transport is simulated by resolving the coupled three-dimensional Nernst-Planck, Poisson, and Navier-Stokes equations. In the modeled system, the electric double layer extends into the channel, and consequently, the funnel tip exhibits charge-selective properties, which results in the formation of enriched and depleted concentration polarization (CP) zones within the nanofunnel in the high- and low-conductance states, respectively. This scenario is similar to the one observed for ion transport through a charged conical nanopore connecting two macroscopic reservoirs. However, the presence of the adjacent straight nanochannels allows the CP zones to propagate out of the funnel into the adjoining channels. The condition for propagation of the CP zones is determined by several parameters, including the electroosmotic flow velocity. We demonstrate that in the high-conductance regime the modeled system is characterized by increased ionic concentrations in the entire cathodic nanochannel, whereas in the low-conductance state the depleted CP zone does not propagate out of the funnel and remains localized. The required three-dimensional modeling scheme is implemented on a parallel computational platform, is general as well as numerically efficient, and will be useful in the study of more advanced nanofluidic device designs for tailoring ionic current rectification.

Ion transport and sensing in nanofluidic devices are receiving a great deal of attention because of their unique transport properties and potential analytical applications. Some aspects of microscale transport transfer directly to the nanoscale, but nanofluidic systems can be significantly influenced by phenomena such as double-layer overlap, surface charge, ion-current rectification, diffusion, and entropic forces, which are either insignificant or absent in larger microchannels. Micro- and nanofabrication techniques create features with a wide range of well-defined geometries and dimensions in synthetic and solid-state substrates. Moreover, these techniques permit coupling of multiple nano- and microscale elements, which can execute various functions. We discuss basic nanofluidic architectures, material transport properties through single and multiple nanochannels, and characterization of single particles by resistive-pulse sensing.

We report characterization of hepatitis B virus (HBV) capsids by resistive-pulse sensing through single track-etched conical nanopores formed in poly(ethylene terephthalate) membranes. The pores were ∼40 nm in diameter at the tip, and the pore surface was covalently modified with triethylene glycol to reduce surface charge density, minimize adsorption of the virus capsids, and suppress electroosmotic flow in the pore. The HBV capsids were assembled in vitro from Cp149, the assembly domain of HBV capsid protein. Assembled T = 3 (90 Cp149 dimer) and T = 4 (120 dimer) capsids are 31 and 36 nm in diameter, respectively, and were easily discriminated by monitoring the change in current as capsids passed through an electrically biased pore. The ratio of the number of T = 3 to T = 4 capsids transiting a pore did not reflect actual concentrations, but favored transport of smaller T = 3 capsids. These results combined with longer transit times for the T = 4 capsids indicated that the capsids must overcome an entropic barrier to enter a pore.

We report fabrication of nanofluidic channels with asymmetric features (e.g., funnels) that were cast in high modulus poly(dimethylsiloxane) and had well-defined geometries and dimensions. Masters used to cast the funnels were written in the negative tone resist SU-8 by electron beam lithography. Replicated funnels had taper angles of 5, 10, and 20 degrees and were 80 nm wide at the tip, 1 microm wide at the base, and 120 nm deep. The planar format permitted easy coupling of the funnels to microfluidic channels and simultaneous electrical and optical characterization of ion transport. All three designs rectified ion current, and the 5 degrees funnel exhibited the highest rectification ratio. Fluorescence measurements at the funnel base showed that an anionic probe was enriched and depleted in the high and low conductance states, respectively.

A cyclic particle extractor based on particle diffusion is presented. The extraction realized by the device features simplicity, programmability, and low cost. Although conventional particle separation based on diffusion can be spontaneously realized without any active inputs, the extraction efficiency decreases as the size difference between particles decreases or if the diffusion length is insufficient. In this article, a primary extraction procedure including four operational steps is proposed to facilitate the process. By simply repeating the procedure, the separation scheme is additive, and increased efficiency is observed with each additional cycle. A mixture of 0.5- and 3-μm polystyrene particles was separated in up to 10 extraction cycles. Using a 2.5-Hz phase frequency, the average flow velocity was 2.5 mm/s. An unequal volume ratio of the sample stream to extraction stream (45:55) created a barrier region to help minimize unwanted (large) particles from entering the extraction stream. The initial concentration of the extracted small particles was 7.5% after 2 cycles, but jumped up to 38% after 10 cycles.

We report integrated nanopore/microfluidic devices in which the unique combination of low pore density, conical nanopore membranes with microfluidic channels created addressable, localized high-field regions for electrophoretic and dielectrophoretic trapping of particles. A poly(ethylene terephthalate) track-etched membrane containing conical pores approximately 130 nm in diameter at the tip and approximately 1 microm in diameter at the base was used as an interconnect between two perpendicular poly(dimethylsiloxane) microfluidic channels. Integration of the nanopore membrane with microfluidic channels allowed for easy coupling of the electrical potentials and for directed transport of the analyte particles, 200 nm and 1 microm polystyrene microspheres and Caulobacter crescentus bacteria, to the trapping region. Square waves applied to the device generated electric field strengths up to 1.3 x 10(5) V/cm at the tips of the nanopores in the microchannel intersection. By varying the applied potentials from +/-10 to +/-100 V and exploring frequencies from dc to 100 kHz, we determined the contributions of electrophoretic and dielectrophoretic forces to the trapping and concentration process. These results suggest that tunable filter elements can be constructed in which the nanoporous elements provide a physical barrier and the applied ac field enhanced selectivity.

We present an improved microfluidic design for generating spatial and temporal gradients. The basic functional elements are bifurcated and trifurcated channels used to split flow between two and three channels, respectively. We use bifurcated channels on the exterior of the channel manifold and trifurcated channels in the interior with mixing tees to recombine flows. For N gradient-forming levels, the number of discrete steps in the gradient is 2(N) + 1, allowing a compact gradient-forming structure that is only 1.6 mm long and 0.5 mm wide. Control of the relative sample concentration at the inlets enables generation of gradients with varying slopes and offsets. The small total channel length allows faster switching (only 2.6 s) between gradients of different compositions than did previous designs, allowing complex temporal sequences and reducing total displacement volume and reagent use. The design permits opposing-gradient experiments and generation of complex nonlinear gradients. We fabricated and tested three channel designs with either three or four gradient-forming levels, 20- or 40-microm channel widths, 60- or 120-microm center-to-center channel spacings, and 9 or 17 output steps. These devices produced essentially identical high-quality linear gradients using both pressure-driven and electrokinetic flow.

As fabrication techniques improve, functional fluidic devices with nanometer scale dimensions are rapidly being developed for chemical analysis. Here, we present fluid dispensing in nanochannels with injection volumes ranging from 42 aL to 4.1 fL. Devices with hybrid poly(dimethylsiloxane) and glass nanochannels, 130 nm deep and 580 nm wide or 130 nm deep and 670 nm wide, were used to evaluate two sample dispensing schemes, modified pinched and gated injections. Electrokinetic transport was achieved by applying up to 10 V directly from an analog output board without amplification, producing modest electric field strengths in the nanochannels (0.2-2 kV/cm) and enabling rapid dispensing and analysis (10-100 ms).

Virus assembly is a coordinated process in which typically hundreds of subunits react to form complex, symmetric particles. We use resistive-pulse sensing to characterize the assembly of Hepatitis B Virus core protein dimers into T = 3 and T = 4 icosahedral capsids. This technique counts and sizes intermediates and capsids in real time, with single particle sensitivity, and at biologically relevant concentrations. Other methods are not able to produce comparable real-time, single-particle observations of assembly reactions below, near, and above the pseudocritical dimer concentration, at which the dimer and capsid concentrations are approximately equal. Assembly reactions across a range of dimer concentrations reveal three distinct patterns. At dimer concentrations as low as 50 nM, well below the pseudo-critical dimer concentration of 0.5 µM, we observe a switch in the ratio of T = 3 to T = 4 capsids, which increases with decreasing dimer concentration. Far above the pseudo-critical dimer concentration, kinetically trapped, incomplete T = 4 particles assemble rapidly, then slowly anneal into T = 4 capsids. At all dimer concentrations tested, T = 3 capsids form more rapidly than T = 4 capsids, suggesting distinct pathways for the two forms.