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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.

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.

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.

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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.

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.

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 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.

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.

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).

Static and dynamic acute toxicity assays of cells were performed on microfluidic devices where materials were hydraulically transported. Static assays were performed by incubating cells with an agent in a microchip reservoir and optically interrogating the cells after hydrodynamic focusing at a cross intersection. Dynamic assays were performed on a microchip with a 25-cm-long spiral channel where the cells were mixed with an agent and optically monitored 0.1, 12, and 22 cm from the point of mixing. The incubation time was determined by the time needed for cells to transit from the mixing location to the point of detection. Cell viability was determined using the ratio of fluorescence signals from membrane permeant (calcein) and membrane impermeant (propidium iodide) stains. The model system used in this study was the viability of Jurkat cells in the presence of the agent Triton X-100). An average LC50 value of 138 microM for Triton X-100 was obtained for an incubation period of 7-12 min using the static assay. LC50 values obtained with the dynamic assay for 25- and 47-s incubation times were 290 and 250 microM Triton X-100, respectively. Higher LC50 values for the dynamic assay were expected due to the shorter incubation times.

Fluorescently labeled proteins were electrophoretically concentrated on microfabricated devices prior to separation and laser-induced fluorescence detection on the same device. The proteins were concentrated using a porous silica membrane between adjacent microchannels that allowed the passage of buffer ions but excluded larger migrating molecules. Concentrated analytes were then injected into the separation column for analysis. Two basic microchip designs were tested that allowed sample concentration either directly in the sample injector loop or within the microchannel leading from the sample reservoir to the injector. Signal enhancements of approximately 600-fold were achieved by on-chip preconcentration followed by SDS-CGE separation. Preconcentration for CE analysis in both coated and uncoated open channels was also demonstrated. Fluorescently labeled ovalbumin could be detected at initial concentrations as low as 100 fM by using a combination of field-amplified injection and preconcentration at a membrane prior to CE in coated channels.

A microfluidic device is reported that integrated cell handling, rapid cell lysis, and electrophoretic separation and detection of fluorescent cytosolic dyes. The device function was demonstrated using Jurkat cells that were loaded with the fluorogenic dyes - carboxyfluorescein diacetate, Oregon green carboxylic acid diacetate, or Calcein AM. The loaded cells were hydrodynamically transported from the cell-containing reservoir to a region on the microfluidic device where they were focused and then rapidly lysed using an electric field. Complete lysis was accomplished in33 ms. The hydrolyzed, fluorescent dyes in the cell lysate were automatically injected into a separation channel on the device and detected 3 mm downstream of the injection point. The total separation time was approximately 2.2 s with absolute migration time reproducibilities of1% and efficiencies ranging from 2300 to 4000 theoretical plates. Results from 139 cells are reported. A small fraction of these cells, approximately 9%, were found to enzymatically hydrolyze the loaded dyes in a manner significantly different from the majority of the cells. Cell analysis rates of 7-12 cells/min were demonstrated and are100 times faster than those reported using standard bench-scale capillary electrophoresis.

Microfabricated devices integrating sample filtration, solid-phase extraction, and chromatographic separation with solvent programming were demonstrated. Filtering of the sample was accomplished at the sample inlet with an array of seven channels each 1 microm deep and 18 microm wide. Sample concentration and separation were performed on channels 5 microm deep and 25 microm wide coated with a C18 phase, and elution was achieved under isocratic, step, or linear gradient conditions. For the solid-phase extraction, signal enhancement factors of 400 over a standard injection of 1.0 s were observed for a 320-s injection. Four polycyclic aromatic compounds were resolved by open channel electrochromatography in under 50 s. Chip operation was unaffected by the presence of the 5-microm silica particles at the filter entrance.

Flow cytometry of the bacterium Escherichia coli was demonstrated on a microfabricated fluidic device (microchip). The channels were coated with poly(dimethylacrylamide) to prevent cell adhesion, and the cells were transported electrophoretically by applying potentials to the fluid reservoirs. The cells were electrophoretically focused at the channel cross and detected by coincident light scattering and fluorescence. The E. coli were labeled with a membrane-permeable nucleic acid stain (Syto15), a membrane-impermeable nucleic acid stain (propidium iodide), or a fluorescein-labeled antibody and counted at rates from 30 to 85 Hz. The observed labeling efficiencies for the dyes and antibody were greater than 94%.

Electroosmotic manipulation of fluids was demonstrated using thin metal electrodes integrated within microfluidic channels at the substrate and cover plate interface. Devices were fabricated by photolithographically patterning electrodes on glass cover plates that were then bonded to polymeric substrates into which the channels were cast. Polymeric substrates were used to provide a permeable membrane for the transport and removal of gaseous electrolysis products generated at the electrodes. Electroosmotic flow between interdigitated electrodes was demonstrated and provided electric field-free pumping of fluids in sections of the channel outside of the electrode pairs. The resultant pumping velocities were shown to be dependent on the applied voltage, not on the applied field strength, and independent of the length of the electroosmotically pumped region.

A two-dimensional separation system on a microfabricated device was demonstrated using open-channel electrochromatography as the first dimension and capillary electrophoresis as the second dimension. The first dimension was operated under isocratic conditions, and the effluent from the first dimension was repetitively injected into the second dimension every few seconds. A 25-cm separation channel with spiral geometry for open-channel electrochromatography was chemically modified with octadecylsilane and coupled to a 1.2-cm straight separation channel for capillary electrophoresis. Fluorescently labeled products from tryptic digests of beta-casein were analyzed in 13 min with this system.

We have fabricated a 25-cm-long spiral-shaped separation channel on a glass microchip with a footprint of only 5 cm x 5 cm. Electrophoretic separation efficiencies for dichlorofluoroscein (DCF) on this chip exceeded 1,000,000 theoretical plates and were achieved in under 46 s at a detection point 22.2 cm from the injection cross. The number of theoretical plates increased linearly with the applied voltage, and at a separation field strength of 1,170 V/cm, the rate of plate generation was approximately 21,000 plates/s. The large radii of curvature of the turns minimized the analyte dispersion introduced by the channel geometry as evidenced by the fact that the effective diffusion coefficient of DCF was within a few percent of that measured on a microchip with a straight separation channel over a wide range of electric field strengths. A micellar electrokinetic chromatography separation of 19 tetramethylrhodamine-labeled amino acids was accomplished in 165 s with an average plate number of 280,000. The minimum resolution between adjacent peaks for this separation was 1.2.

We have developed and characterized cellular optoporation with visible wavelengths of light using standard uncoated glass cover slips as the absorptive media. A frequency-doubled Nd:YAG laser pulse was focused at the interface of the glass surface and aqueous buffer, creating a stress wave and transiently permeabilizing nearby cells. Following optoporation of adherent cells, three spatial zones were present which were distinguished by the viability of the cells and the loading efficiency (or number of extracellular molecules loaded). The loading efficiency also depended on the concentration of the extracellular molecules and the molecular weight of the molecules. In the zone farthest from the laser beam (60 microns under these conditions), nearly all cells were both successfully loaded and viable. To illustrate the wider applicability of this optoporation method, cells were loaded with a substrate for protein kinase C and the cellular contents then analyzed by capillary electrophoresis. In contrast to peptides loaded by microinjection, optoporated peptide showed little proteolytic degradation, suggesting that the cells were minimally perturbed. Also demonstrating the potential for future work, cells were optoporated and loaded with a fluorophore in the enclosed channels of microfluidic devices.

A microfabricated device for flow injection analysis and electrophoretic separation of acetylcholinesterase (AChE) inhibitors is described. Solutions of inhibitor, enzyme, substrate, and derivitizing agent were mixed within the channels of the microchip using computer-controlled electrokinetic transport. AChE-catalyzed hydrolysis of acetylthiocholine to thiocholine was measured in an on-chip reaction of thiocholine with coumarinylphenylmaleimide, and the resulting thioether was detected by laser-induced fluorescence. Inhibitors reduced the fluorescence signal and produced a negative peak diagnostic for the type of inhibition. A Gaussian peak was observed for competitive inhibitors, whereas a broad negative peak was observed for irreversible inhibitors. From a microchip assay for tacrine, an inhibition constant, Ki, of 1.5 ± 0.2 nM was derived, which compared well with a standard cuvette assay. A flow injection assay of two irreversible inhibitors, carbofuran and eserine, was performed. With a 5-min stoppe...

Cross-correlation electrophoresis with fluorescence detection on a microchip device was used to resolve a mixture of 63 pM fluorescein and 70 pM dichlorofluorescein. The signal-to-noise ratios of analyte peaks in a correlogram derived from a 9-bit pseudorandom injection sequence were ∼17 for dichlorofluorescein and ∼6 for fluorescein. In contrast, neither peak was detected in a conventional electropherogram obtained on the microchip. Detection limits estimated from a correlogram derived from the average of 12 7-bit correlograms were 6.5 pM dichlorofluorescein and 21 pM fluorescein. These are the lowest detection limits reported for a microchip separation device using analog detection and the first example of cross-correlation electrophoresis on a monolithic device. The small injection volumes, injection reproducibility, and rapid separations enabled by microchip devices make cross-correlation multiplex injection schemes a viable approach for achieving enhanced sensitivity.

Microfabricated devices for parallel and serial mixing of fluids are demonstrated. To simplify the voltage control hardware, electrokinetic mixing is effected using a single voltage source with the channels dimensioned to perform the desired voltage division. In addition, the number of fluid reservoirs is reduced by terminating multiple buffer, sample, or analysis channels in single reservoirs. The parallel mixing device is designed with a series of independent T-intersections, and the serial mixing device is based on an array of cross intersections and sample shunting. These devices were tested by mixing a sample with buffer in a dilution experiment. Sample fractions of 1.0, 0.84, 0.67, 0.51, 0.36, 0.19, and 0 were generated for the parallel mixing device, and sample fractions of 1.0, 0.36, 0.21, 0.12, and 0.06 for the serial mixing device.

A microchip gated valve is demonstrated that uses a single voltage source and three fluid reservoirs. The fluidic valve is a cross intersection, and the channels are dimensioned to perform the appropriate voltage division, simplifying the voltage control hardware. A single voltage source is applied directly to the sample reservoir and through a high-voltage relay to the buffer reservoir, and the waste reservoir is grounded. The volume of sample dispensed is determined by the duration that the high-voltage relay is open. Volumetric reproducibility is demonstrated to be

Polymerase chain reactions (PCRs) were carried out on as many as four DNA samples at a time on a microchip device. The PCR products were then analyzed, either individually or together on the same device, by microchip gel electrophoresis. A standard PCR protocol was used to amplify 199- and 500-base pair (bp) regions of bacteriophage lambda DNA and 346- and 410-bp regions of E. coli genomic and plasmid DNAs, respectively. Thermal lysis of the bacteria was integrated into the PCR cycle. A product sizing medium, poly(dimethylacrylamide), and an intercalating dye for fluorescence detection were used in the electrophoretic analysis of the products. PCR product sizes were determined by coelectrophoresis with marker DNA.

A mathematical model describing electrokinetically driven mass transport phenomena in microfabricated chip devices is presented in this paper. The model accounts for principal material transport mechanisms such as electrokinetic migration (electrophoresis and electroosmosis) and diffusion. A computer code that implements the model is capable of simulating transport of materials during electrokinetic manipulation in 2-D channel structures. The computer code allows arbitrary channel geometries with various boundary conditions for the electric field and the sample concentration. Two fundamental microfluidic chip elements, a cross and a mixing tee, are of particular interest. An electrokinetic focusing experiment using a cross structure and mixing in a tee structure are simulated. Simulations revealed an optimum focusing voltage for which the ratio of sample concentration to sample width is maximized. They also verified that the mixing tee provides very accurate dilution/mixing characteristics for both charged and neutral samples. Good agreement between simulated and experimental data verified the accuracy of the mathematical model.

Folding the separation channel in a microchip device generally introduces an additional geometrical contribution to analyte dispersion through lateral variations in both migration distance and field strength. The geometrical dispersion generated depends on the ratio of the analyte transverse diffusion time to the time that the analyte band spends traversing the turn. A one-dimensional model has been developed which predicts the amount of excess dispersion introduced by turns in microchip channels. This model accounts for migration length differences, field strength differences, and transverse diffusion effects and accurately describes the experimental data. The introduction of turns into the channel on a microchip is shown to reduce separation efficiency compared to the same separation length in a straight channel, especially for molecules with small diffusion coefficients. Several methods to reduce geometrical dispersion are examined including the manipulation of channel width and analyte velocity and th...

Microchip electrophoresis was used to resolve a binary mixture in 0.8 ms using a separation field strength of 53 kV cm-1 and a separation length of 200 μm. The microchip design permitted an electric field strength of 6.1 V cm-1 in the separation channel per volt applied to the microchip. Concurrently, the spatial extent of the injection plug and the detector observation region were minimized to increase separation efficiency.

A monolithic micromachined device is presented which allows on-chip adjustments of the content of organic modifier in the run buffer for fast, efficient MEKC separations. Isocratic and gradient conditions are controlled by proper setting of voltages applied to the buffer reservoirs of the microchip. The precision of this control is tested for gradients of various shapes (linear, concave, convex) by mixing pure buffer and buffer doped with a fluorescent dye. The effect of isocratic and gradient solvent changes on the MEKC separation of a mixture of coumarin dyes is demonstrated using methanol and acetonitrile as modifiers. Separations were carried out using a column length of 25 mm and a field strength of 660 V/cm with high resolution. Analysis times were as short as 33 s for methanol and under 22 s for acetonitrile. Gradients with both modifiers were executed within 10 s. Of the two modifiers, acetonitrile proved to have a more pronounced impact on the elution pattern of the test mixture. Only slight band...

Glass microchips are fabricated to demonstrate two-dimensional spatial confinement of ions and fluids using electrokinetic forces. Neutral and charged molecular samples can be electrokinetically focused to widths as narrow as 3.3 μm in a 18 μm long by 18 μm wide chamber. The electrokinetic focusing characteristics are tested using cationic, neutral, and anionic samples to evaluate the influence of electric fields on the sample stream. Focusing is achieved using electrophoresis, electroosmosis, or a combination of the two. In addition, concentration enhancement is observed in the focusing chamber by employing sample stacking, and the focusing chamber is multiplexed to confine spatially more than one sample stream simultaneously.

Micellar electrokinetic capillary chromatography (MECC) of three neutral coumarin dyes was performed on glass microchips. Manifolds of channels for analyte injection and separation were machined into one surface of the glass substrates using standard photolithographic, etching, and deposition techniques. Cover plates were then directly bonded over these channels to form capillary networks, with fluid flow in these networks controlled by varying the applied high-voltage potentials at the outlets. The separation capillary was 16.5 cm long for a serpentine channel chip and 1.3 cm long for a straight channel chip. Detection of analyte zones was accomplished by laser-induced fluorescence using the UV lines (nearly 350 nm) of an argon ion laser. At low applied electric field strengths, MECC analyses with on-chip injections gave high reproducibilities in peak areas and migration times ( 400 V/cm), analysis times were shorter, but separation efficiency decreased at later migration times. These peaks showed significant broadening, consistent with mass transfer effects. 14 refs., 6 figs., 2 tabs.

A fused quartz microchip is fabricated to perform capillary electrophoresis of metal ions complexed with 8-hydroxyquinoline-5-sulfonic acid (HQS). The channel manifold on the quartz substrate is fabricated using standard photolithographic, etching, and deposition techniques. By incorporating a direct bonding technique during the fabrication of the microchip, the substrate and cover plate can be fused together below the melting temperature for fused quartz. To enhance the resolution for the separation, the electroosmotic flow is minimized by covalently bonding polyacrylamide to the channel walls. A separation length of 16.5 mm and separation field strength of 870 V/cm enable separations to be performed in {

A glass microchip was constructed to perform chemical reactions and capillary electrophoresis sequentially. The channel manifold on the glass substrate was fabricated using standard photolithographic, etching, and deposition techniques. The microchip has a reaction chamber with a 1 nL reaction volume and a separation column with a 15.4 mm separation length. Electrical control of the buffer, analyte, and reagent streams made possible the precise manipulation of the fluids within the channel manifold. The microchip was operated under a continuous reaction mode with gated injections to introduce the reaction product onto the separation column with high reproducibility (

A glass microchip with a postcolumn reactor was fabricated to conduct postseparation derivatization using o-phthaldialdehyde as a fluorescent «tag» for amino acids. This miniaturized separation device was constructed using standard photolithographic, wet chemical etching, and bonding techniques. Effects of the reagent stream on separation efficiency were investigated. In addition, a novel gated injector was demonstrated which maintains the integrity of the analyte, buffer, and reagent streams

A glass microchip having a channel with a cross section of 5.6 [mu]m high and 66 [mu]m wide was fabricated using standard photolithographic and etching techniques. The surface of the channel was chemically modified with octadecylsilane to function as the stationary phase for open channel chromatography. Electroosmotic flow was used to [open quotes]load[close quotes] the sample into the microchip and to [open quotes]pump[close quotes] the mobile phase during the experiments. For electric field strengths in the separation column from 27 to 163 V/cm, the linear velocity for the electroosmotic flow ranged from 0.13 to 0.78 mm/s. Detection was performed using direct fluorescence for separation monitoring and indirect fluorescence for void time measurements. Plate heights as low as 4.1 and 5.0 [mu]m were generated for unretained and retained components, respectively. 28 refs., 6 figs., 2 tabs.

A glass microchip column was fabricated for free-solution electrophoresis. The channels were wet chemically etched on a substrate using standard photolithographic techniques and were sealed using a direct bonding technique. Two methods of sample introduction are evaluated employing a cross-type channel geometry. With the preferred sample loading method, the volume of the sample plug can be accurately controlled and is time independent, enabling a constant volume to be injected. The injection can be controlled electronically and does not induce any electrophoretic mobility based bias. Separations were performed on a compact chip with a serpentine column geometry that has a 165-mm separation channel in an area of less than 10 mm x 10 mm. Band-broadening effects in a serpentine column pattern were studied. 18 refs., 9 figs.

Fast, efficient separations are sought in liquid-phase analyses which incorporate a nondiscriminatory sample injection scheme and can implement a variety of detection modes. A glass microchip device for free solution electrophoresis was fabricated using standard photolithographic procedures and chemical wet etching. Separations were performed at several separation lengths from the injector to the detector with electric field strengths from 0.06 to 1.5 kV/cm. For a separation length of 0.9 mm, electrophoretic separations with baseline resolution are achieved in less than 150 ms with an electric field strength of 1.5 kV/cm and an efficiency of 1820 plates/s. For a separation length of 11.1 mm, a minimum plate height of 0.7 [mu]m and a maximum number of plates per second of 18,600 were achieved. 19 refs., 5 figs., 2 tabs.

We report fabrication and characterization of nanochannel devices with two nanopores in series for resistive-pulse sensing of hepatitis B virus (HBV) capsids. The nanochannel and two pores are patterned by electron beam lithography between two microchannels and etched by reactive ion etching. The two nanopores are 50-nm wide, 50-nm deep, and 40-nm long and are spaced 2.0-μm apart. The nanochannel that brackets the two pores is 20× wider (1 μm) to reduce the electrical resistance adjacent to the two pores and to ensure the current returns to its baseline value between resistive-pulse events. Average pulse amplitudes differ by

Flow cytometry of fluorescently labeled and unlabeled latex particles is demonstrated on a microfabricated device. The latex particles were detected and counted using laser light scattering and fluorescence coincidence measurements. Sample confinement was accomplished using electrokinetic focusing at a cross intersection, and detection occurred 50 μm downstream from the intersection. Particles with diameters of 1 and 2 μm were analyzed and distinguished from each other based on their light scattering intensity and fluorescence. A maximum sample throughput of 34 particles/s was achieved. Sample mixtures with varying proportions of fluorescently labeled and unlabeled particles were also analyzed and found to be within experimental error of the expected ratios.

Separations of 15 pM rhodamine 6G and 30 pM rhodamine B performed in a micromachined electrophoresis channel were detected by counting fluorescence bursts from individual molecules. The migration times, peak widths, and analyte concentrations were estimated from the number and the migration time distribution of the detected molecules. Concentration detection limits estimated at >99% confidence were 1.7 pM rhodamine 6G and 8.5 pM rhodamine B. The separations required

Motile bacteria bias the random walk of their motion in response to chemical gradients by the process termed chemotaxis, which allows cells to accumulate in favorable environments and disperse from less favorable ones. In this work, we describe a simple microchannel-nanopore device that establishes a stable chemical gradient for chemotaxis assays in ≤ 1 min. Chemoattractant is dispensed by diffusion through 10 nm diameter pores at the intersection of two microchannels. This design requires no external pump and minimizes the effect of transmembrane pressure, resulting in a stable, reproducible gradient. The microfluidic platform facilitates microscopic observation of individual cell trajectories, and chemotaxis is quantified by monitoring changes in cell swimming behavior in the vicinity of the intersection. We validate this system by measuring the chemotactic response of an aquatic bacterium, Caulobacter crescentus, to xylose concentrations from 1.3 μM to 1.3 M. Additionally, we make an unanticipated observation of increased turn frequency in a chemotaxis-impaired mutant which provides new insight into the chemotaxis pathway in C. crescentus.

As the field of nanofluidics matures, fundamental discoveries are being applied to lab-on-a-chip analyses. The unique behavior of matter at the nanoscale is adding new functionality to devices that integrate nanopores or nanochannels. (To listen to a podcast about this feature, please go to the Analytical Chemistry website at pubs.acs.org/journal/ancham.).

We report serial-to-parallel interfaces for rapidly and efficiently transferring samples from a single microfluidic channel to multiple parallel channels. Three designs and operation modes were evaluated to determine the most efficient transfer process. All designs employed two routing channels to direct the sample into the parallel channels and to prevent sample from leaking into adjacent channels. For two of the three designs, a tee valve and gated valve were added to the interface prior to routing the samples to assist with sample injections into the parallel channels. Injection times as short as 20 ms and injection frequencies up to 10 Hz were achieved with relative standard deviations less than 0.5% for the injected area. With an injection time of 50 ms and injection frequency of 10 Hz, up to 50% of the sample is efficiently transferred. Among the three designs, the interface with the gated valve provided the highest performance and reproducibility.