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Extracellular vesicles (EVs) are cell-derived particles that exhibit diverse sizes, molecular contents, and clinical implications for various diseases depending on their specific subpopulations. However, fractionation of EV subpopulations with high resolution, efficiency, purity, and yield remains an elusive goal due to their diminutive sizes. In this study, we introduce a novel strategy that effectively separates EV subpopulations in a gel-free and label-free manner, using two-dimensional (2D) electrophoresis in a microfluidic artificial sieve. The microfabricated artificial sieve consists of periodically arranged micro-slit-well structures in a 2D array and generates an anisotropic electric field pattern to size fractionate EVs into discrete streams and steer the subpopulations into designated outlets for collection within a minute. Along with fractionating EV subpopulations, contaminants such as free proteins and short nucleic acids can be simultaneously directed to waste outlets, thus accomplishing both size fractionation and purification of EVs with high performance. Our platform offers a simple, rapid, and versatile solution for EV subpopulation isolation, which can potentially facilitate the discovery of biomarkers for specific EV subtypes and the development of EV-based therapeutics.

MicroRNAs (miRNAs) are a class of biomolecules that have high clinical and pharmaceutical significance because of their ability to regulate protein expression. Better methods are needed to quantify target miRNAs, but their similar sequence lengths and low concentrations in biomedical samples impede analysis. This report aimed to develop a simple, rapid method to directly quantify multiple miRNAs using microfluidic thermal gel electrophoresis (TGE). Fluorescent probes were designed complementarily in sequence to four target miRNAs that also contained variable DNA overhangs to alter their electrophoretic mobilities. Samples and probes were directly added into thermal gel and loaded throughout a microchannel. Applying voltage resulted in an inline preconcentration and separation of the miRNAs that did not require a sample injection nor user intervention to switch between modes. Baseline resolution was achieved between four double-stranded miRNA-probe hybrids and four excess single-stranded probes. Analytical performance was then improved by designing an innovative microfluidic device with a tapered channel geometry. This device exhibited superior detection limits and separation resolution compared to standard channel devices without increasing the complexity of microfabrication or device operation. A proof-of-concept demonstration was then performed, showing that target miRNAs could be detected from cell extracts. These results demonstrate that TGE provides a simple, inexpensive means of conducting multiplexed miRNA measurements, with the potential for automation to facilitate future clinical and pharmaceutical analyses.

Electric fields are commonly used to trap and separate micro- and nanoparticles near channel constrictions in microfluidic devices. The trapping mechanism is attributed to the electrical forces arising from the nonhomogeneous electric field caused by the constrictions, and the phenomenon is known as insulator-based-dielectrophoresis (iDEP). In this paper, we describe stationary electroosmotic flows of electrolytes around insulating constrictions induced by low frequency AC electric fields (below 10 kHz). Experimental characterization of the flows is described for two different channel heights (50 and 10 μm), together with numerical simulations based on an electrokinetic model that considers the modification of the local ionic concentration due to surface conductance on charged insulating walls. We term this phenomenon concentration-polarization electroosmosis (CPEO). The observed flow characteristics are in qualitative agreement with the predictions of this model. However, for shallow channels (10 μm), trapping of the particles on both sides of the constrictions is also observed. This particle and fluid behavior could play a major role in iDEP and could be easily misinterpreted as a dielectrophoretic force.

Chemical stimulus-induced neurotransmitter release from neuronal cells is well-documented. However, the dynamic changes in neurochemical release remain to be fully explored. In this work, a three-layered microfluidic chip was fabricated and evaluated for studying the dynamics of neurotransmitter release from PC-12 cells. The chip features integration of a nanoliter sized chamber for cell perfusion, pneumatic pressure valves for fluidic control, a microfluidic channel for electrophoretic separation, and a nanoelectrospray emitter for ionization in MS detection. Deploying this platform, a microchip electrophoresis-mass spectrometric method (MCE-MS) was developed to simultaneously quantify important neurotransmitters, including dopamine (DA), serotonin (5-HT), aspartic acid (Asp), and glutamic acid (Glu) without need for labeling or enrichment. Monitoring neurotransmitter release from PC-12 cells exposed to KCl (or alcohol) revealed that all four neurotransmitters investigated were released. Two release patterns were observed, one for the two monoamine neurotransmitters (i.e., DA and 5-HT) and another for the two amino acid neurotransmitters. Release dynamics for the two monoamine neurotransmitters was significantly different. The cells released DA most quickly and heavily in response to the stimulation. After exposure to the chemical stimulus for 4 min, the DA level in the perfusate from the cells was 86% lower than that at the beginning. Very interestingly, the cells started to release 5-HT in large quantities when they stopped releasing DA. These results suggest that DA and 5-HT are packaged into different vesicle pools and they are mobilized differently in response to chemical stimuli. The microfluidic platform proposed is proven useful for monitoring cellular release in biological studies., Competing Interests: Notes The authors declare no competing financial interest.

High-throughput screening (HTS) using multiwell plates and fluorescence plate readers is a powerful tool for drug discovery and evaluation by allowing tens of thousands of assays to be completed in 1 day. Although this method has been successful, electrophoresis-based methods for screening are also of interest to avoid difficulties associated fluorescence assays such as requirements to engineer fluorogenic reactions and false positives. We have developed a method using droplet microfluidics to couple multiwell plate-based assays to microchip electrophoresis (MCE) to screen enzyme modulators. Samples contained in multiwell plates are reformatted in to plugs with a sample volume of 8 nL segmented by an immiscible oil. The segmented flow sample streams are coupled to a hybrid polydimethylsiloxane-glass microfluidic device capable of selectively extracting the aqueous samples from the droplet stream and rapidly analyzing by MCE with laser-induced fluorescence detection. This system was demonstrated by screening a test library of 140 compounds against using protein kinase A. For each sample in the screen, two droplets are generated, allowing approximately 6 MCE injections per sample. Using a 1 s separation at 2000 V/cm, we are able to analyze 96 samples in 12 min. Separation resolution between the internal standard, substrate, and product is 1.2 and average separation efficiency is 16,000 plates/s using real samples. Twenty-five compounds were identified as modulators during primary screening and verified using dose-response curves.

We report rapid and efficient electrophoretic separations of N-glycans on microfluidic devices. Using a separation length of 22 cm and an electric field strength of 750 V/cm, analysis times were less than 3 min, and separation efficiencies were between 400,000 and 655,000 plates for the N-glycans and up to 960,000 plates for other sample components. These high efficiencies were necessary to separate N-glycan positional isomers derived from ribonuclease B and linkage isomers from asialofetuin. Structural isomers of N-glycans derived from a blood serum sample of a cancer patient were also analyzed to demonstrate that clinically relevant, complex samples could be separated on-chip with efficiencies similar to those derived from model glycoproteins. In addition, we compared microchip and capillary electrophoresis under similar separation conditions, and the microchips performed as well as the capillaries. These results confirmed that the noncircular cross section of the microchannel did not hamper separation performance. For all experiments, the glycan samples were derivatized with 8-aminopyrene-1,3,6-trisulfonic acid to impart needed charge for electrophoresis and a fluorescent label for detection.

This study presents a new cell manipulation method using a moving dielectrophoretic force to transport or fractionate cells along a microfluidic channel. The proposed moving dielectrophoresis (mDEP) is generated by sequentially energizing a single electrode or an array of electrodes to form an electric field that moves cells continuously along the microchannel. Cell fractionation is controlled by the applied electrical frequency, and cell transportation is controlled by the interelectrode activation time. The applicability of this method was demonstrated to simultaneously fractionate and transport Saccharomyces cerevisiae yeast cells, both viable and nonviable, by operating at conditions under which the cells were subjected to positive and negative dielectrophoresis, respectively. Compared to the conventional dielectrophoresis (cDEP and traveling wave dielectrophoresis (twDEP), moving dielectrophoresis allows cells to be separated on the basis of the real part of the Clausius-Mossotti factor, as in cDEP, but yet allows the direct transportation of separated cells without using fluid flow, as in twDEP. This dielectrophoresis technique provides a new way to manipulate cells and can be readily implemented on programmable multielectrode devices.

For domestic and military security, an autonomous system capable of continuously monitoring for airborne biothreat agents is necessary. At present, no system meets the requirements for size, speed, sensitivity, and selectivity to warn against and lead to the prevention of infection in field settings. We present a fully automated system for the detection of aerosolized bacterial biothreat agents such as Bacillus subtilis (surrogate for Bacillus anthracis) based on protein profiling by chip gel electrophoresis coupled with a microfluidic sample preparation system. Protein profiling has previously been demonstrated to differentiate between bacterial organisms. With the goal of reducing response time, multiple microfluidic component modules, including aerosol collection via a commercially available collector, concentration, thermochemical lysis, size exclusion chromatography, fluorescent labeling, and chip gel electrophoresis were integrated together to create an autonomous collection/sample preparation/analysis system. The cycle time for sample preparation was approximately 5 min, while total cycle time, including chip gel electrophoresis, was approximately 10 min. Sensitivity of the coupled system for the detection of B. subtilis spores was 16 agent-containing particles per liter of air, based on samples that were prepared to simulate those collected by wetted cyclone aerosol collector of approximately 80% efficiency operating for 7 min.

A biomimetic surface has been formed on the poly(methyl methacrylate) (PMMA) microfluidic chips for biofouling resistance on the basis of a simple modification. Accordingly, an amphiphilic phospholipid copolymer of 2-methacryloyloxyethyl phosphorylcholine and n-butyl methacrylate (PMB) was developed to introduce the phosphorylcholine functional groups onto the PMMA surface via the anchoring of hydrophobic n-butyl methacrylate units. The 2-methacryloyloxyethyl phosphorylcholine segments could form hydrophilic domains, considered to be located on the surface, to provide a biocompatible surface. X-ray photoelectron spectroscopy and Fourier transform infrared spectra confirmed the success of surface functionalization. The PMB-modified microchips containing phosphorylcholine moieties exhibited more stable electroosmotic mobility compared with the untreated one. In addition to being characterized for minimized nonspecific adhesion of serum proteins and plasma platelets, the PMB-functionalized microchannels have been exemplified by electrophoresis of proteins. This one-step procedure offers an effective approach for a biomimetic surface design on microfluidic chips, which is promising in high-throughput and complex biological analysis.

Within the context of microfluidic systems, it has been difficult to devise pumping systems that can deliver adequate flow rates at high pressure for applications such as HPLC. An on-chip electrochemical pumping system based on electrolysis that offers certain advantages over designs that utilize electroosmotic driven flow has been fabricated and tested. The pump was fabricated on both silicon and glass substrates using photolithography. The electrolysis electrodes were formed from either platinum or gold, and SU8, an epoxy-based photoresist, was used to form the pump chambers. A glass cover plate and a poly(dimethylsiloxane) (PDMS) gasket were used to seal the chambers. Filling of the chambers was accomplished by using a syringe to inject liquid via filling ports, which were later sealed using a glass cover plate. The current supplied to the electrodes controlled the rate of gas formation and, thus, the resulting fluid flow rate. At low backpressures, flow rates >1 microL/min have been demonstrated using <1 mW of power. Pumping at backpressures as high as 200 psi have been demonstrated, with 20 nL/min having been observed using <4 mW. By integrating two electrochemical pumps with a polymer electrospray nozzle, we have confirmed the successful generation of a solvent gradient via a mass spectrometer.

We present an experimental study of the microfluidic electrophoresis of long DNA in self-assembling matrixes of magnetic bead columns. Results are presented for the rapid separation of lambda-phage, 2lambda-DNA, and bacteriophage T4 DNA, where separation resolutions greater than 2 between lambda and T4 are achieved in times as short as 150 s. The use of a computer-piloted flow control system and injection results in high reproducibility between separations. We compare the experimentally measured mobility and dispersion with an exactly solvable lattice Monte Carlo model. The theory predicts that the mean velocity scales linearly with the field, the band broadening scales with the inverse of the field, and the resolution is independent of the field for intermediate fields-all of which are in accord with the experimental results. Moreover, reasonable quantitative agreement is achieved for band broadening for longer DNA (2lambda and T4) when the average postengagement time is measured experimentally. This work demonstrates the possibility of achieving fast microfluidic separation of large DNA on a routine basis.

For miniaturization and integration of chemical synthesis and analytics on small length scales, the development of complex lab-on-chip (LOC) systems is in the focus of many current research projects. While application specific synthesis and analytic modules and LOC devices are widely described, the combination and integration of different modules is intensively investigated. Problems for in-line processes such as solvent incompatibilities, e.g., for a multistep synthesis or the combination of an organic drug synthesis with a cellbased biological activity testing system, require a solvent exchange between serialized modules. Here, we present a continuously operating microfluidic solvent exchanger based on the principle of free-flow electrophoresis for miscible organic/ aqueous fluids. We highlight a proof-of-principle and describe the working principle for the model compound fluorescein, where the organic solvent DMSO is exchanged against an aqueous buffer. The DMSO removal performance could be significantly increased to 95% by optimization of the microfluidic layout. Moreover, the optimization of the inlet flow ratio resulted in a minimized dilution factor of 5, and we were able to demonstrate that a reduction of the supporting instrumentation is possible without a significant decrease of the DMSO removal performance. Finally, the compatibility of the developed solvent exchanger for cell based downstream applications was proven. The impedimetric monitoring of HEK293A cells in a continuously operating microfluidic setup revealed no adverse effects of the residual DMSO after the solvent replacement. Our solvent exchanger device demonstrates the power of micro-free-flow electrophoresis not only as a powerful technique for separation and purification of compound mixtures but also for solvent replacement. [ABSTRACT FROM AUTHOR]

While protein electrophoresis conducted in capillaries and microchannels offers high-resolution separations, such formats can be cumbersome to parallelize for single-cell analysis. One approach for realizing large numbers of concurrent separations is open microfluidics (i.e., no microchannels). In an open microfluidic device adapted for single-cell electrophoresis, we perform 100s to 1000s of simultaneous separations of endogenous proteins. The microscope slide-sized device contains cells isolated in microwells located in a ~40 µm polyacrylamide gel. The gel supports protein electrophoresis after concurrent in situ chemical lysis of each isolated cell. During electrophoresis, Joule (or resistive) heating degrades separation performance. Joule heating effects are expected to be acute in open microfluidic devices, where a single, high-conductivity buffer expedites the transition from cell lysis to protein electrophoresis. Here, we test three key assertions. First, Joule heating substantially impacts analytical sensitivity due to diffusive losses of protein out of the open microfluidic electrophoretic (EP) cytometry device. Second, increased analyte diffusivity due to autothermal runaway Joule heating is a dominant mechanism that reduces separation resolution in EP cytometry. Finally, buffer exchange reduces diffusive losses and band broadening, even when handling single-cell lysate protein concentrations in an open device. We develop numerical simulations of Joule heating-enhanced diffusion during electrophoresis and observe ~50% protein loss out of the gel, which is reduced using the buffer exchange. Informed by analytical model predictions of separation resolution (with Joule heating), we empirically demonstrate nearly fully resolved separations of proteins with molecular mass differences of just 4 kDa or 12% (GAPDH, 36 kDa; PS6, 32 kDa) in each of 129 single cells. The attained separation performance with buffer exchange is relevant to detection of currently unmeasurable protein isoforms responsible for cancer progression. [ABSTRACT FROM AUTHOR]

A breadboard approach for electrophoretic separations with contactless conductivity detection is presented. This is based on miniature off-the-shelf components such as syringe pumps, valves, and pressure controllers which could be set up in a very compact overall arrangement. It has a high flexibility for different tasks at hand, and the common operations of hydrodynamic injection and capillary flushing are automated. For demonstration of the versatility of the proposition, several very diverse configurations and modes of electrophoresis were successfully implemented, namely, standard capillary zone electrophoresis, pressure assisted zone electrophoresis, the simultaneous separation of cations and anions by dual-capillary zone electrophoresis, the separation of cationic amino acids by isotachophoresis, as well as the separation of small carboxylic acids by gradient elution moving boundary electrophoresis. The system also allows fast separations, as demonstrated by the analysis of six inorganic cations within 35 s. The approach addresses respective limitations of either conventional capillary electrophoresis instruments as well as electrophoretic lab-on-chip devices, while maintaining a performance in terms of detection limits and reproducibility comparable to standard instrumentation. [ABSTRACT FROM AUTHOR]

We describe a microfluidic Western blot assay (μWestern) using a Tris tricine discontinuous buffer system suitable for analyses of a wide molecular mass range (6.5-116 kDa). The Tris tricine μWestern is completed in an enclosed, straight glass microfluidic channel housing a photopattemed polyacrylamide gel that incorporates a photoactive benzophenone methacrylamide monomer. Upon brief ultraviolet (UV) light exposure, the hydrogel toggles from molecular sieving for size-based separation to a covalent immobilization scaffold for in situ antibody probing. Electrophoresis controls all assay stages, affording purely electronic operation with no pumps or valves needed for fluid control. Electrophoretic introduction of antibody into and along the molecular sieving gel requires that die probe must traverse through (i) a discontinuous gel interface central to the transient isotachophoresis used to achieve high-performance separations and (ii) the full axial length of the separation gel. In-channel antibody probing of small molecular mass species is especially challenging, since the gel must effectively sieve small proteins while permitting effective probing with large-molecularmass antibodies. To create a well-controlled gel interface, we introduce a fabrication method that relies on a hydrostatic pressure mismatch between the buffer and polymer precursor solution to eliminate the interfacial pore-size control issues that arise when a polymerizing polymer abuts a nonpolymerizing polymer solution. Combined with a new swept antibody probe plug delivery scheme, the Tris tricine μWestern blot enables 40% higher separation resolution as compared to a Tris glycine system, destacking of proteins down to 6.5 kDa, and a 100-fold better signal-to-noise ratio (SNR) for small pore gels, expanding the range of applicable biological targets. [ABSTRACT FROM AUTHOR]

Herein, the fabrication, characterization, calibration, and application of integrated microfluidic platforms for fast isoelectric point (pI) determinations via free-flow electrophoresis with integrated inkjet-printed fluorescent pH sensor microstructures are presented. These devices allow one to determine the pI of a biomolecule from a sample mixture with moderately good precision and without addition of markers in typically less than 10 s total separation and analysis time. Polyhydroxyethyl methacrylate (pHEMA) hydrogels were covalently coupled with fluorescein and hydroxypyrene trisulfonic acid (HPTS)-based pH probes. These were piezoelectrically jet-dispensed onto acrylate-modified glass as pH sensor microarrays with a diameter of 300-600 µm and thicknesses of 0.4-2.4 µm with high spatial accuracy. Microchip fabrication and integration of these pH sensor arrays was realized by multistep liquid-phase photolithography from oligoethylene glycol precursors resulting in glass-based microfluidic free-flowisoelectric focusing (μFFIEF) chips with integrated pH observation capabilities. The microchips were characterized with regard to pH sensitivity, response times, photo-, and flow stability. Depending on the sensor matrix, they allowed IEF within a pH range of roughly 5.5-10.5 with good sensitivity and fast response times. These microchips were used for FFIEF of small molecule markers and several protein mixtures with simultaneous monitoring of local pH. This allowed the determination of their pI via multispectral imaging of protein and pH sensor fluorescence without addition of external markers. Obtained pI's were generally in good agreement with known data, demonstrating the applicability of the method for pI determination in micropreparative procedures within a time frame of a few seconds only. [ABSTRACT FROM AUTHOR]

In this paper, we evaluate the strategy of using self-assembled microbeads to build a robust and tunable membrane for free-flow zone electrophoresis in a PDMS microfluidic chip. To fabricate a porous membrane as a salt bridge for free-flow zone electrophoresis, we used silica or polystyrene microbeads between 3--6 μm in diameter and packed them inside a microchannel. After complete evaporation, we infiltrated the porous microbead structure with a positively or negatively charged hydrogel to modify its surface charge polarity. Using this device, we demonstrated binary sorting (separation of positive and negative species at a gfven pH) of peptides and dyes in standard buffer systems without using sheath flows. The sample loss during sorting could be mininuzed by using ion selectivity of hydVogel-iniiltrated microbead membranes. Our fabrication method enables building a robust membrane for pressure-driven free-flow zone electrophoresis with tunable pore size as well as surface charge polarity. [ABSTRACT FROM AUTHOR]

Isoelectric focusing (IEF) is the first step for two-dimensional (2D) gel electrophoresis and plays an important role in sample purification for proteomics. However, biases in protein size and p/ resolution, as well as limitations in sample volume, gel capacity, sample loss, and experimental time, remain challenges. In order to address some of the limitations of traditional IEF, we present a microfluidic free flow IEF (FF-IEF) device for continuous protein separation into 24 fractions. The device reproducibly establishes a nearly linear pH gradient from 4 to 10. Optimized dynamic coatings of 4% poly(vinyl alcohol) (PVA) minimize peak broadening by transverse electrokinetic flows. Even though the device operates at high electric fields (up to 370 V/cm), efficient cooling maintains solution temperature inside the separation channel controllably in the range 2-25 °C. Protein samples with a dynamic concentration range from μg/mL to mg/mL can be loaded into the microdevice at a flow rate of 1 mL/h and residence time of ∼12 min. By using a protein complex of nine proteins and 13 isoforms, we demonstrate improved separation with the FF-IEF system over traditional 2D gel electrophoresis. Device-to-device reproducibility is also illustrated through the efficient depletion of the albumin and hemoglobin assays. Post-device sample concentrations result in a 10-20-fold increase, which allow for isolation and detection of low abundance proteins. The separation of specific proteins from a whole cell lysate is demonstrated as an example. The microdevice has the further benefits of retaining high molecular weight proteins, providing higher yield of protein that has a broader range in pi, and reducing experimental time compared to conventional IEF IGP gel strip approaches. [ABSTRACT FROM AUTHOR]

Sample-in answer-out analytical tools remain the goal of much lab on a chip research, but miniaturized methods capable of examining minimally prepared samples have proven elusive. Complex samples, including whole milk, various types of dirt and leaves, coal fly ash, and blood serum, were analyzed quantitatively for dissolved potassium, calcium, sodium, maesium, lithium,and melamine using gradient elution moving boundary electrophoresis (GEMBE) and contactless conductivity detection with the single preparatory step of dilution or suspension in sample buffer. GEMBE is a simple, robust analytical technique, well-suited to microfluidic analysis of complex samples containing material, such as particulates or proteins, that would confound the majority of other microfluidic techniques. GEMBE utilizes electrophoretic flow to drive electrically charged analytes into a microfluidic channel or capillary for detection, while opposing electro-osmotic and variable pressure-driven flows prevent the remainder of the sample from entering the channel. Contactiess conductivity detection further simplifies device construction and operation, positioning GEMBE for inexpensive and facile multiplexed implementation outside laboratory settings. [ABSTRACT FROM AUTHOR]

A method for simultaneously concentrating and separating analytes in a buffer-filled microfluidic channel is reported. The approach is based on modulation of the local electric field within the channel and the corresponding opposition of electrophoretic and electroosmotic flow (EOF) velocities. Dye molecules having different electrophoretic mobilities are focused at different locations within the channel where concentration takes place. At least three species, all small dye molecules, can be simultaneously concentrated and separated, with localized enrichment factors up to ∼600 achieved within 400 s. The enrichment mnes affect the electric field profile, as evidenced by significant differences in focusing of single versus multiple analytes. The EOF could be modulated by modifying the channel walls with an appropriate polymer, and this had the effect of increasing both the enrichment factors and resolution of the separation. Numerical simulations provide insights into the underlying fundamental principles for the experimental findings. [ABSTRACT FROM AUTHOR]

The performance of microfluidic glass devices with a monolithically integrated nanospray tip have been evaluated. The nanospray tip is generated directly on the edge of a microfluidic glass chip by a pulling step followed by HF-etching for directed formation of a sharp tip with defined emitter area. For a fair judgment of the MS- detection sensitivity, we compared the detection performance with commercial nanospray needles. For that purpose the effect of the emitter opening on the sensitivity was studied in detail for different microfluidic chips as well as for commercial nanospray needles. A comparison of the chip-nanospray device with commercial nanospray needles revealed that a comparable spray performance is obtained at similar emitter diameters. A stable electrospray could be generated at such tapered tips without any need for hydrodynamic or electroosmotic pumping. The nanospray chips were successfully applied for coupling microchip electrophoresis and mass spectrometry. For improved performance, the separation channel of the microdevice was flushed with hydroxypropylmethylcellulose (HPMC) in order to reduce analyte-wall interactions and electroosmotic flow. [ABSTRACT FROM AUTHOR]

A new microfluidic electrophoresis device and technique is described that is designed specifically for multiplexed, high-throughput separations. The device consists of an array of short (3 mm) capillaries connecting individual sample reservoirs to a common buffer reservoir. Each capillary in the array functions as both a separation channel and as a conductivity-based detection cell. The new technique is based upon the recently described gradient elution moving boundary electrophoresis (GEM-BE) technique, which uses a combination of an electric field and buffer counterflow to achieve electrophoretic separations in short capillaries or microfluidic channels. A high voltage drives electrophoresis of the sample analytes through each separation channel. At the start of a separation, the bulk counterflow of buffer through the channel is high, and none of the analytes of interest can enter the channel. The counterflow is then gradually reduced until each analyte, in turn, is able to enter the channel where it is detected as a moving boundary or step. With very short capillaries, only one step at a time is present in each capillary, and the electric current through the channels can then be used as the detector signal, without any extra detector hardware. The current vs time signal for each channel is then smoothed and differentiated to produce a set of simultaneous electropherograms. Because there is no light source or other added hardware required for detection, the system is simple and can be easily and inexpensively scaled up to perform large numbers of simultaneous analyses. As a first demonstration, a 16-channel array device is used for high-throughput, time-series measurements of enzyme activity and inhibition. [ABSTRACT FROM AUTHOR]

Here we describe the fabrication, optimization, and application of a microfluidic device that integrates microdialysis (MD) sampling, microchip electrophoresis (ME), and electrochemical detection (EC). The manner in which the chip is produced is reproducible and enables the fixed alignment of the MD/ME and ME/EC interfaces. Poly(dimethylsioxane) (PDMS)-based valves were used for the discrete injection of sample from the hydrodynamic MD dialysate stream into a separation channel for analysis with ME. To enable the integration of ME with EC detection, a palladium decoupler was used to isolate the high voltages associated with electrophoresis from micrometer-sized carbon ink detection electrodes. Optimization of the ME/EC interface was needed to allow the use of biologically appropriate perfusate buffers containing high salt content. This optimization included changes in the fabrication procedure, increases in the decoupler surface area, and a programmed voltage shutoff. The ability of the MD/ME/EC system to sample a biological system was demonstrated by using a linear probe to monitor the stimulated release of dopamine from a confluent layer of PC 12 cells. To our knowledge, this is the first report of a microchip-based system that couples microdialysis sampling with microchip electrophoresis and electrochemical detection. [ABSTRACT FROM AUTHOR]

A method for sampling and electrophoretic analysis of aqueous plugs segmented in a stream of immiscible oil is described. In the method, an aqueous buffer and oil stream flow parallel to each other to form a stable virtual wall in a microfabricated K-shaped fluidic element. As aqueous sample plugs in the oil stream make contact with the virtual wall, coalescence occurs and sample is electrokinetically transferred to the aqueous stream. Using this virtual wall, two methods of injection for channel electrophoresis were developed. In the first, discrete sample zones flow past the inlet of an electrophoresis channel and a portion is injected by electroosmotic flow, termed the "discrete injector". With this approach at least 800 plugs could be injected without interruption from a continuous segmented stream with 5.1% RSD in peak area. This method generated up to 1,050 theoretical plates, although analysis of the injector suggested that improvements may be possible. In a second method, aqueous plugs are sampled in a way that allows them to form a continuous stream that is directed to a microfluidic cross-style injector, termed the "desegmenting injector". This method does not analyze each individual plug but instead allows periodic sampling of a high-frequency stream of plugs. Using this system at least 1000 injections could be performed sequentially with 5.8% RSD in peak area and 53,500 theoretical plates. This method was demonstrated to be useful for monitoring concentration changes from a sampling device with 10 s temporal resolution. Aqueous plugs in segmented flows have been applied to many different chemical manipulations including synthesis, assays, sampling processing and sampling. Nearly all such studies have used optical methods to analyze plug contents. This method offers a new way to analyze such samples and should enable new applications of segmented flow systems. [ABSTRACT FROM AUTHOR]

We have applied multiple-time-point reaction mapping to generate high-dynamic-range quantitative data from PCR multiplexes. The approach measures, then compensates, numerous PCR slope nonidealities across the multiplex without prejudice. A multilane microelectophoresis device with a novel scanning detector that reports redundantly over more than six decades in signal strength was used to collect data with multiple readings for each amplification point and with double internal calibration (lane standards and gene standards). We investigated scaling properties and sensitivity for readout of l2plex PCR reactions. The sensitive detection, stemming from confocal optics, allowed reduction of the PCR cycle number by approximately five cycles compared to commercial fluorometric readout. This increased sensitivity appears to allow quantitative PCR over a dynamic range of >9 log2 abundance ratio in multiplex reactions exceeding 20plexes. We argue that the combination of mapping, multiplexing, and an internal standard, improves the per-well efficiency of quantitative expression analysis by a factor of 50-100 relative to fluorometric qPCR readout. Therefore, the approach is attractive for analysis of large gene networks at reduced cost. [ABSTRACT FROM AUTHOR]

A new method for performing continuous electrophoretic separation of complex mixtures in microscale devices is proposed. Unlike in free-flow electrophoresis devices, no mechanical pumping is required-both fluid transport and separation are driven electrokinetically. This gives the method great potential for on-a-chip integration in multistep analytical systems. The method enables us to collect fractionated sample and tensfold purification is possible. The model of the operation is presented and a detailed description of the optimal conditions for performing purification is given. The chip devices with 10-μm-deep separation chamber of 1.5 mm × 4 mm in size were fabricated in glass. A standard microchip electrophoresis setup was used. Continuous separation of rhodamine B, rhodamine 6G, and fluorescein was accomplished. Purification was demonstrated on a mixture containing rhodamine B and fluorescein, and the recovery of both fractions was achieved. The results show the feasibility of the method. [ABSTRACT FROM AUTHOR]

Acoustophoresis in microfluidic structures has primarily been reported in silicon microfabricated devices. This paper demonstrates, for the first time, acoustophoresis performed in isotropically etched glass chips providing a performance that matches that of the corresponding silicon microdevices. The resonance mode characteristics of the glass chip were equal to those of the silicon chip at its fundamental resonance. At higher order resonance modes the glass chip displays resonances at lower frequencies than the silicon chip. The cross-sectional profiles of acoustically focused particle streams are also reported for the first time, displaying particles confined in a vertical band in the channel center for both glass and silicon chips. A particle extraction efficiency of 98% at flow rates up to 200 μL/min (2% particle concentration) is reported for the glass chip at the fundamental resonance. The glass and silicon chips displayed equal particle extraction performance when tested for increasing particle concentrations of 2-15%, at a flow velocity of 12.9 cm/s for the glass chip and 14.8 cm/s for the silicon chip. [ABSTRACT FROM AUTHOR]

A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 x 7.62 cm2 glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial charge-coupled device (CCD) camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis—Menten constants. A chip with 36 channels was used for screening for modulators of the G protein—RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. Thirty-six electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4320 assays/h with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11%, respectively, in the 16- and 36-channel designs. [ABSTRACT FROM AUTHOR]

A novel microfluidic device with an array of analytical chambers was developed in order to perform single-cell-based gene-function analysis. A series of analytical processes was carried out using the device, including electrophoretic manipulation of single cells and electrochemical measurement of gene function. A poly(dimethylsioxane) microstructure with a microfluidic channel (150 μm in width, 10 μm in height) and an analytical chamber (100 × 20 × 10 μm³) were fabricated and aligned on a glass substrate with an array of Au microelectrodes. Two microelectrodes positioned in the analytical chamber were employed as a working electrode for the electrophoretic manipulation of cells and electrochemical measurements. A yeast strain (Saccharomyces cerevisiae Y190) carrying the β-galactosidase reporter gene was used to demonstrate that the device could detect the enzyme. Target cells flowing through the main channel were introduced into the chamber by electrophoresis using the ground electrode laid on the main channel. When the cell was treated with 17β-estradiol, gene expression was triggered to produce β-galactosidase, catalyzing the hydrolysis of β-aminophenyl-β-D-galactopyranoside to form p-aminophenol (PAP). The enzymatically generated PAP was detected by cyclic voltammetry and amperometry at the single-cell level in the chamber of the device. Generator- collector mode amperometry was also applied to amplify the current response originating from gene expression in the trapped single cells. After electrochemical measurement, the trapped cells were easily released from the chamber using electrophoretic force. [ABSTRACT FROM AUTHOR]

Methods for fabricating poly(methyl methacrylate) microchips using a novel two-stage embossing technique and solvent welding to form microchannels in microfluidic devices are presented. The hot embossing method involves a two-stage process to create the final microchip design. In its simplest form, a mold made of aluminum is fabricated using CNC machining to create the desired microchannel design. In this work, two polymer substrates with different glass transition temperatures (Tg), polyetherimide (PEI) and poly(methyl methacrylate) (PMMA), were used to make the reusable secondary master and the final chip. First, the aluminum mold was used to emboss the PEI, a polymeric substrate with Tg ~ 216 °C. The embossed PEI was then used as a secondary mold for embossing PMMA, a polymeric substrate with a lower Tg (~105 °C). The resulting PMMA substrate possessed the same features as those of the aluminum mold. Successful feature transfer from the aluminum mold to the PMMA substrate was verified by profilometry. Bonding of the embossed layer and a blank PMMA layer to generate the microchip was achieved by solvent welding. The embossed piece was first filled with water that formed a solid sacrificial layer when frozen. The ice layer prevented channel deformation when the welding solvent (dichloroethane) was applied between the two chips during bonding. Electrophoretic separations of fluorescent dyes, rhodamine B (Rh B) and fluorescein (FL), were performed on PMMA microchips to demonstrate the feasibility of the fabrication process for microreplication of useful devices for separations. The PMMA micro- chip was tested under an electric field strength of 705 V cm-1. Separations of the test mixture of Rh B and FL generated 55 500 and 66 300 theoretical plates/meter, respectively. [ABSTRACT FROM AUTHOR]

We have studied the effects of polymer molar mass and concentration on the electrophoretic migration modalities of individual molecules of DNA in LPA, HEC, and PEO solutions via epifluorescent videomicroscopy. While both transient entanglement coupling (TEC) and reptation have been studied in the past, the transition between them has not. Understanding this transition will allow for polymer network properties to be optimized to enhance the speed and resolution of DNA separations in microfluidic devices. Near the overlap threshold concentration, C*, TEC is the dominant observed mode of DNA migration, and the observation frequency of TEC increases with increasing polymer molar mass. As polymer concentration is increased, observed TEC events reduce to zero while DNA reptation events become the only detected mechanism. Individual DNA molecules undergoing both migration mechanisms were counted in solutions of varying polymer molar masses and concentrations and were plotted against a dimensionless polymer concentration, C/C*. The data for LPA reduce to form universal curves with a sharp increase in DNA reptation at ∼6.5C*. Analogous transition concentrations for PEO and HEC were observed at 5C* and 3.5C*, respectively, reflecting the different physical properties of these polymers. This transition correlates closely with the polymer network entanglement concentration, Ce, as measured by rheological techniques. The electrophoretic mobility of 2-DNA in LPA polymer solutions was also measured and shows how a balance can be struck between DNA resolution and separation speed by choosing the desired prevalence of DNA reptation. [ABSTRACT FROM AUTHOR]

We report rapid and efficient electrophoretic separations of N-glycans on microfluidic devices. Using a separation length of 22 cm and an electric field strength of 750 V/cm, analysis times were less than 3 mm, and separation efficiencies were between 400 000 and 655 000 plates for the N-glycans and up to 960 000 plates for other sample components. These high efficiencies were necessary to separate N-glycan positional isomers derived from ribonuclease B and linkage isomers from asialofetuin. Structural isomers of N-glycans derived from a blood serum sample of a cancer patient were also analyzed to demonstrate that clinically relevant, complex samples could be separated on-chip with efficiencies similar to those derived from model glycoproteins. In addition, we compared microchip and capillary electrophoresis under similar separation conditions, and the microchips performed as well as the capillaries. These results confirmed that the noncircular cross section of the microchannel did not hamper separation performance For all experiments, the glycan samples were derivatized with 8-aminopyrene- 1,3,6-trisullonic acid to impart needed charge for electrophoresis and a fluorescent label for detection. [ABSTRACT FROM AUTHOR]

This paper demonstrates for the first time the use of adaptive functional nickel nanowires for switching on-demand operation of microfluidic devices. Controlled reversible magnetic positioning and orientation of these nanowires at the microchannel outlet offers modulation of the detection and separation processes, respectively. The former facilitates switching between active and passive detection states to allow the microchip to be periodically activated to perform a measurement and reset it to the passive (‘off’) state between measurements. Fine magnetic tuning of the separation process (postchannel broadening of the analyte zone) is achieved by reversibly modulating the nanowire orientation (i.e., detector alignment) at the channel outlet. The concept can be extended to other microchip functions and stimuli-responsive materials and holds great promise for regulating the operation of microfluidic devices in reaction to specific needs or unforeseen scenarios. [ABSTRACT FROM AUTHOR]