Bilinear temperature gradient focusing in a hybrid PDMS/glass microfluidic chip integrated with planar heaters for generating temperature gradients

Temperature gradient focusing (TGF) is a counterflow gradient focusing technique, which utilizes a temperature gradient across a microchannel or capillary to separate analytes. With an appropriate buffer, the temperature gradient creates a gradient in both the electric field and electrophoretic velocity. Combined with a bulk counter flow, ionic species concentrate at a unique point where the total velocity sums to zero and separate from each other. Scanning TGF uses varying bulk flow so that a large number of analytes that have large differences in electrophoretic mobility can be sequentially focused and passed by a single detection point. Up to now, scanning TGF examples have been performed using a linear temperature gradient which has limitations in improving peak capacity and resolution at the same time. In this work, we develop a bilinear temperature gradient along the separation channel that improves both peak capacity and separation resolution simultaneously. The temperature profile along the channel consists of a very sharp gradient used to preconcentrate the sample followed by a shallow gradient that increases separation resolution. A specialized design is developed for the heaters to achieve the bilinear profile using both analytical and numerical modeling. The heaters are integrated onto a hybrid PDMS/glass chip fabricated using conventional sputtering and soft-lithography techniques. Separation performance is characterized by separating several different dyes and amino acids that have close electrophoretic mobilities. Experiments show a dramatic improvement in peak capacity and resolution in comparison to the standard linear temperature gradient.     

Integration of isoelectric focusing with parallel sodium dodecyl sulfate gel electrophoresis for multidimensional protein separations in a plastic microfluidic [correction of microfludic] network

An integrated protein concentration/separation system, combining non-native isoelectric focusing (IEF) with sodium dodecyl sulfate (SDS) gel electrophoresis on a polymer microfluidic chip, is reported. The system provides significant analyte concentration and extremely high resolving power for separated protein mixtures. The ability to introduce and isolate multiple separation media in a plastic microfluidic network is one of two key requirements for achieving multidimensional protein separations. The second requirement lies in the quantitative transfer of focused proteins from the first to second separation dimensions without significant loss in the resolution acquired from the first dimension. Rather than sequentially sampling protein analytes eluted from IEF, focused proteins are electrokinetically transferred into an array of orthogonal microchannels and further resolved by SDS gel electrophoresis in a parallel and high-throughput format. Resolved protein analytes are monitored using noncovalent, environment-sensitive, fluorescent probes such as Sypro Red. In comparison with covalently labeling proteins, the use of Sypro staining during electrophoretic separations not only presents a generic detection approach for the analysis of complex protein mixtures such as cell lysates but also avoids additional introduction of protein microheterogeneity as the result of labeling reaction. A comprehensive 2-D protein separation is completed in less than 10 min with an overall peak capacity of approximately 1700 using a chip with planar dimensions of as small as 2 cm x 3 cm. Significant enhancement in the peak capacity can be realized by simply raising the density of microchannels in the array, thereby increasing the number of IEF fractions further analyzed in the size-based separation dimension.     

Tandem isotachophoresis-zone electrophoresis via base-mediated destacking for increased detection sensitivity in microfluidic systems

Electrophoresis in microfluidic devices is becoming a useful analytical platform for a variety of biological assays. In this report, we present a method that allows for an increased sensitivity of detection of fluorescent molecules in microfluidic electrophoresis devices. This capability is provided by the implementation of a particular buffer system that is designed to initially function in an isotachophoretic (ITP) mode and, then after a controlled amount of electric current has been applied to the system, to transition to a zone electrophoretic mode. In the initial ITP mode, analytes dissolved in a large volume of injected sample are concentrated into a single narrow zone. After application of a sufficient and adjustable amount of electric current, the system switches into a zone electrophoretic mode, where the concentrated analytes are separated according to their electrophoretic mobilities. Application of this tandem ITP-zone electrophoretic strategy to the concentration, separation, and detection of fluorescent reporter molecules in a standard microfluidic device results in an approximately 50-fold increase in detection sensitivity relative to equivalent separations that are obtained with zone electrophoresis alone. Even with very long initial sample plugs (up to 3000 microm), this strategy produces electrophoretic separations with high resolutions and peak efficiencies. This strategy can be implemented to increase detection sensitivity in any standard microfluidic electrophoresis platform and does not require any specialized hardware or microchannel configurations.     

Gradient elution moving boundary electrophoresis for high-throughput multiplexed microfluidic devices

A novel method for performing electrophoretic separations is described-gradient elution moving boundary electrophoresis (GEMBE). The technique utilizes the electrophoretic migration of chemical species in combination with variable hydrodynamic bulk counterflow of the solution through a separation capillary or microfluidic channel. Continuous sample introduction is used, eliminating the need for a sample injection mechanism. Only analytes with an electrophoretic velocity greater than the counterflow velocity enter the separation channel. The counterflow velocity is varied over time so that each analyte is brought into the separation column at different times, allowing for high-resolution separations in very short channels. The new variable of bulk flow acceleration affords a new selectivity parameter to electrophoresis analogous to gradient elution compositions in chromatography. Because it does not require extra channels or access ports to form an injection zone and because separations can be performed in very short channels, GEMBE separations can be implemented in much smaller areas on a micro-fluidic chip as compared to conventional capillary electrophoresis. Demonstrations of GEMBE separations of small dye molecules, amino acids, DNA, and immunoassay products are presented. A low-cost, polymeric, eight-channel multiplexed microfluidic device was fabricated to demonstrate the reduced area requirements of GEMBE; the device was less than 1 in.2 in area and required only n + 1 fluidic access ports per n analyses (in this instance, nine ports for eight analyses). Parallel separations of fluorescein and carboxyfluorescein yielded less than 3% relative standard deviation (RSD) in interchannel migration times and less than 5% RSD in both peak and height measurements. The device was also used to generate a calibration curve for a homogeneous insulin immunoassay using each of the eight channels as a calibration point with less than 5% RSD at each point with replicate analyses.     

Probing electric fields inside microfluidic channels during electroosmotic flow with fast-scan cyclic voltammetry

Fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes was used in microfluidic channels. This method offers the advantage that it can resolve electroactive species not separated in the channel. In addition, this method provides a route to investigate the distribution of applied electrophoretic fields in microfluidic channels. To probe this, microelectrodes were inserted at various distances into channels and cyclic voltammograms recorded at 300 V/s were repeated at 0.1-s intervals. The use of a battery-powered laptop computer and potentiostat provided galvanic isolation between the applied electrophoretic field and the electrochemical measurements. In the absence of an external field, the peak potential for oxidation of the test solute, Ru(bpy)3(2+), was virtually unaltered by insertion of the microelectrode tip into the channel. When an electrophoretic field was applied, the peak potential for Ru(bpy)3(2+) oxidation shifted to more positive potentials in a manner that was directly proportional to the field in the channel. The shifts in peak potential observed with FSCV enabled direct compensation of the applied electrochemical potential. This approach was used to explore the electrophoretic field at the channel terminus. It was found to persist for more than 50 microm from the channel terminus. In addition, the degree of analyte dispersion was found to depend critically on the electrode position outside the channel.     

Simultaneous concentration and separation of enantiomers with chiral temperature gradient focusing

A new technique is demonstrated for the simultaneous concentration and high-resolution separation of chiral compounds. With temperature gradient focusing, a combination of a temperature gradient, an applied electric field, and a buffer with a temperature-dependent ionic strength is used to cause analytes to move to equilibrium, zero-velocity points along a microchannel or capillary. Different analytes are thus separated spatially and concentrated in a manner that resembles isoelectric focusing but that is applicable to a greater variety of analytes including small chiral drug molecules. Chiral separations are accomplished by the addition of a chiral selector, which causes the different enantiomers of an analyte to focus at different positions along a microchannel or capillary. This new technique is demonstrated to provide high performance in a number of areas desirable for chiral separations including rapid separation optimization and method development, facile reversal of peak order (desirable for analysis of trace enantiomeric impurities), and high resolving power (comparable to capillary electrophoresis) in combination with greater than 1000-fold concentration enhancement enabling improved detection limits. In addition, chiral temperature gradient focusing allows for real-time monitoring of the interaction of chiral analyte molecules with chiral selectors that could potentially be applied to the study of other molecular interactions. Finally, unlike CE, which requires long channels or capillaries for high-resolution separations, separations of equivalent resolution can be performed with TGF in very short microchannels (mm); thus, TGF is inherently much more suited to miniaturization and integration into lab-on-a-chip-devices.     

Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system

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 cm(2) 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.     

Integrated microfluidic system enabling protein digestion, peptide separation, and protein identification

An integrated platform is presented for rapid and sensitive protein identification by on-line protein digestion and analysis of digested proteins using electrospray ionization mass spectrometry or transient capillary isotachophoresis/capillary zone electrophoresis with mass spectrometry detection. A miniaturized membrane reactor is constructed by fabricating the microfluidic channels on a poly(dimethylsiloxane) substrate and coupling the microfluidics to a poly(vinylidene fluoride) porous membrane with the adsorbed trypsin. On the basis of he large surface area-to-volume ratio of porous membrane media, adsorbed trypsin onto the poly(vinylidene fluoride) membrane is employed for achieving ultrahigh catalytic turnover. The extent of protein digestion in a miniaturized membrane reactor can be directly controlled by the residence time of protein analytes inside the trypsin-adsorbed membrane, the reaction temperature, and the protein concentration. The resulting peptide mixtures can either be directly analyzed using electrospray ionization mass spectrometry or further concentrated and resolved by electrophoretic separations prior to the mass spectrometric analysis. This microfluidic system enables rapid identification of proteins in minutes instead of hours, consumes very little sample (nanogram or less), and provides on-line interface with upstream protein separation schemes for the analysis of complex protein mixtures such as cell lysates.     

Resolution of multiple ssDNA structures in free solution electrophoresis

By using high concentrations of buffer, electroosmotic flow within uncoated channels of a microfluidic chip was minimized, allowing the free solution electrophoretic separation of DNA. More importantly, because of the ability to efficiently dissipate heat within these channels, field strengths as high as 600 V/cm could be applied with minimal Joule heating (<2 degrees C). As a result of the higher field strengths, separations within an 8-cm-long channel were achieved within a few minutes. However, when the electrophoretic separation of single-stranded DNA (ssDNA) less than 22 bases in length was performed, containing the fluorophore Texas Red as an end label, more than the expected single peak was observed at this high electric field. On the other hand, the free solution electrophoresis of a double-stranded DNA (dsDNA) consisting of a random sequence did exhibit the expected single peak. The appearance of these multiple peaks for ssDNA is shown to be dependent upon the base content and sequence of the ssDNA as well as on the chemical structure of the fluorophore used to tag the DNA for detection. Specifically, the peaks can be attributed to different secondary structures that result either from hydrophobic interactions between the DNA bases and an uncharged fluorescent dye or from G-quadruplexes within guanine-rich strands.     

Immobilization of DNA hydrogel plugs in microfluidic channels

Acrylamide-modified DNA probes are immobilized in polycarbonate microfluidic channels via photopolymerization in a polyacrylamide matrix. The resulting polymeric, hydrogel plugs are porous under electrophoretic conditions and hybridize with fluorescently tagged complementary DNA. The double-stranded DNA can be chemically denatured, and the chip may be reused with a new analytical sample. Conditions for photopolymerization, hybridization, and denaturation are discussed. We also demonstrate the photopolymerization of plugs containing different DNA probe sequences in one microfluidic channel, thereby enabling the selective detection of multiple DNA targets in one electrophoretic pathway.     

A two-channel microfluidic sensor that uses anodic electrogenerated chemiluminescence as a photonic reporter of cathodic redox reactions

This paper describes a new approach for sensing electrochemically active substrates in microfluidic systems. This two-electrode sensor relies on electrochemical detection at one electrode and electrogenerated chemiluminescent (ECL) reporting at the other. Each microfabricated indium tin oxide electrode is located in a separate microfluidic channel, but the channels are connected downstream of the electrodes to maintain a complete electrical circuit. Because of laminar flow, there is no bulk mixing of the fluids in the detecting and reporting channels. This approach allows the ECL reaction to be physically and chemically decoupled from the sensing channel of the device, which greatly expands the number of analytes that can be detected. However, because the cathode and anode are connected, electron-transfer processes occurring at the sensing electrode are electrically coupled to the ECL reaction. Charge balance permits the ECL light output to be quantitatively correlated to electrochemical reductions at the cathode. The system is used to detect Fe(CN)6(3-), Ru(NH3)6(3+), and benzyl viologen and report their presence via Ru(bpy)3(2+) (bpy = bipyridine) luminescence. Each different redox target initiates ECL at a unique potential bias related to its standard redox potential. The influence of the concentrations of Ru(bpy)3(2+) and the target analytes is discussed.     

Development of a multichannel microfluidic analysis system employing affinity capillary electrophoresis for immunoassay

A six-channel microfluidic immunoassay device with a scanned fluorescence detection system is described. Six independent mixing, reaction, and separation manifolds are integrated within one microfluidic wafer, along with two optical alignment channels. The manifolds are operated simultaneously and data are acquired using a singlepoint fluorescence detector with a galvano-scanner to step between separation channels. A detection limit of 30 pM was obtained for fluorescein with the scanning detector, using a 7.1-Hz sampling rate for each of the reaction manifolds and alignment channels (57-Hz overall sampling rate). Simultaneous direct immunoassays for ovalbumin and for anti-estradiol were performed within the microfluidic device. Mixing, reaction, and separation could be performed within 60 s in all cases and within 30 s under optimized conditions. Simultaneous calibration and analysis could be performed with calibrant in several manifolds and sample in the other manifolds, allowing a complete immunoassay to be run within 30 s. Careful chip conditioning with methanol, water, and 0.1 M NaOH resulted in peak height RSD values of 3-8% (N = 5 or 6), allowing for cross-channel calibration. The limit of detection (LOD) for an anti-estradial assay obtained in any single channel was 4.3 nM. The LOD for the cross-channel calibration was 6.4 nM. Factors influencing chip and detection system design and performance are discussed in detail.     

Microfluidic biosensor based on an array of hydrogel-entrapped enzymes

Here we show that a microfluidic sensor based on an array of hydrogel-entrapped enzymes can be used to simultaneously detect different concentrations of the same analyte (glucose) or multiple analytes (glucose and galactose) in real time. The concentration of paraoxon, an acetylcholine esterase inhibitor, can be quantified using the same approach. The hydrogel micropatch arrays and the microfluidic systems are easy to fabricate, and the hydrogels provide a convenient, biocompatible matrix for the enzymes. Isolation of the micropatches within different microfluidic channels eliminates the possibility of cross talk between enzymes.     

Fluorescence correlation spectroscopy for rapid multicomponent analysis in a capillary electrophoresis system

We describe a new technique for performing multicomponent analysis using a combination of capillary electrophoresis (CE) and fluorescence correlation spectroscopy (FCS), which we refer to as CE/FCS. FCS is a highly sensitive and rapid optical technique that is often used to perform multicomponent analysis in static solutions based on the different diffusion times of the analyte species through the detection region of a tightly focused laser beam. In CE/FCS, transit times are measured for a mixture of analytes continuously flowing through a microcapillary in the presence of an electric field. Application of an electric field between the inlet and outlet of the capillary alters the transit times, depending on the magnitude and polarity of the applied field and the electrophoretic mobilities of the analytes. Multicomponent analysis is accomplished without the need to perform a chemical separation, due to the different electrophoretic mobilities of the analytes. This technique is particularly applicable to ultradilute solutions of analyte. We have used CE/FCS to analyze subnanomolar aqueous solutions containing mixtures of Rhodamine 6G (R6G) and R6G-labeled deoxycytosine triphosphate nucleotides. Under these conditions, fewer than two molecules were typically present in the detection region at a time. The relative concentrations of the analytes were determined with uncertainties of ～10%. Like diffusional FCS, this technique is highly sensitive and rapid. Concentration detection limits are below 10(-)(11) M, and analysis times are tens of seconds or less. However, CE/FCS does not require the diffusion coefficients of the analytes to be significantly different and can, therefore, be applied to multicomponent analysis of systems that would be difficult or impossible to study by diffusional FCS.     

Integrated nanopore/microchannel devices for ac electrokinetic trapping of particles

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.     

Temperature gradient focusing with field-amplified continuous sample injection for dual-stage analyte enrichment and separation

We describe the serial combination of temperature gradient focusing (TGF) and field-amplified continuous sample injection (FACSI) for improved analyte enrichment and electrophoretic separation. TGF is a counterflow equilibrium gradient method for the simultaneous concentration and separation of analytes. When TGF is implemented with a low conductivity sample buffer and a (relatively) high conductivity separation buffer, a form of sample enrichment similar to field-amplified sample stacking (FASS) or field-amplified sample injection (FASI) is achieved in addition to the normal TGF sample enrichment. FACSI-TGF differs from FASI in two important respects: continuous sample injection, versus a discrete injection, is utilized; because of the counterflow employed for TGF, the stacking interface exists in a pseudo-stationary region outside of the separation column. Notably, analyte concentration enrichment factors greater than the ratio of separation and sample conductivities (gamma) were achieved in this method. For gamma=6.1, the concentration factor for one model analyte (Oregon Green 488) was found to be 36-fold higher with FACSI-TGF as compared to TGF without FACSI. A separation of five fluorescently labeled amino acids is also demonstrated with the technique, yielding an average enrichment of greater than 1000-fold.     

Quenched phosphorescence detection in cyclodextrin-based electrokinetic chromatography

Quenched phosphorescence detection is a sensitive detection method recently introduced in capillary zone electrophoresis. It is based on the dynamic quenching interaction of the analytes (quenchers) with a phosphorophore, 1-bromo-4-naphthalenesulfonate (BrNS), present in the separation buffer. In this study, it is shown that this detection method can also be used in cyclodextrin-based electrokinetic chromatography (CD-EKC) despite the presence in the buffer solution of cyclodextrins, which are known to reduce the luminescence quenching rate constants. Experiments indicate that BrNS mainly resides in the aqueous phase, while the analytes are distributed between both phases. In principle, the observed quenching might arise from the interaction of BrNS with uncomplexed as well as complexed analytes. However, from the dependence of the fractional quenching on the capacity factor (the normalized fractional quenching was found to be equal to the fraction of analyte in the aqueous phase), it was concluded that only aqueous-phase quenching contributes significantly to the observed quenching. Nevertheless, separation and detection can be regarded as fully compatible, because the capacity factors encountered in CD-EKC are generally low (in this study they ranged from about 0.1 to 2.5). Indeed, with nitroaromatic compounds as the target analytes, limits of detection in the 10(-8) M range were achieved.     

Near-simultaneous and real-time detection of multiple analytes in affinity microcolumns

A miniaturized immunoassay system based on beads in poly(dimethylsiloxane) microchannels for analyzing multiple analytes has been developed. The method involves real-time detection of soluble molecules binding to receptor-bearing microspheres, sequestered in affinity column format inside a microfluidic channel. Identification and quantitation of analytes occurs via direct fluorescence measurements or fluorescence resonance energy transfer. A preliminary account of this work based on single-analyte format has been published in this journal (Buranda, T.; Huang, J.; Perez-Luna, V. H.; Schreyer, B.; Sklar, L. A.; Lopez, G. P. Anal. Chem. 2002, 74, 1149-1156). We have extended the work to a multianalyte model system composed of discrete segments of beads that bear distinct receptors. Near-simultaneous and real-time detection of diverse analytes is demonstrated. The importance of this work is established in the exploration of important factors related to the design, assessment, and utility of affinity microcolumn sensors. First, beads derivatized with surface chemistry suitable for the attachment of fluorescently labeled biomolecules of interest are prepared and characterized in terms of functionality and receptor site densities by flow cytometry. Second, calibrated beads are incorporated in microfluidic channels. The analytical device that emerges replicates the basic elements of affinity chromatography with the advantages of microscale and real-time direct measurement of bound analyte on beads rather than the indirect determination from eluted sample typical of affinity chromatography. In addition, the two-compartment analysis of the assay data as demonstrated in single-analyte columns provides a template upon which the dynamics of multiple-analyte assays can be characterized using existing theoretical models and be tested experimentally. The assay can potentially detect subfemtomole quantities of protein with high signal-to-noise ratio and a large dynamic range spanning nearly 4 orders of magnitude in analyte concentration in microliter to submicroliter volumes of analyte fluid. The approach has the potential to be generalized to a host of bioaffinity assay methods including analysis of protein complexes (e.g., biomolecular indicators of diseases). Proof-of-principle analytes include FLAG peptide and carcinoembryonic antigen detected at physiologically relevant concentration levels.     

Microfluidic high-resolution free-flow isoelectric focusing

A microfluidic free-flow isoelectric focusing glass chip for separation of proteins is described. Free-flow isoelectric focusing is demonstrated with a set of fluorescent standards covering a wide range of isoelectric points from pH 3 to 10 as well as the protein HSA. With respect to an earlier developed device, an improved microfluidic FFE chip was developed. The improvements included the usage of multiple sheath flows and the introduction of preseparated ampholytes. Preseparated ampholytes are commonly used in large-scale conventional free-flow isoelectric focusing instruments but have not been used in micromachined devices yet. Furthermore, the channel depth was further decreased. These adaptations led to a higher separation resolution and peak capacity, which were not achieved with previously published free-flow isoelectric focusing chips. An almost linear pH gradient ranging from pH 2.5 to 11.5 between 1.2 and 2 mm wide was generated. Seven isoelectric focusing markers were successfully and clearly separated within a residence time of 2.5 s and an electrical field of 20 V mm-1. Experiments with pI markers proved that the device is fully capable of separating analytes with a minimum difference in isoelectric point of Delta(pI) = 0.4. Furthermore, the results indicate that even a better resolution can be achieved. The theoretical minimum difference in isoelectric point is Delta(pI) = 0.23 resulting in a peak capacity of 29 peaks within 1.8 mm. This is an 8-fold increase in peak capacity to previously published results. The focusing of pI markers led to an increase in concentration by factor 20 and higher. Further improvement in terms of resolution seems possible, for which we envisage that the influence of electroosmotic flow has to be further reduced. The performance of the microfluidic free-flow isoelectric focusing device will enable new applications, as this device might be used in clinical analysis where often low sample volumes are available and fast separation times are essential.     

Analysis of lipoproteins by capillary zone electrophoresis in microfluidic devices: assay development and surface roughness measurements

The development of a new assay for lipoproteins by capillary electrophoresis in fused-silica capillaries and in glass microdevices is described in this paper. The separation of low-density (LDL) and high-density (HDL) lipoproteins by capillary zone electrophoresis is demonstrated in fused-silica capillaries with both UV absorption and laser-induced fluorescence detection. This separation was accomplished using Tricine buffer (pH 9.0) with methylglucamine added as a dynamic coating. With UV detection, LDL eluted as a relatively sharp peak with a migration time of approximately 11 min and HDL eluted as a broad peak with a migration time of 12.5 min. Fluorescence detection of lipoproteins stained with NBD-ceramide was used with the same buffer system to give comparable results. Furthermore, fluorescence staining of human serum samples yielded results similar to the fluorescently stained LDL and HDL fractions, showing that this method can be used to quantify lipoproteins in serum samples. The method was also used to detect lipoproteins in glass micro-CE devices. Very similar results were obtained in microdevices although with much faster analysis times, LDL eluted as a sharp peak at approximately 25 s and HDL as a broad peak at slightly longer time. In addition, higher resolution was obtained on chips. To our knowledge, these results show the first separation and detection of lipoproteins in a microfluidic device using native serum samples. Atomic force microscopy was used to characterize the rms surface roughness (Rq) of microfluidic channels directly. Devices with different surface roughness values were fabricated using two different etchants for Pyrex wafers with a polysilicon masking layer. Using 49% HF, the measured roughness is Rq = 10.9 +/- 1.6 nm and with buffered HF (NH4F + HF) the roughness is Rq = 2.4 +/- 0.7 nm. At this level of surface roughness, there is no observable effect on the performance of the devices for this lipoprotein separation.