An electrokinetic/hydrodynamic flow microfluidic CE-ESI-MS interface utilizing a hydrodynamic flow restrictor for delivery of samples under low EOF conditions

A hydrodynamic flow restrictor (HDR) that is used to combine electrokinetic and hydrodynamic flow streams has been fabricated in a microfluidic channel by laser micromachining. Combining electrokinetic and hydrodynamic flow streams is challenging in microfluidic devices, because the hydrodynamic flow often overpowers the electrokinetic flow, making it more difficult to use low electroosmotic flow in the electrokinetic portion of the system. The HDR has been incorporated into a capillary electrophoresis-mass spectrometry interface that provides continuous introduction of a make-up solution and negates the hydrodynamic backpressure in the capillary electrophoresis channel to the extent that low EOF can be utilized. Moreover, the hydrodynamic backpressure is sufficiently minimized to allow coatings that minimize EOF to be used in the electrokinetically driven channel. Such coatings are of great importance for the analysis of proteins and other biomolecules that adsorb to charged surfaces.     

Electroosmotic flow-switchable poly(dimethylsiloxane) microfluidic channel modified with cysteine based on gold nanoparticles

An electroosmotic flow (EOF)-switchable poly(dimethylsiloxane) (PDMS) microfluidic channel modified with cysteine has been developed. The native PDMS channel was coated with poly(diallyldimethylammonium chloride) (PDDA), and then gold nanoparticles by layer-by-layer technique was assembled on PDDA to immobilize cysteine. The assembly was followed by infrared spectroscopy/attenuated total reflection method, contact angle, EOF measurements and electrophoretic separation methods. EOF of this channel can be reversibly switched by varying the pH of running buffer. At low pH, the surface of channels is positively charged, EOF is from cathode to anode. At high pH, the surface is negatively charged, EOF is from anode to cathode. At pH 5.0, near the isoelectric point of the chemisorbed cysteine, the surfaces of channels show neutral. When pH is above 6.0 or below 4.0, the magnitude of EOF varies in a narrow range. And the modified channel surface displayed high reproducibility and good stability, a good reversibility of cathodic-anodic EOF transition under the different pH conditions was observed. Separation of dopamine and epinephrine as well as arginine and histidine were performed on the modified chip.     

Simple 2D-HPLC using a monolithic silica column for peptide separation

Separation of peptides by fast and simple two-dimensional (2D)-HPLC was studied using a monolithic silica column as a second-dimension (2nd-D) column. Every fraction from the first column, 5 cm long (2.1 mm ID) packed with polymer-based cation exchange beads, was subjected to separation in the 2nd-D using an octadecylsilylated (C18) monolithic sillica column (4.6 mm ID, 2.5 cm). A capillary-type monolithic silica C18column (0.1 mm ID, 10 cm) was also employed as a 2nd-D column with split flow/injection. Effluentof the first dimension (1st-D) was directly loaded into an injector loop of 2nd-D HPLC. UV and MS detection were successfully carried out at high linear velocity of mobile phase at 2nd-D using flow splitting for the 4.6 mm ID 2nd-D column, or with directconnection of the capillary column to the MS interface. Two-minute fractionation inthe 1st-D, 118-second loading, and 2-second injection by the 2nd-D injector, allowed one minute for gradient separation in the 2nd-D, resulting in a maximum peak capacity of about 700 within 40 min. The use of a capillary column in solvent consumption and better MS detectability compared to a larger-sized column. This kind of fast and simple 2D-HPLC utilizing monolithic silica columns will be useful for the separation of complex mixtures in a short time.     

Integration of a nanoporous platinum thin film into a microfluidic system for non-enzymatic electrochemical glucose sensing.

In this paper, we describe an amperometric-type enzymeless glucose sensing system based on a nanoporous platinum (Pt) electrode embedded in a microfluidic chip. This microchip system is comprised of a microfluidic transport channel network and a miniaturized electrochemical cell for nonenzymatic glucose sensing. Sample and buffer solutions were transferred to the cell by programmed electroosmotic flow (EOF). A nanoporous Pt electrode with the roughness factor of 200.6 was utilized to determine glucose concentrations in phosphate buffered saline (PBS) by the direct oxidation of glucose, without any separation process. The sensitivity of the developed system is 1.65 microA cm-2 mM-1 in the glucose concentration range from 1-10 mM in PBS.     

Reversed-phase liquid chromatography on a microchip with sample injector and monolithic silica column

In micro total analysis systems, liquid chromatography (LC) works under pressure-driven flow is the essential analysis component. There were not, however, much works on microchip LC. Here we developed a microchip for reversed-phase LC using porous monolithic silica. The chip consisted of a double T-shaped injector and a approximately 40-cm serpentine separation channel. The octadecyl-modified monolithic silica was prepared in the specified part of the channel on the microchip using sol-gel process. Furthermore, the effect of geometry of turn sections on band dispersion at turns was examined under pressure-driven flow. High separation efficiencies of 15,000-18,000 plates/m for catechins were obtained using the LC chip.     

Field-effect flow control in a polydimethylsiloxane-based microfluidic system.

The application of the field-effect for direct control of electroosmosis in a polydimethylsiloxane (PDMS)-based microfluidic system, constructed on a silicon wafer with a 2.0 microm electrically insulating layer of silicon dioxide, is demonstrated. This microfluidic system consists of a 2.0 cm open microchannel fabricated on a PDMS slab, which can reversibly adhere to the silicon wafer to form a hybrid microfluidic device. Aside from mechanically serving as a robust bottom substrate to seal the channel and support the microfluidic system, the silicon wafer is exploited to achieve field-effect flow control by grounding the semiconductive silicon medium. When an electric field is applied through the channel, a radial electric potential gradient is created across the silicon dioxide layer that allows for direct control of the zeta potential and the resulting electroosmotic flow (EOF). By configuring this microfluidic system with two power supplies at both ends of the microchannel, the applied electric potentials can be varied for manipulating the polarity and the magnitude of the radial electric potential gradient across the silicon dioxide layer. At the same time, the longitudinal potential gradient through the microchannel, which is used to induce EOF, is held constant. The results of EOF control in this hybrid microfluidic system are presented for phosphate buffer at pH 3 and pH 5. It is also demonstrated that EOF control can be performed at higher solution pH of 6 and 7.4 by modifying the silicon wafer surface with cetyltrimethylammonium bromide (CTAB) prior to assembly of the hybrid microfluidic system. Results of EOF control from this study are compared with those reported in the literature involving the use of other microfluidic devices under comparable solution conditions.     

Electrochromatography in cyclic olefin copolymer microchips: A step towards field portable analysis

In order to develop a portable and disposable instrument for on-site analysis of neutral compounds, a lauryl methacrylate monolith has been synthesized into a cyclic olefin copolymer microdevice for reversed-phase electrochromatography purposes. This monolith was tested in capillary to evaluate electrochromatographic performances in terms of electroosmotic flow (EOF) mobility, retention and efficiency prior to its transfer into the microfluidic device. The produced monolithic bed exhibited a good run-to-run repeatability, column-to-column reproducibility and batch-to-batch reproducibility, with relative standard deviation (RSD) values below 9% for EOF mobility, retention factors and heights of theoretical plate. The electrochromatographic performances of the monolith were optimized by reducing irradiation time. Photopolymerization time of 10 min was found to be the best process in order to obtain a robust, retentive and efficient system. The on-chip performances of this monolith were evaluated in detail for the reversed-phase electrochromatographic separation of polycyclic aromatic hydrocarbons, with plate heights reaching down to 15 μm when working at optimal velocity. Aiming at the maximum simplification of instrumental fabrication and operation, a direct injection from a 2 μL droplet was compared with more conventional dynamic injection process.     

Characterization of cell lysis events on a microfluidic device for high-throughput single cell analysis

A microfluidic device capable of rapidly analyzing cells in a high-throughput manner using electrical cell lysis is further characterized. In the experiments performed, cell lysis events were studied using an electron multiplying charge coupled device camera with high frame rate (>100 fps) data collection. It was found that, with this microfluidic design, the path that a cell follows through the electric field affects the amount of lysate injected into the analysis channel. Elimination of variable flow paths through the electric field was achieved by coating the analysis channel with a polyamine compound to reverse the electroosmotic flow (EOF). EOF reversal forced the cells to take the same path through the electric field. The improved control of the cell trajectory will reduce device-imposed bias on the analysis and maximizes the amount of lysate injected into the analysis channel for each cell, resulting in improved analyte detection capabilities.     

Direct coupling of polymer-based microchip electrophoresis to online MALDI-MS using a rotating ball inlet

We report on the coupling of a polymer-based microfluidic chip to a MALDI-TOF MS using a rotating ball interface. The microfluidic chips were fabricated by micromilling a mold insert into a brass plate, which was then used for replicating polymer microparts via hot embossing. Assembly of the chip was accomplished by thermally annealing a cover slip to the embossed substrate to enclose the channels. The linear separation channel was 50 microm wide, 100 microm deep, and possessed an 8 cm effective length separation channel with a double-T injector (V(inj) = 10 nL). The exit of the separation channel was machined to allow direct contact deposition of effluent onto a specially constructed rotating ball inlet to the mass spectrometer. Matrix addition was accomplished in-line on the surface of the ball. The coupling utilized the ball as the cathode transfer electrode to transport sample into the vacuum for desorption with a 355 nm Nd:YAG laser and analyzed on a TOF mass spectrometer. The ball was cleaned online after every rotation. The ability to couple poly(methylmethacrylate) microchip electrophoresis devices for the separation of peptides and peptide fragments produced from a protein digest with subsequent online MALDI MS detection was demonstrated.     

Study of the electroosmotic flow as a means to propel the mobile phase in capillary electrochromatography in view of further miniaturization of capillary electrochromatography systems

In this paper, we investigate the phenomenon of electroosmosis as a means to propel a mobile phase, in particular in view of an application in microfluidic systems, which are characterized by significantly smaller volumes of the reservoirs and the separation channels compared to conventional instrumentation. In the microfluidic chip, pH changes due to water electrolysis quickly showed an effect on the electroosmotic flow (EOF), which could be counteracted by either regularly exchanging or buffering the mobile phase. Surface treatment was of no effect in regard to EOF stabilization in empty channels but may have an influence in channels filled with a charged monolith. In fused-silica capillaries the EOF was generally found to decrease from 'naked' to surface-treated to monolith-filled capillaries. The EOF tended to be higher when an organic solvent (acetonitrile) was added to the mobile phase and could be further increased by substituting the water with equal amounts of methanol. In addition, the hydrostatic pressure exerted by the EOF was investigated. In a microfluidic chip with empty (cross-)channels such an effect could be responsible for a redirection of the flow. In capillaries partially filled with a noncharged (non-EOF-generating) monolith, a linear relationship could be established between the EOF created in the empty section of the capillary (apparent mobility) and the length of the monolith (backpressure). In capillaries partially filled with a charged (EOF-producing) monolith, flow inhomogeneities must be expected as a consequence of a superimposition of hydrodynamic pressure and EOF as mobile phase driving force.     

Poly(vinyl alcohol)-coated microfluidic devices for high-performance microchip electrophoresis

The channels of microfluidic glass chips have been coated with poly(vinyl alcohol) (PVA). Applied for microchip electrophoresis, the coated devices exhibited a suppressed electroosmotic flow and improved separation performance. The superior performance of PVA-coated channels could be demonstrated by electrophoretic separations of labeled amines and by video microscopy. While a distorted sample zone is injected using uncoated channels the application of PVA-coated channels results in an improved shape of the sample zone with less band broadening. Applying PVA-coated microchips for the separation of amines labeled with Alexa Fluor 350 even sub-second separations, utilizing a separation length of only 650 microm, could be obtained, while this was not possible using uncoated devices. By using PVA-coated devices rather than an uncoated chip a threefold increase in separation efficiencies could be observed. As the electroosmotic flow (EOF) was suppressed, the anionic compounds were detected at the anode whereas the dominant EOF in uncoated devices resulted in an effective mobility to the cathode. Besides improved separation performance another important feature of the PVA-coated channels was the suppressed adsorption of fluorescent compounds in repetitive runs which results in an improved robustness and detection sensitivity. Applying PVA-coated channels, rinsing or etching steps could be omitted while this was necessary for a reliable operation of uncoated devices.     

Fabrication and performance of poly(methyl methacrylate) microfluidic chips with fiber cores

In this work, a piece of glass fiber was inserted into the channel of a poly(methyl methacrylate) (PMMA) electrophoresis microchip to enhance the electroosmotic flow (EOF) and the separation efficiency. The EOF value of the glass fiber-containing microchannel at pH 8.2 was determined to be 4.17 x 10(-4)cm2 V(-1)s(-1). The performance of the new microchip was demonstrated by its ability to separate and detect three purines coupled with end-column amperometric detection. In addition, a piece of trypsin-immobilized glass fiber was inserted into the channel of a PMMA microchip to fabricate a core-changeable microfluidic bioreactor that can be regenerated by changing the fiber. The in-channel fiber bioreactor has been coupled with matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the digestion and peptide mapping of bovine serum albumin and myoglobin.     

Integration of immobilized trypsin bead beds for protein digestion within a microfluidic chip incorporating capillary electrophoresis separations and an electrospray mass spectrometry interface

A microfluidic device is described in which an electrospray interface to a mass spectrometer is integrated with a capillary electrophoresis channel, an injector and a protein digestion bed on a monolithic substrate. A large channel, 800 microm wide, 150 microm deep and 15 mm long, was created to act as a reactor bed for trypsin immobilized on 40-60 microm diameter beads. Separation was performed in channels etched 10 microm deep, 30 microm wide and about 45 mm long, feeding into a capillary, attached to the chip with a low dead volume coupling, that was 30 mm in length, with a 50 microm i.d. and 180 microm o.d. Sample was pumped through the reactor bed at flow rates between 0.5 and 60 microL/min. The application of this device for rapid digestion, separation and identification of proteins is demonstrated for melittin, cytochrome c and bovine serum albumin (BSA). The rate and efficiency of digestion was related to the flow rate of the substrate solution through the reactor bed. A flow rate of 1 or 0.5 microL/min was found adequate for complete consumption of cytochrome c or BSA, corresponding to a digestion time of 3-6 min at room temperature. Coverage of the amino acid sequence ranged from 92% for cytochrome c to 71% for BSA, with some missed cleavages observed. Melittin was consumed within 5 s. In contrast, a similar extent of digestion of melittin in a cuvet took 10-15 min. The kinetic limitations associated with the rapid digestion of low picomole levels of substrate were minimized using an integrated digestion bed with hydrodynamic flow to provide an increased ratio of trypsin to sample. This chip design thus provides a convenient platform for automated sample processing in proteomics applications.     

微流控芯片电泳/电化学检测人单个肝癌细胞中的谷胱甘肽

采用微流控装置结合电化学检测研究了测定人单个血红细胞中谷胱甘肽(GSH)的方法。在该方法中,细胞的进样、定位、溶膜以及细胞中谷胱甘肽的转移和检测都在配有通道端安培检测器的双T形芯片中完成。单个细胞用液压导入到双T的交界面,在电泳缓冲液中毛地黄皂苷的作用下,细胞膜被穿孔。再施加直流电压,细胞被溶膜。释放出来的GSH被此直流电压电迁移至通道端并在Au/Hg电极上被检测。用校正曲线法可以定量测定单个细胞中的GSH。     

A split microchannel design and analytical model to compensate for electroosmotic instabilities in micro-separations

Organic polymers offer many advantages as materials for the construction of microfluidic devices but suffer frequently from the limitation that the electrodynamic flow they support can exhibit considerable instability. This article describes a split-channel microfluidic device that can be used to compensate for changes in electroosmotic flow. The design of the separation system divides an analyte plug after injection between two separation channels of differing length. The two channels are later recombined for single point detection, eliminating the need for a scanning optical detection system. The utility of this simple design lies in the fact that the migration time of any analyte can be referenced to its twin in the parallel separation channel. This eliminates the need for a separate electroosmotic marker and allows mobilities measured in multiple devices to be compared quantitatively. Using a model adopted from the literature, the data from the split channel system can be used to precisely account for the drift that characterizes electrophoretic separations made in a polymer chip. The relative standard deviations of the analyte mobilities measured for replicate runs on multiple devices were reduced from values as high as 20% to ca. 1% RSD. This internal standardization procedure also appears to address other sources of drift in the electroosmotic flow (EOF) supported by the polymer microchannel, eliminating the need for careful monitoring of either the temperature or reservoir pH between separation runs.     

A microfluidic device for performing pressure-driven separations

Microchannels in microfluidic devices are frequently chemically modified to introduce specific functional elements or operational modalities. In this work, we describe a miniaturized hydraulic pump created by coating selective channels in a glass microfluidic manifold with a polyelectrolyte multilayer (PEM) that alters the surface charge of the substrate. Pressure-driven flow is generated due to a mismatch in the electroosmotic flow (EOF) rates induced upon the application of an electric field to a tee channel junction that has one arm coated with a positively charged PEM and the other arm left uncoated in its native state. In this design, the channels that generate the hydraulic pressure are interconnected via the third arm of the tee to a field-free analysis channel for performing pressure-driven separations. We have also shown that modifications in the cross-sectional area of the channels in the pumping unit can enhance the hydrodynamic flow through the separation section of the manifold. The integrated device has been demonstrated by separating Coumarin dyes in the field-free analysis channel using open-channel liquid chromatography under pressure-driven flow conditions.     

Use of electrokinetic measurements for characterization of columns used in capillary electrochromatography

The separation mechanism in capillary electrochromatography (CEC) is a hybrid differential migration process, which entails the features of both high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE), i.e., chromatographic retention and electrophoretic migration. The focus of this paper is on the use of electrokinetic data, such as current, electroosmotic flow (EOF) and column efficiency measurements, that are readily available, for an improved understanding of CEC separations. A framework is presented here for the use of this data for evaluation of a variety of performance parameters including, conductivity ratio, interstitial EOF mobility, porosity, and zeta potential. This framework is applied for characterization of two monolithic columns with different chemistry that were manufactured in-house. The above-mentioned performance parameters were calculated for the two columns and it is found that the poly(VBC-EGDMA-SWNT) monolithic column with the GPTMS-PEI coating offers a significantly improved flow distribution in comparison to the poly(VBC-EGDMA) monolithic column. This observation is confirmed by performing separation of peptides on the two columns and height equivalent of a theoretical plate (HETP) measurements on the resulting peaks. It is shown that following our approach leads to an improved understanding of the separations achieved with the columns and to better column design.     

Chemically L-prolinamide-modified monolithic silica column for enantiomeric separation of dansyl amino acids and hydroxy acids by capillary electrochromatography and mu-high performance liquid chromatography

A silica-based chiral monolithic column prepared by sol-gel process and chemical modification of chiral selector was used for enantioseparation of dansyl amino acids and hydroxy acids by capillary electrochromatography (CEC) and mu-high-performance liquid chromatography (mu-HPLC). L-Prolinamide was modified as a chiral selector. The chiral stationary phase (CSP), the chiral complex of Cu(II) with L-prolinamide, provides an anodic electroosmotic flow (EOF) in CEC. The EOF was found to be dependent on applied electric field strength, the pH, and the composition of mobile phases. Scanning electron micrograph showed that monolithic columns have the morphology of continuous skeleton and large through-pore. D-Enantiomers migrated before L-enantiomers except for dansyl-(Dns)-DL-Ser. The separation efficiencies of up to 17600 (D) and 13,200 plates m(-1) (L) were achieved for the separation of DL-indole-3-lactic acid.     

Controlling retention, selectivity and magnitude of EOF by segmented monolithic columns consisting of octadecyl and naphthyl monolithic segments--applications to RP-CEC of both neutral and charged solutes

Monolithic capillaries made of two adjoining segments each filled with a different monolith were introduced for the control and manipulation of the electroosmotic flow (EOF), retention and selectivity in reversed phase-capillary electrochromatography (RP-CEC). These columns were called segmented monolithic columns (SMCs) where one segment was filled with a naphthyl methacrylate monolith (NMM) to provide hydrophobic and π-interactions, while the other segment was filled with an octadecyl acrylate monolith (ODM) to provide solely hydrophobic interaction. The ODM segment not only provided hydrophobic interactions but also functioned as the EOF accelerator segment. The average EOF of the SMC increased linearly with increasing the fractional length of the ODM segment. The neutral SMC provided a convenient way for tuning EOF, selectivity and retention in the absence of annoying electrostatic interactions and irreversible solute adsorption. The SMCs allowed the separation of a wide range of neutral solutes including polycyclic aromatic hydrocarbons (PAHs) that are difficult to separate using conventional alkyl-bonded stationary phases. In all cases, the k' of a given solute was a linear function of the fractional length of the ODM or NMM segment in the SMCs, thus facilitating the tailoring of a given SMC to solve a given separation problem. At some ODM fractional length, the fabricated SMC allowed the separation of charged solutes such as peptides and proteins that could not otherwise be achieved on a monolithic column made from NMM as an isotropic stationary phase due to the lower EOF exhibited by this monolith.     

Continuous dielectrophoretic cell separation microfluidic device

We present a prototype microfluidic device developed for the continuous dielectrophoretic (DEP) fractionation and purification of sample suspensions of biological cells. The device integrates three fully functional and distinct units consisting of an injector, a fractionation region, and two outlets. In the sheath and sample injection ports, the cell sample are hydrodynamically focused into a stream of controlled width; in the DEP fractionation region, a specially shaped nonuniform (isomotive) electric field is synthesized and employed to facilitate the separation, and the sorted cells are then delivered to two sample collection ports. The microfluidic behavior of the injector region was simulated and then experimentally verified. The operation and performance of the device was evaluated using yeast cells as model biological particles. Issues relating to the fabrication and operation of the device are discussed in detail. Such a device takes a significant step towards an integrated lab-on-a-chip device, which could interface/integrate to a number of other on-chip components for the device to undertake the whole laboratory procedure.