Suppression of non-specific adsorption using sheath flow

The use of a confining sheath fluid within a microfluidic channel in order prevent non-specific adsorption of analytes to the walls of microchannels is demonstrated. A sheath-flow channel fabricated using laser cutting of Mylar films is developed. Numerical simulations of convective and diffusive mass transport within the channel are presented. The device is characterized experimentally using epifluorescence microscopy. It is demonstrated that the device is capable of preventing the adsorption of Rhodamine B to the walls of the channel for a period that would allow for adsorption-free T-sensor measurements to be made within the core of the flow channel. Generalized scaling rules based on the diffusion coefficient, sheath thickness and affinity of the potential adsorbant for the surface material are discussed. The controlled adsorption of the protein bovine serum albumin (BSA) to a gold surface is also demonstrated using SPR microscopy.     

Two simple and rugged designs for creating microfluidic sheath flow

A simple design capable of 2-dimensional hydrodynamic focusing is proposed and successfully demonstrated. In the past, most microfluidic sheath flow systems have often only confined the sample solution on the sides, leaving the top and bottom of the sample stream in contact with the floor and ceiling of the channel. While relatively simple to build, these designs increase the risk of adsorption of sample components to the top and bottom of the channel. A few designs have been successful in completely sheathing the sample stream, but these typically require multiple sheath inputs and several alignment steps. In the designs presented here, full sheathing is accomplished using as few as one sheath input, which eliminates the need to carefully balance the flow of two or more sheath inlets. The design is easily manufactured using current microfabrication techniques. Furthermore, the sample and sheath fluid can be subsequently separated for recapture of the sample fluid or re-use of the sheath fluid. Designs were demonstrated in poly(dimethylsiloxane) (PDMS) using soft lithography and poly(methyl methacrylate) (PMMA) using micromilling and laser ablation.     

A PDMS sheath flow cuvette for high‐sensitivity LIF measurements in CE

A simple method for producing a sheath flow cuvette in PDMS suitable for post‐column detection in CE is described. Two types of cuvette were investigated. In the first, the sheath flow channel had a round cross‐section of approximately 635 μm diameter, whereas the second cuvette had a 300×300 μm² square channel. Both cuvettes produced laminar flows that ensheathed the separation capillary's effluent allowing sensitive fluorescence measurements. The elasticity of the PDMS allowed the 300×300 μm² square sheath flow channel to expand uniformly and accommodate the larger 330–340 μm od round separation capillary, producing a self‐aligning cuvette with robust mechanical properties. With this cuvette, linear calibrations of over five orders of magnitude and 15–30 zmol fluorescein detection limits were obtained for 12 and 50 μm id capillaries.     

Modular approach to fabrication of three-dimensional microchannel systems in PDMS-application to sheath flow microchips

A modular approach to fabrication of three-dimensional microchannel systems in polydimethylsiloxane (PDMS) is presented. It is based on building blocks with microstructuring on up to three faces. The assembled 3D-microchip consists of three building blocks in two layers. For assembly of the bottom layer two building blocks are joined horizontally, whereby the side structuring of the first is sealed against the flat side surface of the other. This results in the formation of a vertical interconnection opening between the building blocks to supplement the microstructuring on the lower faces. The 3D microchannel system is completed by placing a third building block, with microstructuring only on its lower face, on top of the assembled layer. While plasma assisted bonding is used between the two building blocks of the bottom layer, inherent adhesion is sufficient between the layers and for attaching the assembled 3D-microchip to a substrate. This modular approach was applied to the fabrication of a 3D-sheath flow microchip. It comprises a 20 microm deep microchannel system with sample inlet, open sensing area and outlet in the bottom layer and sheath flow inlet in the top layer. 100 microM fluorescein at 6 microL min(-1) was used as sample flow and water at increasing flow rates as sheath flow. With ratios of sheath to sample flow up to 20:1 sample layers down to 1 microm thickness could be generated. Sample layer thickness was determined via volume detection on an epi-fluorescence microscope followed by image analysis.     

Microelectrospray interface with coaxial sheath flow for high-resolution capillary electrophoresis/mass spectrometry separation

We have fabricated a coaxial sheath liquid flow microelectrospray ionization (microESI) interface for capillary electrophoresis coupled with mass spectrometry (CE/MS). The ESI interface, which features a reduced probe diameter (130 microm i.d. x 174 microm o.d.) with a nebulizer-free format, can relatively easily electrospray a large amount of make-up sheath liquid (5-10 microL/min) over the long term (more than 80 runs) with a high degree of stability. The interface also provides higher separation qualities and improved detection sensitivities compared with a conventional ion spray (IS) interface.     

Integrated flow-cells for novel adjustable sheath flows

In this paper two integrated flow-cells are presented that can generate novel sheath flows. The flow-cells allow for dynamic orthogonal control of the sample flow dimensions. In addition to this, the sample flow can be freely positioned inside the channel. The flow-cells are attractive, because they are very simple to fabricate and are compatible with the integration of sensors. Experiments have been carried out demonstrating that the sample flow dimensions can be controlled over a wide range; also the results show good agreement with finite element simulation results.     

A hard microflow cytometer using groove-generated sheath flow for multiplexed bead and cell assays

With a view toward developing a rugged microflow cytometer, a sheath flow system was micromachined in hard plastic (polymethylmethacrylate) for analysis of particles and cells using optical detection. Six optical fibers were incorporated into the interrogation region of the chip, in which hydrodynamic focusing narrowed the core stream to ∼35 μm × 40 μm. The use of a relatively large channel at the inlet as well as in the interrogation region (375 μm × 125 μm) successfully minimized the risk of clogging. The device could withstand pressures greater than 100 psi without leaking. Assays using both coded microparticles and cells were demonstrated using the microflow cytometer. Multiplexed immunoassays detected nine different bacteria and toxins using a single mixture of coded microspheres. A549 cancer cells processed with locked nucleic acid probes were evaluated using fluorescence in situ hybridization.     

Capillary gel electrophoresis for DNA sequencing. Laser-induced fluorescence detection with the sheath flow cuvette

Capillary polyacrylamide gel electrophoresis separation of dideoxycytidine chain-terminated DNA fragments is reported. A post-column laser-induced fluorescence detector based on the sheath flow cuvette was used to minimize background signals due to light scatter from the gel and capillary. A preliminary mass detection limit of 10(-20) mol of fluorescein-labeled DNA fragments was obtained. The system was used to analyze an actual DNA sequencing sample. Theoretical plate counts of 2 x 10(6) were produced. Gel stability limits the performance of the current system.     

Post-column fluorescence derivatization of proteins and peptides in capillary electrophoresis with a sheath flow reactor and 488 nm argon ion laser excitation

We report the use of a sheath flow reactor for post-column fluorescence derivatization of proteins. The derivatization reaction employed naphthalene-2,3-dicarboxaldehyde (NDA) and beta-mercaptoethanol, which were added in the sheath buffer. The labeled proteins were detected by laser-induced fluorescence with an argon-ion laser beam at 488 nm. The performance of this detection scheme was evaluated by separation of some protein standards. A column efficiency of 450,000 plates/m was obtained without stacking. The limits of detection for those standard proteins were determined to be from 8 to 32 nM. Excellent linear relationship was obtained with correlation coefficient of 0.9998 for alpha-lactalbumin concentration ranging from 3.91 x 10(-7) to 1.25 x 10(-5) M. Separation of protein standards at low pH was also demonstrated by reversing the electroosmotic flow (EOF) with addition of cetyltrimethylammonium bromide (CTAB) to the running buffer. Different separation selectivity was achieved, but the sensitivity is poorer than that at high pH. This post-column derivatization detection system was applied successfully to analyze the protein extract from HT29 human colon cancer cells as well as tryptic peptides.     

Enhancing resolution of free‐flow zone electrophoresis via a simple sheath‐flow sample injection

In this work, a simple and novel sheath‐flow sample injection method (SFSIM) is introduced to reduce the band broadening of free‐flow zone electrophoresis separation in newly developed self‐balance free‐flow electrophoresis instrument. A needle injector was placed in the center of the separation inlet, into which the BGE and sample solution were pumped simultaneously. BGE formed sheath flow outside the sample stream, resulting in less band broadening related to hydrodynamics and electrodynamics. Hemoglobin and C‐phycocyanin were successfully separated by the proposed method in contrast to the poor separation of free‐flow electrophoresis with the traditional injection method without sheath flow. About 3.75 times resolution enhancement could be achieved by sheath‐flow sample injection method.     

A beveled tip sheath liquid interface for capillary electrophoresis-electrospray ionization-mass spectrometry

A simple and durable sheath liquid interface for capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) has been developed. This interface utilized a beveled tip emitter and was found to be more sensitive than the conventional sheath liquid interface. The use of a beveled tip reduces the optimal flow rate and therefore decreases sample dilution. The interface utilized a 380 microm inner diameter and 400 microm outer diameter beveled tapered tip. Because of the large inner diameter and outer diameter of the tip, the interface is robust and can be easily implemented. The performance of this interface for CZE-ESI-MS and micelle electrokinetic capillary electrophoresis-electrospray-mass spectrometry, as demonstrated by the analysis of synthetic drugs and triazine mixtures, was significantly better than results obtained using a conventional sheath liquid interface.     

Coupling of a large-size capillary column with an electrospray mass spectrometer. A reliable and sensitive sheath flow capillary electrophoresis-mass spectrometry interface

The concept of interfacing a large-size column for capillary electrophoresis (CE) to electrospray ionization mass spectrometry (ESI-MS) for robust and automatic CE-MS operation is reported. Both standard ionspray interface and microionspray interface have been modified to operate in a sheath flow pattern to overcome the common stability problem in CE-MS coupling. To make the interface sensitive, a step-down stainless steel tube with smaller inner diameter and tapered tip was incorporated onto a larger tube embracing the CE column via cold soldering. The devices were evaluated for quantitative analysis of nucleotides at femtomole level and stable analytical performance in peptide profiling.     

Lactone cleavage reaction kinetics of rhodamine dye at liquid/liquid interfaces studied by micro-two-phase sheath flow/two-photon excitation fluorescence microscopy

Two-photon excitation fluorescence microscopy was combined with the two-phase microflow system in order to measure the fast interfacial reaction rate at liquid/liquid interfaces. The lactone cleavage kinetics of octadecylrhodamine B (C(18)RB) at the toluene/water and heptane/water interfaces was studied by this new method. The organic solution containing the nonfluorescent lactone of C(18)RB was made to flow as an inner flow with an aqueous outer sheath flow. The diameter of the inner flow was <20 microm. A focused fundamental beam of a Ti:sapphire pulse laser of 780 nm was irradiated to the interface, and emitted fluorescence from the fluorescent product was detected by a charge-coupled device (CCD) camera or a streakscope. The increase in the concentration of the fluorescent form of C(18)RB was measured along the interface of the inner flow of the toluene/water and heptane/water systems for 80 micros just after the contact of two phases. The analysis made by the time-dependent Langmuir adsorption model with the aid of the digital simulation method gave the cleavage reaction rate constants of the lactone form of C(18)RB at the liquid/liquid interfaces.     

Characteristics of a liquid/liquid optical waveguide using sheath flow and its application to detect molecules at a liquid/liquid interface.

The formation conditions and characteristics of a liquid/liquid optical waveguide (LLW) were studied using a two-phase sheath flow, where the inner organic phase flow acted as the core and the outer aqueous flow acted as the clad. In immiscible solvent systems, i.e., toluene/water and diethyl ether/water systems, the LLWs were formed in the range of higher than ca. 600 of the Reynolds number (Re), where the linear velocity of the organic solvent was much higher than that of the aqueous solution. On the other hand, in a miscible solvent system, i.e., a tetrahydrofuran/water system, a stable LLW was formed in the range of a much lower Re than in immiscible systems. Moreover, the molecules at the toluene/water interface of the LLW were observed with both fluorescence and absorbance measurement systems. In particular, the change in the fluorescence spectrum of 1-anilino-8-naphthalenesulfonate (ANS) at the interface within 1 ms was observed by this method, indicating the usefulness of the LLW for a fast kinetic study of a liquid/liquid interface.     

A continuous-flow,microfluidic fraction collection device

A microfluidic device is presented that performs electrophoretic separation coupled with fraction collection. Effluent from the 3.5 cm separation channel was focused via two sheath flow channels into one of seven collection channels. By holding the collection channels at ground potential and varying the voltage ratio at the two sheath flow channels, the separation effluent was directed to either specific collection channels, or could be swept past all channels in a defined time period. As the sum of the voltages applied to the two sheath flow channels was constant, the electric field remained at 275 V/cm during the separation regardless of the collection channel used. The constant potential in the separation channel allowed uninterrupted separation for late-migrating peaks while early-migrating peaks were being collected. To minimize the potential for carryover between fractions, the device geometry was optimized using a three-level factorial model. The optimum conditions were a 22.5° angle between the sheath flow channels and the separation channel, and a 350 μm length of channel between the separation outlet and the fraction channels. Using these optimized dimensions, the device performance was evaluated by separation and fraction collection of a fluorescently labeled amino acid mixture. The ability to fraction collect on a microfluidic platform will be especially useful during automated or continuous operation of these devices or to collect precious samples.     

A sheath flow gating interface for the on-line coupling of solid-phase extraction with capillary electrophoresis

A sheath flow gating interface (SFGI) is presented for the on-line coupling of solid-phase extraction (SPE) with capillary electrophoresis (CE). The design, construction and operation of the SFGI are described in detail. After operating conditions were investigated and selected, the SFGI was evaluated on a SPE–CE–UV setup using hydroxylated poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith as the absorbent and using three phenols as the test analytes. The preconcentration factors obtained with the SPE–CE–UV system and the SPE–UV part are 530 and 550, respectively. The plate numbers obtained using the SPE–CE–UV system are slightly better than or comparable to those with the CE–UV part. The precisions (RSDs) of 100 consecutive injections are 2.43%, 3.86%, and 4.25% for peak height, peak area and migration time, respectively. The measured recoveries for the river water samples spiked at three different levels are in the range of 93.6–102.8% with the interday RSD values ranging from 2.0 to 4.5% (n = 3). These data collectively demonstrate that the SFGI has the ability to exactly and reproducibly transfer nanoliters of fractions from SPE onto CE with no degradation of the efficiencies of SPE and CE, suggesting a great potential to be routinely used for the coupling of SPE, microcolumn LC or FIA with CE.     

Design, optimisation, and evaluation of a sheath flow interface for automated capillary electrophoresis-electrospray-mass spectrometry

A sheath-flow capillary electrophoresis-mass spectrometry (CE-MS) system utilizing a fully integrated large-bore stainless-steel emitter electrode tapered at the end for micro-ionspray operation has been developed and evaluated. A separation capillary with an outer diameter of up to 360 microm was inserted into the electrode thus forming a void volume of less than 15 nL between the capillary end and the electrospray ionisation (ESI) tip. The sheath liquid, usually methanol-water (80:20) with 0.1% formic acid for positive ion mode or methanol for negative ion mode, was delivered at 0.5-1.0 microL/min. Unlike previously reported CE-MS interfaces, the CE-MS probe was incorporated directly onto an Applied Biosystems/MDS SCIEX orthogonal-spray Turbo "V" ion source for ease of use and automatic operation. This integration enables fast and facile coupling and replacement of the separation capillary without interrupting the ion source configuration, and the sheath liquid supply. The reusable electrospray electrode was precisely fabricated and aligned with the length of the nebulizing gas tube for improved reproducibility. Automation was achieved through software control of both CE and tandem MS (MS/MS) for unattended batch sample analysis. The system was evaluated for attomole- to low femtomole-level profiling of model peptides and protein mixtures, bisphosphates, as well as antiviral nucleosidic drugs in cellular extracts.     

Development and characterization of an integrated silicon micro flow cytometer

This paper describes an innovative integrated micro flow cytometer that presents a new arrangement for the excitation/detection system. The sample liquid, containing the fluorescent marked particles/cells under analysis, is hydrodynamically squeezed into a narrow stream by two sheath flows so that the particles/cells flow individually through a detection region. The detection of the particles/cells emitted fluorescence is carried out by using a collection fiber placed orthogonally to the flow. The device is based on silicon hollow core antiresonant reflecting optical waveguides (ARROWs). ARROW geometry allows one to use the same channel to guide both the sample stream and the fluorescence excitation light, leading to a simplification of the optical configuration and to an increase of the signal-to-noise ratio. The integrated micro flow cytometer has been characterized by using biological samples marked with standard fluorochromes. The experimental investigation confirms the success of the proposed microdevice in the detection of cells. An erratum to this article can be found at     

Field and flow programming in frit-inlet asymmetrical flow field-flow fractionation

The separation of wide molecular mass (Mr) ranges of macromolecules using frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF) has been improved by implementing a combination of field and flow programming. In this first implementation, field strength (governed by the cross flow-rate through the membrane-covered accumulation wall) is decreased with time to obtain faster elution and improved detection of the more strongly retained (high Mr) materials. The channel outlet flow-rate is optionally held constant, increased, or decreased with time. With circulation of the flow exiting the accumulation wall to the inlet frit, the dual programming of cross flow and channel outlet flow could be implemented using just two pumps. With this flow configuration, the channel outlet flow-rate is always equal to the channel inlet flow-rate, and these may be programmed independently of the cross flow-rate through the membrane. FI-AFlFFF retains its operational advantage over conventional asymmetrical flow FFF (AFlFFF). Unlike conventional AFlFFF, FI-AFlFFF does not require time consuming, and experimentally inconvenient, sample focusing and relaxation steps involving valve switching and interruption of sample migration. The advantages of employing dual programming with FI-AFlFFF are demonstrated for sets of polystyrene sulfonate standards in the molecular mass range of 4 to 1000 kDa. It is shown that programmed FI-AFlFFF successfully expands the dynamic separation range of molecular mass.     

Fiber optic illumination of a poly(dimethylsiloxane) sheath flow cuvette for diode laser induced fluorescence detection in capillary electrophoresis

A Tee configuration sheath flow cuvette with square cross‐section channels has been produced in PDMS for CE detection. The output of a 1.4 W laser diode operating at 450 nm was focused onto the 300 μm core of a 370 μm od fiber optic whose end was inserted into one arm of the Tee for LIF. The optimal configuration had the fiber optic positioned 500 μm downstream from the intersection and the end of the 35 cm 50 μm id 365 μm od capillary just outside the intersection and in the leg of the Tee, resulting in a 90° configuration. Detection limits of 50 and 3 pM and linear calibrations of at least three orders of magnitude were obtained for Lucifer Yellow and fluorescein, respectively.