Flow injection analysis: from beaker to microfluidics.
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Flow Injection Analysis↗
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Determination of cell viability in single or mixed samples using capillary electrophoresis laser-induced fluorescence microfluidic systems.
The advent of high-efficiency microbial separations will have a profound effect on both chemistry and microbiology. For the first time, it appears that it may be possible to obtain qualitative and quantitative information on microbial systems with the accuracy, precision, speed, and throughput that currently is found for chemical systems. Recently it was suggested that an analytical separations-based approach for determining the viability of cells would be advantageous. The feasibility of such an approach is demonstrated using CE-LIF of two bacteria and yeast. The analytical procedures and figures of merit are outlined. High-throughput analyses and evaluation of microorganisms now appear to be possible.
Bifidobacterium↗
Flow cytometry of Escherichia coli on microfluidic devices.
Flow cytometry of the bacterium Escherichia coli was demonstrated on a microfabricated fluidic device (microchip). The channels were coated with poly(dimethylacrylamide) to prevent cell adhesion, and the cells were transported electrophoretically by applying potentials to the fluid reservoirs. The cells were electrophoretically focused at the channel cross and detected by coincident light scattering and fluorescence. The E. coli were labeled with a membrane-permeable nucleic acid stain (Syto15), a membrane-impermeable nucleic acid stain (propidium iodide), or a fluorescein-labeled antibody and counted at rates from 30 to 85 Hz. The observed labeling efficiencies for the dyes and antibody were greater than 94%.
Cell Membrane Permeability↗
Design and characterization of immobilized enzymes in microfluidic systems.
Herein we report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes. Streptavidin-conjugated alkaline phosphatase was linked to biotinylated phospholipid bilayers coated inside poly(dimethylsiloxane) microchannels and borosilicate microcapillary tubes. Rapid determination of enzyme kinetics at many different substrate concentrations was made possible by carrying out laminar flow-controlled dilution on-chip. This allowed Lineweaver-Burk analysis to be performed from a single experiment with all the data collected simultaneously. The results revealed an enzyme turnover number of 51.1 +/- 3.2 s(-1) for this heterogeneous system. Furthermore, the same enzyme immobilization strategy was extended to demonstrate that multiple chemical reactions could be performed in sequence by immobilizing various enzymes in series. Specifically, the presence of glucose was detected by two coupled steps employing immobilized avidinD-conjugated glucose oxidase and streptavidin-conjugated horseradish peroxidase.
Bacterial Proteins↗
Rapid microfluidic mixing.
A preformed T-microchannel imprinted in polycarbonate was postmodified with a pulsed UV excimer laser (KrF, 248 nm) to create a series of slanted wells at the junction. The presence of the wells leads to a high degree of lateral transport within the channel and rapid mixing of two confluent streams undergoing electroosmotic flow. Several mixer designs were fabricated and investigated. All designs were relatively successful at low flow rates (0.06 cm/s, > or = 75% mixing), but had varying degrees of success at high flow rates (0.81 cm/s, 45-80% mixing). For example, one design operating at high flow rates was able to split an incoming fluorescent stream into two streams of varying concentrations depending on the number of slanted wells present. The final mixer design was able to overcome stream splitting at high flow rates, and it was shown that the two incoming streams were 80% mixed within 443 microm of the T-junction for a flow rate of 0.81 cm/s. Without the presence of the mixer and at the same high flow rate, a channel length of 2.3 cm would be required to achieve the same extent of mixing when relying upon molecular diffusion entirely, while 6.9 cm would be required for 99% mixing.
Equipment Design↗
Contact conductivity detection in poly(methyl methacrylate)-based microfluidic devices for analysis of mono- and polyanionic molecules.
An on-column contact conductivity detector was developed for the analysis of various mono- and polyanionic compounds separated by electrophoresis chips fabricated in poly(methyl methacrylate) (PMMA) using hot embossing techniques from Ni electroforms. The detector consisted of a pair of Pt wires (127 microm diameter) with an end-to-end spacing of approximately 20 microm and situated within the fluidic channel. The waveform applied to the electrode pair was a bipolar pulse with a frequency of 5.0 kHz and was used to reduce the charging current from measurement so that the current recorded at the end of one pulse is more representative of the solution conductivity. Using the detector, separations of amino acids, peptides, proteins, and oligonucleotides were demonstrated. For the amino acids and peptides, free-solution zone electrophoresis was performed. A calibration plot for the amino acid alanine was found to be linear from approximately 10 to 100 nM in a carrier electrolyte consisting of 10 mM triethylamonium acetate. The concentration detection limit was found to be 8.0 nM, with the corresponding mass detection limit equal to 3.4 amol (injection volume = 425 pL). The protein separations with conductivity detection were performed using MEKC, in which the carrier electrolyte contained the anionic surfactant sodium dodecyl sulfate (SDS) above its cmc. Near baseline resolution was achieved in the PMMA microchip for a solution containing 8 different proteins. In the case of the DNA fragments, capillary electrochromatography was used with a C18-modified PMMA chip and a carrier electrolyte containing an ion-pairing agent.
Amino Acids↗
Micropreparative fraction collection in microfluidic devices.
Micropreparative fraction collection following microchip-based electrophoretic analysis of biomolecules is of major importance for a variety of biomedical applications. In this paper, we present a microfabricated device-based fraction collection system. Various size DNA fragments were separated and collected by simply redirecting the desired portions of the detected sample zones to corresponding collection wells using appropriate voltage manipulations. The efficiency of sampling and collection of the fractions was enhanced by placing a cross channel at or downstream of the detection point. Following the detection of the band of interest, the potentials were reconfigured to sampling/collection mode, so that the selected sample zone migrated to the appropriate collection well of the microdevice. The potential distribution assured that the rest of the analyte components in the separation column was retarded, stopped, or reversed, increasing in this way the spacing between the sample zone being collected and the immediately following one. By this means, a precise collection of spatially close consecutive bands could be facilitated. Once the target sample fraction reached the corresponding collection well, the potentials were switched back to separation mode. Alternation of the separation/detection and sampling/collection cycles was repeated until all required sample zones were physically isolated. The integrated device consists of a sample introduction, separation, fraction sampling, and fraction collection compartments. The feasibility of the fraction collection technique was tested on a mixture of dsDNA fragments. The amounts of DNA collected in this way were enough for further downstream sample processing, such as conventional PCR-based analysis.
Chemical Fractionation↗
Microfluidics: controlling fluids in small places.
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Journal Article↗
Gradients from a microfluidic "Christmas tree".
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News↗
SERRS. In situ substrate formation and improved detection using microfluidics.
Surface-enhanced resonance Raman scattering (SERRS) of a model derivative of TNT was detected using a microflow cell designed within the framework of the lab-on-a-chip concept, using only the analyte and readily available reagents. The SERRS substrate, silver colloid, was prepared in situ, on-chip, by borohydride reduction of silver nitrate. The silver colloid was imaged within the chip using a white light microscope in either transmission or, due to the high reflectivity of the colloid, reflection mode. A fine stream of colloid approximately 30 microm in width was formed in a 250-microm-wide channel at the point where the colloid preparation reagents met. The chip was designed to produce a concentrated stream of colloid within a laminar regime, such that particles did not readily disperse into the fluid. One result of this was to reduce the effective volume of analysis. Attempts to deliberately disrupt this stream with microstructured pillars, fabricated in the fluidic channels, were unsuccessful. The chip was also designed to have the appropriate dimensions for detection using a modern Raman microscope system, which collects scattering from a very small volume. A dye derived from TNT was used as a model analyte. Quantitative behavior was obtained over 4 orders of magnitude with a detection limit of 10 fmol. This performance is between 1 and 2 orders of magnitude better than that achieved using a macroflow SERRS cell. The technique has the added advantage that both reagent consumption and effluent production are greatly reduced, leading to reduced operating costs and a decreased environmental impact
Journal Article↗
Principles of surface-directed liquid flow in microfluidic channels.
To direct liquid flow inside microchannels, surface free energies were patterned by use of self-assembled monolayers (SAMs) in combination with either multistream laminar flow or photolithography. For the photolithographic method, two photocleavable SAMs were designed and synthesized. Carboxylic acid-terminated monolayers were obtained by photodeprotection, which was confirmed by contact angle and X-ray photoelectron spectroscopy. Using either of these patterning methods, we show that aqueous liquids flow only along the hydrophilic pathways when the pressure is maintained below a critical value; the liquids are referred to as being confined by virtual walls. Several principles of liquid flow in surface-patterned channels were derived analytically and verified experimentally. These principles include the maximum pressure that virtual walls can withstand, the critical width of the hydrophilic pathway that can support spontaneous flow, the smallest width of the liquid streams under an external pressure, the critical radius of curvature of turns that can be introduced into the hydrophilic pathway without liquid crossing the hydrophilic-hydrophobic boundary, and the minimal distance for two liquid streams to remain separated under the maximum pressure. Experimental results are in good agreement with the analytical predictions.
Biotechnology↗
Selective electroless and electrolytic deposition of metal for applications in microfluidics: fabrication of a microthermocouple.
This paper describes a general strategy for the fabrication of a microthermocouple based on the spatially defined electroless deposition of metal, followed by annealing and electroplating. We present scanning electron microscopy and atomic force microscopy characterizations of the deposition and annealing process, as well as the performance of the microfabricated Ni-Ag thermocouple. The temperature-voltage curve for this Ni-Ag microthermocouple is linear over the range 0-50 degrees C with a slope of 61.9 degrees C mV(-1). The sensitivity of our temperature measurement, which is limited by the uncertainty of our calibration curve, is approximately 1 degrees C. The optimum figure of merit (Z(opt)) is 1.0 x 10(-5) for this type of Ag-Ni thermocouple. We have fabricated microthermocouples ranging in size from 50 to 300 microm. The microthermocouple was integrated into microchannels and used to measure the in-channel temperature rise caused by the following: (1) a simple acid-base reaction, HCl + NaOH --> H2O + NaCl, and (2) an enzyme-catalyzed biochemical reaction, H2O2 + catalase --> H2O + 1/2 O2. We have also profiled the temperature increase in the presence of electroosmotic flow for a 100-, 200-, and 300-microm channel.
Journal Article↗
Plastic advances microfluidic devices.
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Chemistry Techniques, Analytical↗
Sample filtration, concentration, and separation integrated on microfluidic devices.
Microfabricated devices integrating sample filtration, solid-phase extraction, and chromatographic separation with solvent programming were demonstrated. Filtering of the sample was accomplished at the sample inlet with an array of seven channels each 1 microm deep and 18 microm wide. Sample concentration and separation were performed on channels 5 microm deep and 25 microm wide coated with a C18 phase, and elution was achieved under isocratic, step, or linear gradient conditions. For the solid-phase extraction, signal enhancement factors of 400 over a standard injection of 1.0 s were observed for a 320-s injection. Four polycyclic aromatic compounds were resolved by open channel electrochromatography in under 50 s. Chip operation was unaffected by the presence of the 5-microm silica particles at the filter entrance.
Journal Article↗
Microfluidic temperature gradient focusing.
A new technique is described for the concentration and separation of ionic species in solution within microchannels or capillaries. Concentration is achieved by balancing the electrophoretic velocity of an analyte against the bulk flow of solution in the presence of a temperature gradient. With an appropriate buffer, the temperature gradient can generate a corresponding gradient in the electrophoretic velocity, so that the electrophoretic and bulk velocities sum to zero at a unique point, and the analyte will be focused at that point. The technique is demonstrated for a variety of analytes, including fluorescent dyes, amino acids, DNA, proteins, and particles, and is shown to be capable of greater than 10,000-fold concentration of a dilute analyte.
Journal Article↗
Three-dimensional microfluidic confinement for efficient sample delivery to biosensor surfaces. application to immunoassays on planar optical waveguides.
A microchip-based flow confinement method for rapid delivery of small sample volumes to sensor surfaces is described. For flow confinement, a sample flow is joined with a perpendicular makeup flow of water or sample medium. Under laminar flow conditions, the makeup flow confines the sample into a thin layer above the sensing area and increases its velocity. This can benefit mass transport limited processes such as DNA hybridization or heterogeneous immunoassays. For proof of concept, this method was applied to a high-affinity immunoassay with excess capture antibody. Rabbit IgG was immobilized onto a silicon nitride waveguide. Cy5-labeled anti-rabbit IgG was hydrodynamically pumped over the immobilized zone through an attached 3D-PDMS flow cell with 20-microm-deep microchannels. The degree of confinement was adjusted through the volume flow rate of the confining flow. Evanescent field-based fluorescence detection enabled monitoring of the binding event. Assays were allowed to reach equilibrium to enable sensorgram normalization for inter-run comparison. The corresponding assay completion times could be reduced from 55 min for static drop conditions to 13 min for 25:1 flow confinement (ratio of confining to sample flow). For typical analytical applications, where equilibrium formation is not required, the faster response should translate to very short analysis times. Concurrently with the faster binding, sample consumption was reduced by 96% compared to conventional whole-channel sample delivery. Diffusional loss of analyte into the confining layer was identified as the main limitation of flow confinement, particularly for long sensing pads.
Biosensing Techniques↗
Solid-phase reversible immobilization in microfluidic chips for the purification of dye-labeled DNA sequencing fragments.
In this manuscript, we discuss the use of photoactivated polycarbonate (PC) for purification of dye-labeled terminator sequencing fragments using solid-phase reversible immobilization (SPRI) prior to gel electrophoretic sorting of these DNAs. An immobilization bed for the DNA purification was produced by exposing a posted microchannel to UV radiation, which induced a surface photooxidation reaction, resulting in the production of carboxylate groups. The immobilization microchannel contained microposts to increase the loading level of DNAs to improve signal intensity without the need for preconcentration. By suspending the sequencing cocktail in an immobilization buffer (TEG/ethanol), the DNA fragments demonstrated a high affinity for this carboxylated surface. The loading density of DNAs to this activated surface was found to be 3.9 pmol cm(-2). The captured DNA could be subsequently released from the surface by incubation with ddH2O. SPRI cleanup of dye-terminator sequencing fragments using the photoactivated PC chip and slab gel electrophoresis produced a read length comparable to the conventional SPRI format, which utilized carboxylated magnetic beads and a magnetic field. The read length for the PC-SPRI format was found to be 620 bases with a calling accuracy of 98.9%. The PC-SPRI cleanup format was also integrated to a capillary gel electrophoresis (CGE) system. The PC-SPRI method was shown to effectively remove excess dye terminator from the CGE tract, but yielded lower plate numbers, as compared to a direct injection method with purification accomplished off-chip. The loss in efficiency was found to result primarily from the extended injection time associated with the microchip purification method.
Base Sequence↗