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Biomedical subjects

Laurie E Locascio

Publications and source records attributed to Laurie E Locascio.

At least 19 recordsLinked to original sources
PubMed2006

Cellular immobilization within microfluidic microenvironments: dielectrophoresis with polyelectrolyte multilayers.

The development of biomimetic microenvironments will improve cell culture techniques by enabling in vitro cell cultures that mimic in vivo behavior; however, experimental control over attachment, cellular position, or intercellular distances within such microenvironments remains challenging. We report here the rapid and controllable immobilization of suspended mammalian cells within microfabricated environments using a combination of electronic (dielectrophoresis, DEP) and chemical (polyelectrolyte multilayers, PEMS) forces. While cellular position within the microsystem is rapidly patterned via intermittent DEP trapping, persistent adhesion after removal of electronic forces is enabled by surface treatment with PEMS that are amenable to cellular attachment. In contrast to DEP trapping alone, persistent adhesion enables the soluble microenvironment to be systematically varied, facilitating the use of soluble probes of cell state and enabling cellular characterization in response to various soluble stimuli.

Animals↗
PubMed2006

Facilitating the culture of mammalian nerve cells with polyelectrolyte multilayers.

When neuron-like cells (NLCs) derived from pluripotent embryonal carcinoma cells (P19) are cultured on bare tissue culture substrates, they require a monolayer of fibroblast cells to exhibit normal neurite outgrowth, behavior typical of neuronal cultures. However, substrate treatment with polyelectrolyte multilayers (PEMs) composed of poly(allylamine hydrochloride) (PAH) and poly(styrenesulfonic acid) (PSS) significantly improved these cultures. Cell morphology was more spread, indicative of healthy cells, and direct attachment of neuronal cell bodies to the treated surface was observed. Neuronal outgrowth across the surface was not dependent on an underlying fibroblast monolayer with the PEMs surface treatment. Additionally, the PEMs surface treatment can be used to condition various surfaces, facilitating neuronal cultures on surfaces which are natively hydrophilic (tissue culture polystyrene) or hydrophobic (poly(dimethylsiloxane), PDMS). Microfluidic networks were used to micropattern the PEMs onto PDMS, resulting in confined regions of cellular attachment and directed neuronal outgrowth. The ability of PEMs to encourage NLC attachment without supporting cells to a variety of surfaces and surface geometries greatly simplifies neuronal culture methodology and enables neuronal investigations in new environments.

Animals↗
PubMed2006

Capillarity induced solvent-actuated bonding of polymeric microfluidic devices.

Rapid, robust, and economical fabrication of fluidic microchannels is of fundamental importance for the successful development of disposable lab-on-a-chip devices. In this work, we present a solvent-actuated bonding method for fabricating polymeric microfluidic devices at room temperature. A PMMA sheet with an imprinted microchannel was clamped to a blank PMMA sheet, and then 80 +/- 5 muL of acetone (bonding solvent) was introduced at one end of the fluidic channel and aspirated out at the other end. As the solvent moved down the channel, capillary forces drew a fraction of the solvent into the interstitial space between the two polymeric substrates. After aspiration, the assembly was incubated in the clamp for 5 min for effective bond formation. The quantity of the bonding solvent, its water content and flow rate, along with residence time in the channel were found to have significant impact on the bond quality and the channel integrity. Microfluidic electrophoretic separations of a 400-base DNA ladder were performed in devices fabricated using this method in less than 8 min with efficiencies routinely between 2 x 10(6) and 3 x 10(6) plates/m. The simplicity and economy of this technique make it amenable for automation and mass production, which could make polymeric substrates more attractive for single-use chemical analysis devices.

Journal Article↗
PubMed2006

Surface modification of poly(methyl methacrylate) for improved adsorption of wall coating polymers for microchip electrophoresis.

The development of rapid and simple wall coating strategies for high-efficiency electrophoretic separation of DNA is of crucial importance for the successful implementation of miniaturized polymeric DNA analysis systems. In this report, we characterize and compare different methods for the chemical modification of poly(methyl methacrylate) (PMMA) surfaces for the application of wall coating polymers. PMMA surfaces coated with 40 mol% diethylacrylamide and 60 mol% dimethylacrylamide are compared to the PMMA surfaces first oxidized and then coated with hydroxypropylmethyl-cellulose or poly(vinyl alcohol) (PVA). PMMA oxidation was accomplished with UV/ozone or an aqueous solution of HNO(3) to yield hydrogen-bond donors for the spontaneous adsorption of the coating polymers. Contact angle measurements of UV/ozone exposed PMMA surfaces indicate increase in hydrophilicity, and polymer coated surfaces show a strong dependence on the coating polymer and the oxidation method. Fast and repeatable electrophoretic separations of a 10-base and 20-base DNA ladder were performed in PMMA micro CE devices. All analyses were completed in less than 10 min, resulting in the number of theoretical plates as high as 583 000 in a 7.7 cm long separation channel. The duration of UV/ozone treatment was found to have a considerable impact on separation performance. The microchips irradiated with UV for 10 min and coated with PVA as well as the microchips treated with HNO(3) and coated with HPMC were found to have the best separation performance. These results demonstrate facile and robust methods for the surface modification of PMMA enabling low-cost single use devices for electrophoretic DNA separations.

Acrylamides↗
PubMed2006

Hydrogel-immobilized antibodies for microfluidic immunoassays: hydrogel immunoassay.

The integration of immunoassays in microfluidic devices is a rapidly developing research area combining the power of immunoassays with the inherent benefits of microfluidics. Here, a general overview of microfluidic-based immunoassays is presented along with a method for immobilizing antibodies in polyacrylamide gel plugs set in microfluidic channels. These antigen-specific hydrogels can be rapidly formed by photopolymerizing monomer solutions mixed with antibodies or other large proteins. The resulting antigen-specific hydrogels contain pore sizes appropriate for physical entrapment of large antibodies while remaining permeable to smaller proteins. The open structure of these hydrogels enables the capture and concentration of target antigens present at low concentrations. Such physical entrapment provides a conceptually simple method of immobilization compared with immobilization of proteins on surfaces and offers advantages such as resistance to chemical and thermal denaturation.

Acrylic Resins↗
PubMed2006

Fabrication of polymer microfluidic systems by hot embossing and laser ablation.

Fabrication of microfluidic channels in common commercially available thermoplastic materials can be easily accomplished using hot embossing or ultraviolet (UV) laser ablation. Hot embossing involves replication of a microfluidic network in a polymer substrate from a stamp (or template) fabricated in silicon or metal. UV laser ablation is performed by either exposing the polymer substrate through a mask or by using a laser direct-write process. The resulting polymer microfluidic channels are most often sealed with another polymer piece using thermal bonding or solvent bonding to complete the fabrication procedure. Unlike their silicon and glass counterparts, polymer microfluidic systems can be fabricated by these methods in less than 1 h, making the materials attractive for both research prototyping and commercialization.

Equipment Design↗
PubMed2005

Directed growth of pure phosphatidylcholine nanotubes in microfluidic channels.

The morphology of self-assembled phospholipid membranes (e.g., micelles, vesicles, rods, tubes, etc.) depends on the method of formation, secondary manipulation, temperature, and storage conditions. In this contribution, microfluidic systems are used to create pure phosphatidylcholine (PC) micro- and nanotubes with unprecedented lengths. Tubes up to several centimeters in length and aligned with the long axis of the microchannel were created from spots of dry films of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). These high aspect ratio structures, which, to our knowledge, represent the first examples of extended tubes formed from pure PC lipids, were examined by fluorescence microscopy, electron and optical microscopy, and optical manipulation tools (i.e., a laser trap and laser scalpel) to characterize structure and stability. In particular, the tubular structure was confirmed by observation of fluorescent dyes that were sequestered within the aqueous cavity or within the phospholipid tube. Compared to other phospholipid tubes, the tubes formed from PC lipids in microfluidic channels show high mechanical stability and rigidity that depend on tube size, age, and storage conditions.

Microfluidics↗
PubMed2004

Micropatterning neuronal cells on polyelectrolyte multilayers.

This paper describes an approach to adhere retinal cells on micropatterned polyelectrolyte multilayer (PEM) lines adsorbed on poly(dimethylsiloxane) (PDMS) surfaces using microfluidic networks. PEMs were patterned on flat, oxidized PDMS surfaces by sequentially flowing polyions through a microchannel network that was placed in contact with the PDMS surface. Polyethyleneimine (PEI) and poly(allylamine hydrochloride) (PAH) were the polyions used as the top layer cellular adhesion material. The microfluidic network was lifted off after the patterning was completed and retinal cells were seeded on the PEM/PDMS surfaces. The traditional practice of using blocking agents to prevent the adhesion of cells on unpatterned areas was avoided by allowing the PDMS surface to return to its uncharged state after the patterning was completed. The adhesion of rat retinal cells on the patterned PEMs was observed 5 h after seeding. Cell viability and morphology on the patterned PEMs were assayed. These materials proved to be nontoxic to the cells used in this study regardless of the number of stacked PEM layers. Phalloidin staining of the cytoskeleton revealed no apparent morphological differences in retinal cells compared with those plated on polystyrene or the larger regions of PEI and PAH; however, cells were relatively more elongated when cultured on the PEM lines. Cell-to-cell communication between cells on adjacent PEM lines was observed as interconnecting tubes containing actin that were a few hundred nanometers in diameter and up to 55 microm in length. This approach provides a simple, fast, and inexpensive method of patterning cells onto micrometer-scale features.

Animals↗
PubMed2004

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.

Journal Article↗
PubMed2004

Controlled vesicle self-assembly in microfluidic channels with hydrodynamic focusing.

Traditional liposome preparation methods are based on mixing of bulk phases, leading to inhomogeneous chemical and/or mechanical conditions during formation; hence liposomes are often polydisperse in size and lamellarity. Here we show the formation of liposomes that encapsulate reagents in a continuous two-phase flow microfluidic network with precision control of size from 100 to 300 nm by manipulation of liquid flow rates. We demonstrate that by creating a solvent-aqueous interfacial region in a microfluidic format that is homogeneous and controllable on the length scale of a liposome, we can facilitate the fine control of liposome size and polydispersity.

Fluoresceins↗
PubMed2004

Peak compression and resolution for electrophoretic separations in diverging microchannels.

We report the results of experiments and simulations on electrokinetic flow in diverging microchannels (with cross-sectional area that increases with distance along the channel). Because of conservation of mass and charge, the velocity of an analyte in the channel decreases as the channel cross-section increases. Consequently, the leading edge of a band of sample moves more slowly than the trailing edge and the sample band is compressed. Sample peak widths, rather than increasing diffusively with time, can then be controlled by the geometry of the channel and can even be made to decrease with time. We consider the possibility of using this peak compression effect to improve the resolution of electrophoretic separations. Our results indicate that for typical separations that are dispersion limited, this peak compression effect is more than offset by the decreased distance between peaks, and the separation resolution in diverging channels is worse than that found for straight channels at the same applied voltage. For separations in very short channels or at very high field strengths, however, when the separation efficiency is injection limited, the peak compression effect is dominant and diverging channels can then be used to achieve improved separation resolution.

Electrophoresis, Microchip↗
PubMed2003

Using bioinspired thermally triggered liposomes for high-efficiency mixing and reagent delivery in microfluidic devices.

High-efficiency mixing is of fundamental importance for the successful development and application of lab-on-a-chip devices. In this report, we present the use of bioinspired thermally triggered liposomes for the controlled delivery and subsequent rapid mixing of reagents in a microfluidic device. In this technique, reagents are encapsulated inside the aqueous interior of liposomes that are dispersed evenly throughout a microfluidic system. Mixing of the encapsulated reagent and reaction do not occur until the reagent is released by a thermal trigger. This approach takes advantage of the dramatically increased lipid membrane permeability of liposomes near the gel-to-liquid phase transition temperature (T(m)) to deliver reagents at a precise location in the microfluidic device through the modulation of temperature. Implementation of this technique requires the encapsulation of the desired reagent in a liposome whose formulation has an appropriate T(m), as well as accurate spatial control of the temperature in the microfluidic device. As the liposomes are uniformly dispersed through the microfluidic channel, mixing occurs quite rapidly upon the release of the reagent. We demonstrate this technique by using several formulations of thermally triggered liposomes to release the hydrophilic fluorescent dyes at controlled locations in a polycarbonate microfluidic device. Additionally, we demonstrate a DNA labeling reaction using liposomes in a capillary-based microfluidic device. Under the conditions studied here, mixing and reaction are complete in approximately 200 microm of channel length. We believe this approach holds great promise for the performance of rapid high-throughput assays and in particular for biological analytes whose native environment is mimicked by the liposome.

DNA↗
PubMed2003

Effect of caged fluorescent dye on the electroosmotic mobility in microchannels.

We report on measurements of electroosmotic mobility in polymer microchannels and silica capillaries with and without the addition of a caged fluorescein dye to the buffer. For PMMA microchannels, the mobility was found to increase from (2.6 +/- 0.1) x 10(-4) cm2 V(-1) s(-1) to (4.6 +/- 0.1) x 10(-4) cm2 V(-1) s(-1) upon addition of 1.2 mmol/L of caged dye. For PC microchannels, the mobility increased from (4.3 +/- 0.2) x 10(-4) cm2 V(-1) s(-1) to (5.4 +/- 0.1) x 10(-4) cm2 V(-1) s(-1) upon addition of caged dye. For PDMS microchannels, the mobility increased from (4.3 +/- 0.2) x 10(-4) cm2 V(-1) s(-1) to (6.4 +/- 0.5) x 10(-4) cm2 V(-1) s(-1) upon addition of caged dye. For fused-silica capillaries, the mobility ((5.5 +/- 0.2) x 10(-4) cm2 V(-1) s(-1)) was unaffected by the addition of the caged dye.

Journal Article↗
PubMed2002

Characterization and optimization of slanted well designs for microfluidic mixing under electroosmotic flow.

Recently, a series of slanted wells on the floor of a microfluidic channel were experimentally shown to successfully induce off-axis transport and mixing of two confluent streams when operating under electroosmotic (EO) flow. This paper will further explore, through numerical simulations, the parameters that affect off-axis transport under EO flow with an emphasis on optimizing the mixing rate of (a). two confluent streams in steady-state or (b). the transient scenario of two confluent plugs of material, which simulates mixing after an injection. For the steady-state scenario, the degree of mixing was determined to increase by changing any of the following parameters: (1). increasing the well depth, (2). decreasing the well angle relative to the axis of the channel, and (3). increasing the EO mobility of the well walls relative to the mobility of the main channel. Also, it will be shown that folding of the fluid can occur when the well angle is sufficiently reduced and/or when the EO mobility of the wells is increased relative to the channel. The optimum configuration for the transient problem of mixing two confluent plugs includes shallow wells to minimize the well residence time, and an increased EO mobility of the well walls relative to the main channel as well as small well angles to maximize off-axis transport. The final design reported here for the transient study reduces the standard deviation of the concentration across the channel by 72% while only increasing the axial dispersion of the injected plug by 8.6 % when compared to a plug injected into a channel with no wells present. These results indicate that a series of slanted wells on the wall of a microchannel provides a means for controlling and achieving a high degree of off-axis transport and mixing in a passive manner for micro total analysis system (microTAS) devices that are driven by electroosmosis.

Journal Article↗
PubMed2002

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↗
PubMed2002

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↗