Biomedical subjects

Wyatt N Vreeland

Publications and source records attributed to Wyatt N Vreeland.

12 recordsLinked to original sources

PubMed2006

Scanning temperature gradient focusing.

Temperature gradient focusing (TGF) is a recently developed technique for the simultaneous concentration and electrophoretic separation of ionic analytes in microfluidic channels. One drawback to TGF as it has previously been described is the limited peak capacity; only a small number of analyte peaks (approximately 2-3) can be simultaneously focused and separated. In this paper, we report on a variation of the TGF method whereby the bulk flow rate is varied over time so that a large number of analytes can be sequentially focused, moved past a fixed detection point, and flushed to waste. In addition to improved peak capacity, the detection limits of the scanning TGF method can be adjusted on-the-fly, as needed for different samples. Finally, scanning TGF provides a technique by which high-resolution, high-peak-capacity electrophoretic separations can be performed in simple, straight, and short microfluidic channels.

Journal Article↗

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↗

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

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.

Buffers↗

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

Micellar affinity gradient focusing: a new method for electrokinetic focusing.

This report describes a new method for the concentration and separation of neutral and/or hydrophobic analytes based on a combination of the analytes' electrophoretic mobility, and affinity for partitioning into a micellar phase. Micellar affinity gradient focusing (MAGF) works by creating a gradient in the micellar retention factor. An electric field is applied along the channel to cause the (negatively charged) micelles to move from the region of high retention to the region of low retention, and the mobile phase is forced to move from the region of low retention to the region of high retention. Consequently, the analyte moves into the gradient region from both directions where it is concentrated at a point where its total velocity is zero. Different analytes, which interact differently with the micelles, will have zero total velocity at different points along the gradient, and will thereby be simultaneously concentrated and separated.

Journal Article↗

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

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.

Buffers↗

PubMed2002

Multiplexed, high-throughput genotyping by single-base extension and end-labeled free-solution electrophoresis.

Technologies that allow for high-throughput, economical, and accurate single nucleotide polymorphism (SNP) genotyping are becoming crucial for modern genomic efforts. Here, we present a method for multiplexed single-base extension (SBE) genotyping that takes advantage of the unique separation modalities made possible via end-labeled free-solution electrophoresis (ELFSE). Three unique SBE oligonucleotide primers, which probe for mutations of clinical importance in the human p53 gene, were covalently conjugated to three unique polypeptoid frictional end labels and mixed together. This primer-polypeptoid conjugate cocktail was then used in a multiplexed SBE reaction followed by free-solution separation in a 96-capillary array electrophoresis (CAE) instrument. The study was designed to demonstrate multiplexed SNP genotyping of several loci in a single reaction and a single subsequent analysis. Further, the electrophoretic analysis was conducted without any viscous polymeric separation media, was complete in less than 10 min, and can be implemented in any capillary or microfluidic electrophoretic system with four-color fluorescent detection capabilities. Multiplexed SBE-ELFSE genotyping analysis resulted in the simultaneous and accurate genotyping of three p53 loci on five different DNA templates in a single reaction set and single CAE analysis. With the implementation of this method in 96 or more capillaries in parallel, high-throughput screening of SNPs will be accessible to a large number of laboratories.

DNA Primers↗

PubMed2002

Functional materials for microscale genomic and proteomic analyses.

The design of functional materials for genomic and proteomic analyses in microscale systems has begun to mature, from materials designed for capillary-based electrophoresis systems to those tailored for microfluidic-based or 'chip-based' platforms. In particular, recent research has focused on evaluating different polymer chemistries for microchannel surface passivation and improved DNA separation matrix performance. Additionally, novel bioconjugate materials designed specifically for electrophoretic separations in microscale channels are facilitating new separation modalities.

Acrylamides↗

PubMedDate not supplied

Profiling solid-phase synthesis products by free-solution conjugate capillary electrophoresis.

Solid-phase synthesis of oligomers, both natural and nonnatural, has proved to be invaluable for the development of many areas of biotechnology. A critical step in the solid-phase synthesis of any oligomer is determining the number and concentration of different constituents present in the product mixture resulting from the synthesis, both before and after purification. Most typically, this analysis is performed by reversed-phase high performance liquid chromatography (RP-HPLC), with the separated components detected by UV absorbance. Recently, we described a novel technique, free-solution conjugate electrophoresis (FSCE), for the high-resolution separation and sensitive laser-induced fluorescence (LIF) detection of uncharged, synthetic polymers, PEG in particular. In this report, we apply this bioconjugate capillary electrophoresis technique to analyze products of the solid-phase synthesis of oligomeric polyamides, namely poly(N-substituted glycines), or polypeptoids. When compared to more traditional RP-HPLC analysis, FSCE analysis of oligomeric peptoids results in separation resolutions that are approximately five times higher and separation efficiencies that are increased by 150%. Moreover, when FSCE with LIF detection is applied to the analysis of oligomeric polyamides after HPLC purification, impurities that are not detectable in RP-HPLC analysis are readily separated and detected. With the advent of capillary array electrophoresis (CAE), which allows for automated, parallel analysis of many different samples, we believe that FSCE will be especially applicable to the analysis of combinatorial synthesis products, by allowing researchers to evaluate many different samples in a single, highly parallel, fully automated analysis. This is in contrast to RP-HPLC analysis, in which samples must be analyzed in series.

Chromatography, High Pressure Liquid↗