Biomedical subjects

A T Woolley

Publications and source records attributed to A T Woolley.

11 recordsLinked to original sources

PubMed2005

Planar thin film device for capillary electrophoresis.

Hollow tubular microfluidic channels were fabricated on quartz substrates using sacrificial layer, planar micromachining processes. The channels were created using a bottom-up fabrication technique, namely patterning a photoresist/aluminum sacrificial layer and depositing SiO(2) over the substrate. The photoresist/aluminum layer was removed by etching first with HCl/HNO(3), followed by etching in Nano-Strip, a more stable form of piranha (H(2)SO(4)/H(2)O(2)) stripper. Rapid separation of fluorescently labeled amino acids was performed on a device made with these channels. The fabrication process presented here provides unique control over channel composition and geometry. Future work should allow the fabrication of highly complex and precise devices with integrated analytical capabilities essential for the development of micro-total analysis systems.

Acids↗

PubMed2001

Structural and functional imaging with carbon nanotube AFM probes.

Atomic force microscopy (AFM) has great potential as a tool for structural biology, a field in which there is increasing demand to characterize larger and more complex biomolecular systems. However, the poorly characterized silicon and silicon nitride probe tips currently employed in AFM limit its biological applications. Carbon nanotubes represent ideal AFM tip materials due to their small diameter, high aspect ratio, large Young's modulus, mechanical robustness, well-defined structure, and unique chemical properties. Nanotube probes were first fabricated by manual assembly, but more recent methods based on chemical vapor deposition provide higher resolution probes and are geared towards mass production, including recent developments that enable quantitative preparation of individual single-walled carbon nanotube tips [J. Phys. Chem. B 105 (2001) 743]. The high-resolution imaging capabilities of these nanotube AFM probes have been demonstrated on gold nanoparticles and well-characterized biomolecules such as IgG and GroES. Using the nanotube probes, new biological structures have been investigated in the areas of amyloid-beta protein aggregation and chromatin remodeling, and new biotechnologies have been developed such as AFM-based haplotyping. In addition to measuring topography, chemically functionalized AFM probes can measure the spatial arrangement of chemical functional groups in a sample. However, standard silicon and silicon nitride tips, once functionalized, do not yield sufficient resolution to allow combined structural and functional imaging of biomolecules. The unique end-group chemistry of carbon nanotubes, which can be arbitrarily modified by established chemical methods, has been exploited for chemical force microscopy, allowing single-molecule measurements with well-defined functionalized tips.

Animals↗

PubMed2000

Structural biology with carbon nanotube AFM probes.

Carbon nanotubes represent ideal probes for high-resolution structural and chemical imaging of biomolecules with atomic force microscopy. Recent advances in fabrication of carbon nanotube probes with sub-nanometer radii promise to yield unique insights into the structure, dynamics and function of biological macromolecules and complexes.

Animals↗

PubMed2000

Direct haplotyping of kilobase-size DNA using carbon nanotube probes.

We have implemented a method for multiplexed detection of polymorphic sites and direct determination of haplotypes in 10-kilobase-size DNA fragments using single-walled carbon nanotube (SWNT) atomic force microscopy (AFM) probes. Labeled oligonucleotides are hybridized specifically to complementary target sequences in template DNA, and the positions of the tagged sequences are detected by direct SWNT tip imaging. We demonstrated this concept by detecting streptavidin and IRD800 labels at two different sequences in M13mp18. Our approach also permits haplotype determination from simple visual inspection of AFM images of individual DNA molecules, which we have done on UGT1A7, a gene under study as a cancer risk factor. The haplotypes of individuals heterozygous at two critical loci, which together influence cancer risk, can be easily and directly distinguished from AFM images. The application of this technique to haplotyping in population-based genetic disease studies and other genomic screening problems is discussed.

Alleles↗

PubMed1998

Covalently functionalized nanotubes as nanometre-sized probes in chemistry and biology.

Carbon nanotubes combine a range of properties that make them well suited for use as probe tips in applications such as atomic force microscopy (AFM). Their high aspect ratio, for example, opens up the possibility of probing the deep crevices that occur in microelectronic circuits, and the small effective radius of nanotube tips significantly improves the lateral resolution beyond what can be achieved using commercial silicon tips. Another characteristic feature of nanotubes is their ability to buckle elastically, which makes them very robust while limiting the maximum force that is applied to delicate organic and biological samples. Earlier investigations into the performance of nanotubes as scanning probe microscopy tips have focused on topographical imaging, but a potentially more significant issue is the question of whether nanotubes can be modified to create probes that can sense and manipulate matter at the molecular level. Here we demonstrate that nanotube tips with the capability of chemical and biological discrimination can be created with acidic functionality and by coupling basic or hydrophobic functionalities or biomolecular probes to the carboxyl groups that are present at the open tip ends. We have used these modified nanotubes as AFM tips to titrate the acid and base groups, to image patterned samples based on molecular interactions, and to measure the binding force between single protein-ligand pairs. As carboxyl groups are readily derivatized by a variety of reactions, the preparation of a wide range of functionalized nanotube tips should be possible, thus creating molecular probes with potential applications in many areas of chemistry and biology.

Biology↗

PubMed1998

High-throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplates.

Capillary array electrophoresis (CAE) microplates that can analyze 96 samples in less than 8 min have been produced by bonding 10-cm-diameter micromachined glass wafers to form a glass sandwich structure. The microplate has 96 sample wells and 48 separation channels with an injection unit that permits the serial analysis of two different samples on each capillary. An elastomer sheet with an 8 by 12 array of holes is placed on top of the glass sandwich structure to define the sample wells. Samples are addressed with an electrode array that makes up the third layer of the assembly. Detection of all lanes with high temporal resolution was achieved by using a laser-excited confocal fluorescence scanner. To demonstrate the functionality of these microplates, electrophoretic separation and fluorescence detection of a restriction fragment marker for the diagnosis of hereditary hemochromatosis were performed. CAE microplates will facilitate all types of high-throughput genetic analysis because their high assay speed provides a throughput that is 50 to 100 times greater than that of conventional slab gels.

Capillary Action↗

PubMed1998

Capillary electrophoresis chips with integrated electrochemical detection.

Capillary electrophoresis systems with integrated electrochemical detection have been microfabricated on glass substrates. Photolithographic placement of the working electrode just outside the exit of the electrophoresis channel provides high-sensitivity electrochemical detection with minimal interference from the separation electric field. Microchip electrophoretic separations of neurotransmitters in under 100 s exemplify the good resolution and attomole detection sensitivity of these devices. Using indirect electrochemical detection, high-sensitivity DNA restriction fragment and PCR product sizing has also been performed. These microdevices match the detector's size to that of microfabricated separation and reaction devices, bringing to reality the lab-on-a-chip concept.

DNA↗

PubMed1997

High-speed DNA genotyping using microfabricated capillary array electrophoresis chips.

Capillary array electrophoresis (CAE) chips have been designed and fabricated with the capacity to rapidly (< 160 s) analyze 12 different samples in parallel. Detection of all lanes with 0.3 s temporal resolution was achieved using a laser-excited confocal-fluorescence scanner. The operation and capabilities of these CAE microdevices were first determined by performing electrophoretic separations of pBR322 MspI DNA samples. Genotyping of HLA-H, a candidate gene for the diagnosis of hereditary hemochromatosis, was then performed to demonstrate the rapid analysis of biologically relevant samples. Two-color multiplex fluorescence detection of HLA-H genotypes was accomplished by prelabeling the standard pBR322 MspI DNA ladder with a red emitting bis-intercalation dye (butyl TOTIN) and on-column labeling of the HLA-H DNA with thiazole orange. This work establishes the feasibility of using CAE chips for high speed, high-throughput genotyping.

Benzothiazoles↗

PubMed1996

Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device.

Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10 degrees C/s heating, 2.5 degrees C/s cooling) with the high-speed (< 120 s) DNA separations provided by microfabricated CE chips. The PCR chamber and the CE chip were directly linked through a photolithographically fabricated channel filled with hydroxyethylcellulose sieving matrix. Electrophoretic injection directly from the PCR chamber through the cross injection channel was used as an "electrophoretic valve" to couple the PCR and CE devices on-chip. To demonstrate the functionality of this system, a 15 min PCR amplification of a beta-globin target cloned in M13 was immediately followed by high-speed CE chip separation in under 120 s, providing a rapid PCR-CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was performed in under 45 min, demonstrating that challenging amplifications of diagnostically interesting targets can also be performed. Real-time monitoring of PCR target amplification in these integrated PCR-CE devices is also feasible. Amplification of the beta-globin target as a function of cycle number was directly monitored for two different reactions starting with 4 x 10(7) and 4 x 10(5) copies of DNA template. This work establishes the feasibility of performing high-speed DNA analyses in microfabricated integrated fluidic systems.

DNA↗

PubMed1995

Ultra-high-speed DNA sequencing using capillary electrophoresis chips.

DNA sequencing has been performed on microfabricated capillary electrophoresis chips. DNA separations were achieved in 50 x 8 microns cross-section channels microfabricated in a 2 in. x 3 in. glass sandwich structure using a denaturing 9% T, 0% C polyacrylamide sieving medium. DNA sequencing fragment ladders were produced and fluorescently labeled using the recently developed energy transfer dye-labeled primers. Sequencing extension fragments were separated to approximately 433 bases in only 10 min using a one-color detection system and an effective separation distance of only 3.5 cm. Using a four-color labeling and detection format, DNA sequencing with 97% accuracy and single-base resolution to approximately 150 bases was achieved in only 540 s. A resolution of greater than 0.5 was obtained out to 200 bases for both the one- and four-color separations. The prospects for enhancing the resolution and sensitivity of these chip separations are discussed. This work establishes the feasibility of high-speed, high-throughput DNA sequencing using capillary array electrophoresis chips.

Base Sequence↗

PubMed1994

Ultra-high-speed DNA fragment separations using microfabricated capillary array electrophoresis chips.

Capillary electrophoresis arrays have been fabricated on planar glass substrates by photolithographic masking and chemical etching techniques. The photolithographically defined channel patterns were etched in a glass substrate, and then capillaries were formed by thermally bonding the etched substrate to a second glass slide. High-resolution electrophoretic separations of phi X174 Hae III DNA restriction fragments have been performed with these chips using a hydroxyethyl cellulose sieving matrix in the channels. DNA fragments were fluorescently labeled with dye in the running buffer and detected with a laser-excited, confocal fluorescence system. The effects of variations in the electric field, procedures for injection, and sizes of separation and injection channels (ranging from 30 to 120 microns) have been explored. By use of channels with an effective length of only 3.5 cm, separations of phi X174 Hae II DNA fragments from approximately 70 to 1000 bp are complete in only 120 sec. We have also demonstrated high-speed sizing of PCR-amplified HLA-DQ alpha alleles. This work establishes methods for high-speed, high-throughput DNA separations on capillary array electrophoresis chips.

Cellulose↗