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Biomedical subjects

David R Walt

Publications and source records attributed to David R Walt.

12 recordsLinked to original sources

Enhancing vapor sensor discrimination by mimicking a canine nasal cavity flow environment.

This paper discusses the use of flow dynamics in a model nasal cavity as a novel way to expand the information available from a fiber optic vapor sensor. Nasal cavities are known to play an important role in vertebrate olfaction; however, they have not previously been incorporated into gas-phase sensor technology. By exposing identical sensors to different flow environments, we found that measurements of the combined sensor responses provided increased discrimination of a series of alcohols as compared to measurements obtained from a single sensor at the anterior of the cavity. This methodology is potentially useful for improving the discriminatory power of sensors employed in "artificial noses" and could be used to model the effects of flow dynamics in vertebrate olfaction.

1-Propanol↗

Optical imaging fiber-based live bacterial cell array biosensor.

A live cell array biosensor was fabricated by immobilizing bacterial cells on the face of an optical imaging fiber containing a high-density array of microwells. Each microwell accommodates a single bacterium that was genetically engineered to respond to a specific analyte. A genetically modified Escherichia coli strain, containing the lacZ reporter gene fused to the heavy metal-responsive gene promoter zntA, was used to fabricate a mercury biosensor. A plasmid carrying the gene coding for the enhanced cyan fluorescent protein (ECFP) was also introduced into this sensing strain to identify the cell locations in the array. Single cell lacZ expression was measured when the array was exposed to mercury and a response to 100nM Hg(2+) could be detected after a 1-h incubation time. The optical imaging fiber-based single bacterial cell array is a flexible and sensitive biosensor platform that can be used to monitor the expression of different reporter genes and accommodate a variety of sensing strains.

Adenosine Triphosphatases↗

The use of optical fiber bundles combined with electrochemistry for chemical imaging.

The present Review describes the progress made in using imaging optical fiber bundles for fluorescence and electrochemical-initiated chemiluminescence imaging. A novel optoelectrochemical micro-ring array has been fabricated and demonstrated for concurrent electrochemical and optical measurements. The device comprises optical fibers coated with gold via electroless gold deposition and assembled in a random array format. The design yielded an array of approximately 200 micro-ring electrodes, where interdiffusional problems were minimized. The inner diameter of the ring electrode is fixed by the diameter of the individual optical fibers (25 microns), while the outer radius is determined by the thickness of the deposited gold. While all the fibers are optically addressable, they are not all electrochemically addressable. The resolution of this device is in the tens of micrometers range, determined by the diameter of the optical fiber (25 microns) and by the spacing between each electrically connected fiber. For the purpose of having well-behaved microelectrode characteristics, this spacing was designed to be larger than 60 microns. The array was characterized using ferrocyanide in aqueous solution as a model electroactive species to demonstrate that this microelectrode array format exhibits steady-state currents at short response times. This device has potential application to be used as an optoelectronic sensor, especially for the electrolytic generation and transmission of electrochemiluminescence, and was used to demonstrate that electrochemically generated luminescent products can be detected with the fiber assembly.

Diagnostic Imaging↗

High-density, microsphere-based fiber optic DNA microarrays.

A high-density fiber optic DNA microarray has been developed consisting of oligonucleotide-functionalized, 3.1-microm-diameter microspheres randomly distributed on the etched face of an imaging fiber bundle. The fiber bundles are comprised of 6000-50000 fused optical fibers and each fiber terminates with an etched well. The microwell array is capable of housing complementary-sized microspheres, each containing thousands of copies of a unique oligonucleotide probe sequence. The array fabrication process results in random microsphere placement. Determining the position of microspheres in the random array requires an optical encoding scheme. This array platform provides many advantages over other array formats. The microsphere-stock suspension concentration added to the etched fiber can be controlled to provide inherent sensor redundancy. Examining identical microspheres has a beneficial effect on the signal-to-noise ratio. As other sequences of interest are discovered, new microsphere sensing elements can be added to existing microsphere pools and new arrays can be fabricated incorporating the new sequences without altering the existing detection capabilities. These microarrays contain the smallest feature sizes (3 microm) of any DNA array, allowing interrogation of extremely small sample volumes. Reducing the feature size results in higher local target molecule concentrations, creating rapid and highly sensitive assays. The microsphere array platform is also flexible in its applications; research has included DNA-protein interaction profiles, microbial strain differentiation, and non-labeled target interrogation with molecular beacons. Fiber optic microsphere-based DNA microarrays have a simple fabrication protocol enabling their expansion into other applications, such as single cell-based assays.

Equipment Design↗

Fluorescence-based fibre optic arrays: a universal platform for sensing.

Optical fibres provide a universal sensing platform as they are easily integrated with a multitude of different sensing schemes. Such schemes enable the preparation of a multitude of sensors from relatively straightforward pH sensors, to more complex ones, including artificial olfaction sensors, high-density oligonucleotide arrays, and high-throughput cell-based arrays. Imaging fibre bundles comprised of thousands of fused optical fibres are the basis for an optically connected, individually addressable parallel sensing platform. Fibre optic imaging bundles possess miniature feature sizes (3-10 micron diameter fibres), allowing high-density sensor packing (approximately 2 x 10(7) sensors per cm2). Imaging fibre bundles transmit coherent images enabling combined imaging and sensing, relating the responses monitored by the sensor to observable physical changes. The individual fibre cores can also be selectively etched to form a high-density microwell array capable of housing complementary sized microsensors. The miniature feature sizes facilitate a faster response and more sensitive measurement capabilities. The platform is extremely versatile in its sensing design, allowing the sensing scheme to be tailored to fit the experimental design, whether for monitoring single analytes or more complex multiplexed assays. A number of sensing schemes and applications are described in this review.

Biosensing Techniques↗

Automatic decoding of sensor types within randomly ordered, high-density optical sensor arrays.

In this paper automatic sensor identification of sensor classes within a high-density randomized array, without a priori knowledge of sensor locations, is demonstrated. Two different fluorescence-based sensor types, with hundreds of replicates each, were randomly distributed into an optical imaging fiber array platform. The sensor element types were vapor-sensitive microspheres with the environmentally-sensitive fluorescent dye Nile Red adsorbed on their surface. Nile Red undergoes spectral changes when exposed to different microenvironmental polarity conditions, e.g. microsphere surface polarity or odor exposure. These reproducible sensor spectral changes, or sensor-response profiles, enable sensors within a randomized array to be grouped into categories by optical decoding methods. Two computational decoding methods (supervised and unsupervised) are introduced; equal classification rates were achieved for both. By comparing sensor responses from a randomized array with those obtained from known (control) arrays, 587 sensors were correctly classified with 99.32% accuracy. Although both methods were equally effective, the unsupervised method, which uses sensor response changes to odor exposure, is a better decoding model for the vapor-sensitive arrays studied, because it relies only on the odor-response profiles. Another decoding technique employed the emission spectra of the sensors and is more applicable to other types of multiplexed fluorescence-based arrays and assays. The sensor-decoding techniques are compared to demonstrate that sensors within high-density optical chemosensor arrays can be positionally-registered, or decoded, with no additional overhead in time or expense other than collecting the sensor-response profiles.

Journal Article↗

Optical imaging fiber-based single live cell arrays: a high-density cell assay platform.

A high-density, ordered array containing thousands of microwells is fabricated on an optical imaging fiber. Each individually addressable microwell is used to accommodate a single living cell. A charged coupled device (CCD) detector is employed to monitor and spatially resolve the fluorescence signals obtained from each individual cell, allowing simultaneous monitoring of cellular responses of all the cells in the array using reporter genes (lacZ, EGFP, ECFP, DsRed) or fluorescent indicators. Yeast and bacteria cell arrays were fabricated and used to perform multiplexed cell assays with resolution at the single-cell level. Monitoring gene expression in single yeast cells carrying a two-hybrid system was used to detect in vivo protein-protein interactions. The single-cell array technology provides a new platform for monitoring the unique multiple responses of large populations of individual cells from different strains or cell lines. The rich data acquired by the cell array has the potential to be employed as a new tool for cell biology research as well as to improve cell-based high-throughput screening (HTS) applications, such as the validation of new disease-associated cellular targets and the early-stage evaluation of potential drug candidates.

Escherichia coli↗

High-density fiber-optic genosensor microsphere array capable of zeptomole detection limits.

The detection limit of a fiber-optic microsensor array was investigated for simultaneous detection of multiple DNA sequences. A random array composed of oligonucleotide-functionalized 3.1-microm-diameter microspheres on the distal face of a 500-microm etched imaging fiber was monitored for binding to fluorescently labeled complementary DNA sequences. Inherent sensor redundancy in the microarray allows the use of multiple microspheres to increase the signal-to-noise ratio, further enhancing the detection capabilities. Specific hybridization was observed for each of three sequences in an array yielding a detection limit of 10(-21) mol (approximately 600 DNA molecules).

Animals↗

Fabrication of an optoelectrochemical microring array.

In this paper, we describe a novel approach for fabricating an optoelectrochemical microring array. The array was fabricated by coating individual optical fibers of 25-microm diameter with a 1-microm layer of gold nanoparticles via electroless gold deposition. A SAM layer around the individual gold-coated imaging fibers prevented electrical contact with neighboring ring electrodes. To achieve better mechanical stability and to make the device more practical, the electrode/fiber bundle comprising approximately 600 individual gold-coated optical fibers was dipped into epoxy. By polishing the ends of such a device, a ring microelectrode array comprising 600 individual and insulated ring electrodes was formed. To limit diffusional overlap of current, only 20-30% of the microring fiber/electrodes were wired. The inner diameter of the ring electrode is fixed by the diameter of the individual optical fibers (25 microm), while the outer radius is determined by the thickness of the deposited gold. The array was characterized using ferrocyanide in aqueous solution as a model electroactive species to demonstrate that this microelectrode array format exhibits steady-state currents at short response times. In addition, cyclic voltammetry experiments were performed using conventional potentiostats due to the amplification of current inherent in the array format. Finally, electrochemiluminescence at the ring electrode array was demonstrated through the oxidation of Ru(bpy)3(2+) in tri-n-propylamide in a pH 7 phosphate buffer solution, where the light generated was collected and detected via the fiber bundle.

Electrochemistry↗

Aluminum surface corrosion and the mechanism of inhibitors using pH and metal ion selective imaging fiber bundles.

The localized corrosion behavior of a galvanic aluminum copper couple was investigated by in situ fluorescence imaging with a fiber-optic imaging sensor. Three different, but complementary methods were used for visualizing remote corrosion sites, mapping the topography of the metal surface, and measuring local chemical concentrations of H+, OH-, and Al3+. The first method is based on a pH-sensitive imaging fiber, where the fluorescent dye SNAFL was covalently attached to the fiber's distal end. Fluorescence images were acquired as a function of time at different areas of the galvanic couple. In a second method, the fluorescent dye morin was immobilized on the fiber-optic imaging sensor, which allowed the in situ localization of corrosion processes on pure aluminum to be visualized over time by monitoring the release of Al3+. The development of fluorescence on the aluminum surface defined the areas associated with the anodic dissolution of aluminum. We also investigated the inhibition of corrosion of pure aluminum by CeCl3 and 8-hydroxyquinoline. The decrease in current, the decrease in the number of active sites on the aluminum surface, and the faster surface passivation are all consistent indications that cerium chloride and 8-hydroxyquinoline inhibit corrosion effectively. From the number and extent of corrosion sites and the release of aluminum ions monitored with the fiber, it was shown that 8-hydroxyquinoline is a more effective inhibitor than cerium chloride.

Aluminum↗

Imaging optical sensor arrays.

Imaging optical fibres have been etched to prepare microwell arrays. These microwells have been loaded with sensing materials such as bead-based sensors and living cells to create high-density sensor arrays. The extremely small sizes and volumes of the wells enable high sensitivity and high information content sensing capabilities.

Fiber Optic Technology↗