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Biomedical subjects

Donald M Cannon

Publications and source records attributed to Donald M Cannon.

8 recordsLinked to original sources
PubMed2005

Miniaturized lead sensor based on lead-specific DNAzyme in a nanocapillary interconnected microfluidic device.

A miniaturized lead sensor has been developed by combining a lead-specific DNAzyme with a microfabricated device containing a network of microfluidic channels that are fluidically coupled via a nanocapillary array interconnect. A DNAzyme construct, selective for cleavage in the presence of Pb2+ and derivatized with fluorophore (quencher) at the 5' (3') end of the substrate and enzyme strands, respectively, forms a molecular beacon that is used as the recognition element. The nanocapillary array membrane interconnect is used to manipulate fluid flows and deliver the small-volume sample to the beacon in a spatially confined detection window where the DNAzyme is interrogated using laser-induced fluorescence detection. A transformed log plot of the fluorescent signal exhibits a linear response (r2 = 0.982) over a Pb2+ concentration range of 0.1 - 100 microM, and a detection limit of 11 nM. The sensor has been applied to the determination of Pb2+ in an electroplating sludge reference material, the result agreeing with the certified value within 4.9%. Quantitative measurement of Pb2+ in this complex sample demonstrates the selectivity of this sensor scheme and points favorably to the application of such technologies to analysis of environmental samples. The unique combination of a DNAzyme with a microfluidic-nanofluidic hybrid device makes it possible to change the DNAzyme to select for other compounds of interest, and to incorporate multiple sensing systems within a single device for greater flexibility. This work represents the initial steps toward creation of a robust field sensor for lead in groundwater or drinking water.

Biosensing Techniques↗
PubMed2004

Influence of molecular environment on the analysis of phospholipids by time-of-flight secondary ion mass spectrometry.

Understanding the influence of molecular environment on phospholipids is important in time-of-flight secondary ion mass spectrometry (TOF-SIMS) studies of complex systems such as cellular membranes. Varying the molecular environment of model membrane Langmuir-Blodgett (LB) films is shown to affect the TOF-SIMS signal of the phospholipids in the films. The molecular environment of a LB film of dipalmitoylphosphatidylcholine (DPPC) is changed by varying the film density, varying the sample substrate, and the addition of cholesterol. An increase in film density results in a decrease in the headgroup fragment ion signal at a mass-to-charge ratio of 184 (phosphocholine). Varying the sample substrate increases the secondary ion yield of phosphocholine as does the addition of proton-donating molecules such as cholesterol to the DPPC LB film. Switching from a model system of DPPC and cholesterol to one of dipalmitoylphosphatidylethanolamine (DPPE) and cholesterol demonstrates the ability of cholesterol to also mask the phospholipid headgroup ion signal. TOF-SIMS studies of simplistic phospholipid LB model membrane systems demonstrate the potential use of these systems in TOF-SIMS analysis of cells.

Cholesterol↗
PubMed2003

Proton transfer in time-of-flight secondary ion mass spectrometry studies of frozen-hydrated dipalmitoylphosphatidylcholine.

A frozen water matrix, as found in freeze-fractured frozen-hydrated cellular samples, enhances the ionization of phosphatidylcholine lipids with static time-of-flight secondary ion mass spectrometry (TOF-SIMS). Isotopic profiles of the phosphocholine ion from deuterated forms of dipalmitoylphosphatidylcholine (DPPC) have been examined under various sample preparation conditions to show that ionization occurs through protonation from the matrix and is enhanced by the water present in freeze-fractured samples. The ionization of DPPC results in positively charged fragment ions, primarily phosphocholine, with a m/z of 184. Other ions include the M + H ion (m/z 735) and an ion representing the abstraction of the two palmitoyl fatty acid groups (m/z 224). Freeze-fracture techniques have been used to prepare frozen aqueous samples such as liposomes and cells to expose their membranes for static TOF-SIMS imaging. Due to the importance of surface water during SIMS analyses, sources of gas-phase water resulting from freeze-fracture were examined. Under proper fracturing conditions, water vapor, resulting from water in the sample and water condensed onto the outside of the sample, is released into the vacuum but does not condense back onto the surface. Combining the demonstrated enhancement of phosphatidylcholine lipid signal from water with the freeze-fracture preparation techniques described herein demonstrates potential advantages of studying biological samples in a frozen-hydrated state.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2003

Nanocapillary array interconnects for gated analyte injections and electrophoretic separations in multilayer microfluidic architectures.

An electrokinetic injection technique is described which uses a nuclear track-etched nanocapillary array to inject sample plugs from one layer of a microfluidic device into another vertically separated layer for electrophoretic separations. Gated injection protocols for analyte separations, reported here, establish nanocapillary array interconnects as a route to multilevel microfluidic analytical designs. The hybrid nanofluidic/microfluidic gated injection protocol allows sample preparation and separation to be implemented in separate horizontal planes, thereby achieving multilayer integration. Repeated injections and separations of FITC-labeled arginine and tryptophan, using 200-nm pore-diameter capillary array injectors in place of traditional cross injectors are used to demonstrate gated injection with a bias configuration that uses relay switching of a single high-voltage source. Injection times as rapid as 0.3 s along with separation reproducibilities as low as 1% for FITC-labeled arginine exemplify the capability for fast, serial separations and analyses. Impedance analysis of the micro-/nanofluidic network is used to gain further insight into the mechanism by which this actively controlled nanofluidic-interconnect injection method works. Gated sample introduction via a nanocapillary array interconnect allows the injection and separation protocols to be optimized independently, thus realizing the versatility needed for real-world implementation of rapid, serial microchip analyses.

Arginine↗
PubMed2003

Gateable nanofluidic interconnects for multilayered microfluidic separation systems.

The extension of microfluidic devices to include three-dimensional fluidic networks allows complex fluidic and chemical manipulations but requires innovative methods to interface fluidic layers. Externally controllable interconnects, employing nuclear track-etched polycarbonate membranes containing nanometer-diameter capillaries, are described that produce hybrid three-dimensional fluidic architectures. Controllable nanofluidic transfer is achieved by controlling applied bias, polarity, and density of the immobile nanopore surface charge and the impedance of the nanocapillary array relative to the microfluidic channels. Analyte transport between vertically separated microchannels has three stable transfer levels, corresponding to zero, reverse, and forward bias. The transfer can even depend on the properties of the analyte being transferred such as the molecular size, illustrating the flexible character of the analyte transfer. In a specific analysis implementation, nanochannel array gating is applied to capillary electrophoresis separations, allowing selected separated components to be isolated for further manipulation, thereby opening the way for preparative separations at attomole analyte mass levels.

Electrophoresis, Capillary↗
PubMed2002

Imaging of freeze-fractured cells with in situ fluorescence and time-of-flight secondary ion mass spectrometry.

Bioanalytical imaging techniques have been employed to investigate cellular composition at the single-cell and subcellular regimes. Four imaging modes have been performed sequentially in situ to demonstrate the utility of a more integrated approach to imaging cells. The combination of bright-field, scanning ion, and fluorescence microscopy complements TOF-SIMS imaging of native biomolecules. Bright-field microscopy provides a blurred visualization of cells in frozen-hydrated samples, while scanning ion imaging provides a morphological view of freeze-fractured cells after TOF-SIMS analysis is completed. With the use of selective fluorescent labels, fluorescence microscopy allows single mammalian cells to be located in the complex ice matrix of freeze-fractured samples, a task that has not been routine with either bright-field or TOF-SIMS. A fluorescent label, DiI (m/z 834), that does not interfere with the mass spectra of membrane phosphatidylcholine, has been chosen for fluorescence and TOF-SIMS imaging of membrane phospholipids. In this paper, in situ fluorescence microscopy allows the distinction of single cells from ice and other sample debris, previously not possible with bright-field or scanning ion imaging. Once cells are located, TOF-SIMS imaging reveals the localization of membrane lipids, even in the membrane of a single 15-microm rat pheochromocytoma cell. The utility of mapping lipids in the membranes of single cells using this integrated approach will provide more understanding of the functional role of specific lipids in functions of cellular membranes.

Adrenal Gland Neoplasms↗
PubMed2002

Identification of cellular sections with imaging mass spectrometry following freeze fracture.

Freeze-fracture techniques have been used to maintain chemical heterogeneity of frozen-hydrated mammalian cells for static TOF-SIMS imaging. The effects the fracture plane has on scanning electron microscopy and dynamic SIMS images of cells have been studied, but the implications this preparation method has on static SIMS have not been addressed to date. Interestingly, the chemical specificity and surface sensitivity of TOF-SIMS have allowed the identification of unique sections of rat pheochromocytoma cells exposed to the sample surface during freeze fracture. Using the extensive chemical information of the fractured surface, cellular sections have been determined using TOF-SIMS images of water, sodium, potassium, hydrocarbons, phosphocholine, and DiI, a fluorescent dye that remains in the outer leaflet of the cell membrane. Higher amounts of potassium have been imaged inside a cell versus the surrounding matrix in a cross-fractured cell. In other fractures exposing the cell membrane, phosphocholine and DiI have been imaged on the outer leaflet of the cell membrane, while phosphocholine alone has been imaged on the inner leaflet. In this paper, we discuss how imaging mass spectrometry isused to uniquely distinguish three possible sections of cells obtained during freeze fracture. The identification of these sections is important in choosing cells with a region of interest, like the cell membrane, exposed to the surface for a more thorough investigation with imaging static TOF-SIMS.

Adrenal Gland Neoplasms↗