Biomedical subjects

Dana M Spence

Publications and source records attributed to Dana M Spence.

12 recordsLinked to original sources

PubMed2006

Dynamic monitoring of glutathione in erythrocytes, without a separation step, in the presence of an oxidant insult.

A method for the quantitative determination of the antioxidant form of glutathione (GSH) in red blood cells (RBCs) is described that does not require separation of the analyte of interest from the complex cellular matrix. The measurement portion of the analysis is performed using fluorescence spectrophotometry after monochlorobimane (a recognized probe for GSH) is added to a mixture containing RBCs and glutathione transferase (GST). This method was employed to determine the GSH concentration (0.042 +/- 0.002 mM) in a solution of 1% RBCs obtained from rabbits (n = 6). When spiked with authentic GSH (0.50 micromol), 99.8% of the GSH was recovered. Addition of GST to the sample mixture enabled most measurements to be made after 5-10 min of reaction time. Importantly, a decrease in GSH was measured upon the addition of a recognized oxidant (diamide) to the RBC sample followed by a subsequent return to normal levels of GSH. The ability of the GSH to recover from the oxidant attack occurred in a dose-dependent manner, requiring 30 and 90 min to recover from oxidant insults of 20 and 40 microM diamide, respectively. The antioxidant capabilities of the GSH were able to be monitored in real time, thus providing a method to dynamically monitor the ability of the RBC to maintain homeostasis in a complex matrix.

Animals↗

PubMed2006

Microfluidic technologies as platforms for performing quantitative cellular analyses in an in vitro environment.

Quite often, important cellular events occur in environments that are either not amenable to implanted sensors or other types of molecular probes. In such cases, a viable alternative to taking the sensor or probe to the biological sample of interest is to bring the sample of interest out of its natural environment to one that is more conducive to the measurement scheme. The disadvantage of the latter approach is that the sample may not behave in the same manner in vitro as it does in vivo, or that the agonists and other stimuli to which the sample is subjected to in vivo are no longer present. In this Tutorial Review, the authors attempt to provide some guidance, based on their own experiences and those of other scientists, to performing cellular measurements in a quantitative manner under in vitro conditions. Due to the expansive literature on analyses involving cells, the authors have limited this Tutorial Review to those methods involving microfluidic technologies, both in microbore tubing and in microfabricated channels. Initial reports of analyses involving cells in microbore tubing were first reported nearly two decades ago, while those in microfabricated fluidic devices appeared over a decade ago. However, more recently, the complexity of cell analyses using fabricated microfluidic devices (as opposed to microbore tubing) has increased due in part to the improvements in fabrication technologies, fluid handling and delivery capabilities, advances in coatings of the channels within the microfluidic device, and integrated detection schemes. Examples of cellular analyses in microbore tubing and in fabricated microfluidic devices will be given, as well as associated advantages and challenges. Finally, the authors' thoughts on cellular analyses are presented here using the classical steps in an analysis as a guide.

Animals↗

PubMed2006

Fluorescence monitoring of ATP-stimulated, endothelium-derived nitric oxide production in channels of a poly(dimethylsiloxane)-based microfluidic device.

Intracellular nitric oxide (NO) production in a microfluidic endothelium is detected using fluorescence microscopy. Bovine pulmonary artery endothelial cells (bPAECs) were loaded with the fluorescence probe diaminodifluorofluorescein diacetate (DAF-FM DA), and the subsequent fluorescent DAF-FM DA/NO adduct was measured. Solutions of bradykinin, a well-known stimulus of endothelium-derived NO, activated nitric oxide synthase (NOS) in the immobilized bPAECs. This activation was inhibited using l-nitro arginine methyl ester (L-NAME), a competitive inhibitor of NOS. Importantly, the NO production was also stimulated with adenosine triphosphate (ATP) using concentrations as low as 1 microM. Previous reports on stimulating NO production using an immobilized endothelium in microfluidic channels were limited by the requirement of ATP concentrations of at least 100 microM, a value that is not physiologically relevant. The ability to monitor NO production with ATP concentrations that are similar to in vivo levels of ATP in the microcirculation represents a major advance in the use of microfluidic technology as an in vitro model of the microcirculation.

Adenosine Triphosphate↗

PubMed2005

Monitoring erythrocytes in a microchip channel that narrows uniformly: towards an improved microfluidic-based mimic of the microcirculation.

The release of adenosine triphosphate (ATP) from red blood cells (RBCs) flowing through PDMS microchannels has been determined as a function of channel cross-sectional area using a design containing a channel that narrows uniformly. ATP, released from the RBCs in response to the mechanical deformation of their cell membranes, increased as the channel cross-section decreased. One sample of rabbit RBCs released 1.16 +/- 0.11, 1.92 +/- 0.14 and 2.09 +/- 0.10 microM ATP as the median cross-sectional area decreased from 4314 to 3192 to 2052 microm(2), respectively. Numerous samples (n = 6) displayed the same trend. Incubating a sample of RBCs with diamide, a substance known to stiffen cell membranes without harming the cell cytosol, provided evidence that no cell lysis occurred in the microchip device. This novel use of lab-on-a-chip technology allows for channel designs that enable an in vitro study of physiological events that occur in the microcirculation.

Adenosine Triphosphate↗

PubMed2004

Amperometric determination of nitric oxide derived from pulmonary artery endothelial cells immobilized in a microchip channel.

A simple method for immobilizing a confluent layer of bovine pulmonary artery endothelial cells (bPAECs) in microchip-based channels is described. The microchips are prepared from poly(dimethylsiloxane) and have channel dimensions that approximate resistance vessels in vivo. The reversibly sealed channels were coated with fibronectin (100 microg ml(-1)) by aspiration. The bPAECs, which were introduced in the same manner, became attached to the fibronectin coating in about 2 h. The microchip could then be resealed over a micromolded carbon ink electrode (24 microm width x 6 microm height). Coating the carbon microelectrode with a 0.05% Nafion solution selectively blocked nitrite (10 microM) from being transported to the electrode surface while nitric oxide (NO, 10 microM) was amperometrically measured. Upon stimulation with adenosine triphosphate (ATP, 100 microM) the immobilized bPAECs produced and released micromolar amounts of NO. This NO production was effectively inhibited when the immobilized cells were incubated with L-nitro-arginine methyl ester (L-NAME), a competitive inhibitor for nitric oxide synthase. Moreover, once the immobilized bPAECs were no longer able to produce NO, incubation with L-arginine allowed for further ATP-stimulated NO production.

Animals↗

PubMed2004

Deformation-induced release of ATP from erythrocytes in a poly(dimethylsiloxane)-based microchip with channels that mimic resistance vessels.

The ability of nitric oxide to relax smooth muscle cells surrounding resistance vessels in vivo is well documented. Here, we describe a series of studies designed to quantify amounts of adenosine triphosphate (ATP), a known stimulus of NO production in endothelial cells, released from erythrocytes that are mechanically deformed as these cells traverse microbore channels in lithographically patterned microchips. Results indicate that micromolar amounts of ATP are released from erythrocytes flowing through channels having cross sectional dimensions of 60 x 38 micron (2.22 +/- 0.50 microM ATP). Microscopic images indicate that erythrocytes, when being pumped through the microchip channels, migrate toward the center of the channels, leaving a cell-free or skimming layer at the walls of the channel, a profile known to exist in circulatory vessels in vivo. A comparison of the amounts of ATP released from RBCs mechanically deformed in microbore tubing (2.54 +/- 0.15 microM) vs a microchip (2.59 +/- 0.32 microM) suggests that channels in microchips may serve as functional biomimics of the microvasculature. Control studies involving diamide, a membrane-stiffening agent, suggest that the RBC-derived ATP is not due to cell lysis but rather physical deformation.

Adenosine Triphosphate↗

PubMed2004

Fabrication of carbon microelectrodes with a micromolding technique and their use in microchip-based flow analyses.

In this paper, we report a new technique to pattern carbon microelectrodes for use in microfluidics. This technique, termed micromolding of carbon inks, uses poly(dimethylsiloxane)(PDMS) microchannels to define the size of the microelectrode. First, PDMS microchannels of the approximate dimensions desired for the microelectrode are made by soft lithography. The PDMS is then reversibly sealed to a substrate and the microchannels are filled with carbon ink. After a heating step the PDMS mold is removed, leaving a carbon microelectrode with a size slightly smaller than the original PDMS microchannel. The resulting microelectrode (27 microm wide and 6 microm in height) can be reversibly sealed to a PDMS-based flow channel. Fluorescence microscopy showed that no leakage occurred around the chip/electrode seal, even up to flow rates of 10 microL min(-1). The electrode was characterized by microchip-based flow injection analysis. Injections of catechol in Hank's Balanced Salt Solution (pH 7.4), showed a linear response from 2 mM to 10 microM (r(2)= 0.995), with a sensitivity of 56.5 pA microM(-1) and an estimated limit of detection of 2 microM (0.27 picomole, S/N=3). Reproducibility of the electrode response was shown by repeated injections (n= 10) of a 500 microM catechol solution, resulting in a RSD of 4.6%. Finally, selectivity was demonstrated by coating the microelectrode with Nafion, a perfluoronated cation exchange polymer. Dopamine exhibited a response at the modified microelectrode while ascorbic acid was rejected by the Nafion-coating. These electrodes provide inexpensive detectors for microfluidic applications while also being viable alternatives to use of other carbon microelectrode materials, such as carbon fibers. Furthermore, the manner in which the microelectrodes are produced will be of interest to researchers who do not have access to state of the art microfabrication facilities.

Carbon↗

PubMed2004

Bioanalytical challenges for analytical chemists.

Analytical chemistry contributes significantly to the life sciences through developing measurement techniques that provide quantitative chemical information. Developments in biological research present new challenges for analytical chemistry and call for revolutionary methods and new thinking. Dana Spence outlines here some key and unique challenges encountered when making measurements on samples of a biological nature, supported with real examples from his own research.

Chemistry Techniques, Analytical↗

PubMed2003

Flow-based amperometric detection of dopamine in an immobilized cell reactor.

A protocol is described for immobilizing PC 12 cells on to the lumen of fused silica microbore tubing having an inside diameter of 250 microm and coated with a thin layer of poly-L-lysine. Optimization studies of the immobilization procedure indicated that a 50 microg ml(-1) solution of poly-L-lysine was the best material for cell adhesion to the fused silica tubing. In addition, it was found that the cells become attached to the poly-L-lysine in approximately 2 h, after which they could be maintained inside of the tubing for a period up to 5 days. Importantly, the immobilized cells ability to release neurotransmitters was evident by measuring the Ca(2+)-induced release of dopamine with an in column amperometric detection scheme involving a Nafion modified platinum ultramicroelectrode.

Animals↗

PubMed2003

Extracellular ATP signaling in the rabbit lung: erythrocytes as determinants of vascular resistance.

Previously, it was reported that red blood cells (RBCs) are required to demonstrate participation of nitric oxide (NO) in the regulation of rabbit pulmonary vascular resistance (PVR). RBCs do not synthesize NO; hence, we postulated that ATP, present in millimolar amounts in RBCs, was the mediator, which evoked NO synthesis in the vascular endothelium. First, we found that deformation of RBCs, as occurs on passage across the pulmonary circulation with increasing flow rate, evoked increments in ATP release. Here, ATP (300 nM), administered to isolated, salt solution-perfused (PSS) rabbit lungs, decreased total and upstream (arterial) PVR, a response inhibited by NG-nitro-L-arginine methyl ester (L-NAME, 100 microM). In lungs perfused with PSS containing RBCs, L-NAME increased total and upstream PVR. In lungs perfused with PSS containing glibenclamide-treated RBCs, which inhibits ATP release, L-NAME was without effect. Apyrase grade VII (8 U/ml), which degrades ATP to AMP, was without effect on PVR in PSS-perfused lungs. These results are consistent with the hypothesis that ATP, released from RBCs as they traverse the pulmonary circulation, evokes endogenous NO synthesis.

Adenosine Monophosphate↗

PubMed2003

Detection of ATP-induced nitric oxide in a biomimetic circulatory vessel containing an immobilized endothelium.

Conditions for the adhesion of bovine pulmonary artery endothelial cells (bPAECs) in microbore tubing of 250-microm i.d. are described. When immobilized to the lumen of microbore tubing, these cells represent a mimic of a circulatory vessel's endothelium. The microbore tubing is coated with 100 microg mL(-1) fibronectin in order to promote bPAEC adhesion to the lumen of the tubing. A series of micrographs of the cells inside of the tubing indicates that approximately 3.5 h is necessary for cell adhesion. In this study, adenosine triphosphate (ATP) is used to induce the release of nitric oxide from the endothelium mimic. The endothelium-derived NO is detected amperometrically at a parallel flow cell containing a glassy carbon working electrode modified with Nafion. Results indicate that detectable amounts of NO are only produced by the endothelium mimic when ATP is present in the buffer. The typical concentration of NO produced by the endothelium mimic upon the introduction of 100 microM ATP is approximately 0.80 microM. Based on the injection volume of ATP and the estimated number of cells on the tubing lumen, this value corresponds to approximately 1 amol of NO/cell. Moreover, shear stress alone does not provide the agonistic effect required for NO production in the submicromolar range.

Adenosine Triphosphate↗

PubMed2003

Determination of erythrocyte deformability and its correlation to cellular ATP release using microbore tubing with diameters that approximate resistance vessels in vivo.

A novel method is described for measuring the deformability of red blood cells (RBCs) in tubing whose diameters approximate forces encountered in vivo. Here, RBCs from rabbits are loaded into a 50 cm section of 75 microm id microbore tubing and connected to a syringe pump. This section of tubing is then connected to a 15 cm section of 25 microm id tubing. As buffer is pumped through the flow system, the RBCs are evacuated from both sections of tubing. However, the inability of the RBCs to move freely through the 25 mirom id section of tubing results in a buildup of cells at the inlet of this portion of tubing. The continued force output by the syringe pump results in a deformation of the RBCs until all of the cells are eventually evacuated from the flow system. It was found that a measurement of the time required to reach half of the maximum pressure (1/2 P(max)) may be used as an indicator of the RBC deformability. For a given sample, a simple buffer results in less time to reach 1/2 P(max) (6.9 +/- 0.2 s) than deformable RBCs (21.6 +/- 0.8 s). To verify that the increased amount of time to reach 1/2 P(max) is indeed due to the RBCs, various hematocrits of an RBC sample were investigated and, as expected, it was found that a 12% RBC hematocrit had a higher 1/2 P(max) value (26.0 s +/- 2.2 s) when compared to a 7% hematocrit (19.1 +/- 0.3 s). In addition, RBCs chemically stiffened with glutaraldehyde were shown to be 25% less deformable than normal RBCs. Finally, a study was performed to examine the relationship between RBC deformability and ATP release and it was found that ATP release increased as a function of RBC deformability. This method greatly simplifies deformability measurements, employing only a syringe pump and microbore tubing, and may lead to a more complete understanding of the physiological significance of erythrocyte deformability.

Adenosine Triphosphate↗