Biomedical subjects

William R Heineman

Publications and source records attributed to William R Heineman.

18 recordsLinked to original sources

PubMed2005

Bacillus globigii bugbeads: a model simulant of a bacterial spore.

Nonpathogenic microorganisms are often used as simulants of biological pathogens during the initial phase of detection method development. While these simulants approximate the size, shape, and cellular organization of the microorganism of interest, they do not resemble its surface protein content, a factor particularly important in methods based on immunorecognition. Here, we develop and detect an artificial bacterial spore--B. globigii (BG) Bugbead-a particle mimicking the antigenic surface of BG spores. Two methods of spore protein extraction were compared both quantitatively (by protein concentration assay) and qualitatively (by SDS-PAGE and Western blot): extraction by mechanical disruption and extraction by chemical decoating. The former method was more efficient in producing more protein and a greater number of antigens. BG Bugbeads were made by conjugating the extracted proteins to 0.8-microm carboxyl-coated polystyrene particles via carbodiimide coupling. BG Bugbeads were successfully detected by a bead-based enzyme-labeled immunoassay with fluorescence detection with a detection limit of 6.9 x 10(3) particles/mL. Formation of the Bugbead-capture bead complex was confirmed by ESEM. The concept of a harmless artificial spore can be applied to developing improved simulants for pathogenic spore-forming microorganisms such as B. anthracis, C. botulinum, and B. cereus, which can to be used for method validation, instrument calibration, and troubleshooting.

Antigens, Bacterial↗

PubMed2005

Rapid pKa estimation using vacuum-assisted multiplexed capillary electrophoresis (VAMCE) with ultraviolet detection.

A rapid approach for estimating the pK(a) value of small organic molecules was developed using vacuum-assisted multiplexed capillary electrophoresis (VAMCE) with ultraviolet detection. The VAMCE method employed a 96-capillary array, arranged in a standard 8 x 12 microtiter plate configuration, with each row of capillaries filled with 12 individual running buffers of equal ionic strength (I = 50 mM) covering a pH range from 2.2 to 10.7. A separate compound was injected hydrodynamically into each row of capillaries allowing the estimation of pK(a) values for eight compounds in a single run. The application of a vacuum during the separation generated a bulk fluid flow and allowed the electrophoretic separation to be completed within 5 min. The complete VAMCE method, conditioning, and electrophoretic separation was optimized to allow the pK(a) estimation for between 128 to 168 compounds in an 8-h period. The VAMCE method provided a reliable approach for estimating pK(a) values both within- and between-day. The pK(a) values for a series of 96 compounds estimated by VAMCE agreed well with some of literature pK(a) values with an average absolute difference of 0.22 log units.

Electrophoresis, Capillary↗

PubMed2004

Fabrication of comb interdigitated electrodes array (IDA) for a microbead-based electrochemical assay system.

This research is directed towards developing a more sensitive and rapid electrochemical sensor for enzyme labeled immunoassays by coupling redox cycling at interdigitated electrode arrays (IDA) with the enzyme label beta-galactosidase. Coplanar and comb IDA electrodes with a 2.4 microm gap were fabricated and their redox cycling currents were measured. ANSYS was used to model steady state currents for electrodes with different geometries. Comb IDA electrodes enhanced the signal about three times more than the coplanar IDAs, which agreed with the results of the simulation. Magnetic microbead-based enzyme assay, as a typical example of biochemical detection, was done using the comb and coplanar IDAs. The enzymes could be placed close to the sensing electrodes (approximately 10 microm for the comb IDAs) and detection took less than 1 min with a limit of detection of 70 amol of beta-galactosidase. We conclude that faster and more sensitive assays can be achieved with the comb IDA.

Biosensing Techniques↗

PubMed2004

Spectroelectrochemical sensing based on multimode selectivity simultaneously achievable in a single device. 17. Improvement in detection limits using ultrathin perfluorosulfonated ionomer films in conjunction with continuous sample flow.

We report herein an attenuated total reflectance (ATR) absorbance-based spectroelectrochemical sensor for tris(2,2'-bipyridyl)ruthenium(II) ion [Ru(bpy)(3)(2+)] that employs ultrathin (24-50 nm) Nafion films as the charge-selective layer. This film serves to sequester and preconcentrate the analyte at the optically transparent electrode surface such that it can be efficiently detected optically via electrochemical modulation. Our studies indicate that use of ultrathin films in tandem with continuous flow of sample solution through the cell compartment leads to a 100-500-fold enhancement in detection limit (10 nM) compared to earlier absorbance-based spectroelectrochemical sensors ( approximately 1-5 microM); markedly shorter analysis times also result. We report the dependence of the measured absorbance on sample flow rate and Nafion film thickness, and also provide calibration curves that illustrate the linear range and detection limits of the sensor using a 24 nm film at a constant sample flow rate of 0.07 mL/min.

Journal Article↗

PubMed2004

Microbead-based electrochemical immunoassay with interdigitated array electrodes.

The objective of this study was to develop a sensitive and miniaturized immunoassay by coupling a microbead-based immunoassay with an interdigitated array (IDA) electrode. An IDA electrode amplifies the signal by recycling an electrochemically redox-reversible molecule. The microfabricated platinum electrodes had 25 pairs of electrodes with 1.6-microm gaps and 2.4-microm widths. An enzyme-labeled sandwich immunoassay on paramagnetic microbeads with mouse IgG as the analyte and beta-galactosidase as the enzyme label was used as the model system. beta-Galactosidase converted p-aminophenyl beta-D-galactopyranoside to p-aminophenol (PAP). This enzyme reaction was measured continuously by positioning the microbeads near the electrode surface with a magnet. Electrochemical recycling occurred with PAP oxidation to p-quinone imine (PQI) at +290 mV followed by PQI reduction to PAP at -300 mV vs Ag/AgCl. Dual-electrode detection amplified the signal fourfold compared to single-electrode detection, and the recycling efficiency reached 87%. A calibration curve of PAP concentration vs anodic current was linear between 10(-4) and 10(-6)M. A signal from 1000 beads in a 20-microL drop was detectable and the immunoassay was complete within 10 min with a detection limit of 3.5x10(-15)mol mouse IgG.

Aminophenols↗

PubMed2004

Bead-based electrochemical immunoassay for bacteriophage MS2.

Viruses are one of four classes of biothreat agents, and bacteriophage MS2 has been used as a simulant for biothreat viruses, such as smallpox. A paramagnetic bead-based electrochemical immunoassay has been developed for detecting bacteriophage MS2. The immunoassay sandwich was made by attaching a biotinylated rabbit anti-MS2 IgG to a streptavidin-coated bead, capturing the virus, and then attaching a rabbit anti-MS2 IgG-beta-galactosidase conjugate to another site on the virus. beta-Galactosidase converts p-aminophenyl galactopyranoside (PAPG) to p-aminophenol (PAP). PAPG is electroinactive at the potential at which PAP is oxidized to p-quinone imine (PQI), so the current resulting from the oxidation of PAP to PQI is directly proportional to the concentration of antigen in the sample. The immunoassay was detected with rotating disk electrode (RDE) amperometry and an interdigitated array (IDA) electrode. With an applied potential of +290 mV vs Ag/AgCl and a rotation rate of 3000 rpm, the detection limit was 200 ng/mL MS2 or 3.2 x 10(10) viral particles/mL with RDE amperometry. A trench IDA electrode was incorporated into a poly(dimethyl siloxane) channel, within which beads were collected, incubated with PAPG, and PAP generation was detected. The two working electrodes were held at +290 and -300 mV vs Ag/AgCl, and electrochemical recycling of the PAP/PQI couple by the IDA electrode lowered the limit of detection to 90 ng/mL MS2, or 1.5 x 10(10) MS2 particles/mL.

Aminophenols↗

PubMed2004

Bead-based immunoassays with microelectrode detection.

The suitability of a microelectrode as the detector for a small-volume, bead-based enzyme-labeled immunoassay for later use in a microfluidic device was investigated. The microelectrode helps to overcome consumption of the electroactive species by the electrode (depletion) that is encountered with macroelectrodes such as the rotating disk electrode (RDE) and allows the volume of the detection cell to be reduced. Microelectrodes also allow the chemical reactions to be monitored in real time due to the electrodes' close proximity to the assay site. A bead-based sandwich immunoassay for mouse IgG was developed with alkaline phosphatase (AP) as the enzyme label, p-aminophenyl phosphate (PAPP) as the enzyme substrate, and microelectrode detection. The diffusion coefficient of the product of enzymatic hydrolysis, p-aminophenol (PAP), was determined to be 7.2+/-0.9 x 10(-6) cm(2) s(-1). The detection limits were determined for free (0.52 ng mL(-1)) and bead-bound AP (10 ng mL(-1)). The number of binding sites for AP per bead was calculated to be 9.6 x 10(4) molecules/bead, and under saturation conditions the minimum detectable number of beads was 2500. Lower detection limits could be achieved with the microelectrode than the RDE while maintaining similar reproducibility. The microelectrode also made it possible to work with lower sample volumes (down to 10 microL) than with the RDE (minimum volume of 40 microL). Depletion of PAP was not observed with the microelectrode. The results obtained here with a microelectrode showed great promise for later use of microelectrodes in microfluidic devices with limited sample volumes. RDE detection cannot be used in a microfluidic system due to its complex set-up that includes a motor for rotation.

Alkaline Phosphatase↗

PubMed2004

Detection of secretion from pancreatic islets using chemically modified electrodes.

Secretion of insulin from pancreatic islets was monitored indirectly by detecting zinc. Anodic stripping voltammetric measurements of zinc were done on a bismuth-modified electrode. Comparison of the performance of bismuth-modified electrodes and mercury film electrodes showed that bismuth is an appropriate alternative for Zn detection. The bismuth-coated electrode was used to detect zinc in insulin samples and insulin secreted from pancreatic islets upon stimulation with high concentrations of K(+). Detection of zinc released from pancreatic islets was done in the culture medium without any further cleanup. This detection method can be used to monitor secretion from pancreatic islets in their native environment.

Animals↗

PubMed2004

Spectroelectrochemical sensing based on attenuated total internal reflectance stripping voltammetry. 3. Determination of cadmium and copper.

The optical and electrochemical properties of metallic films on ITO surfaces resulting from deposition of copper and cadmium were monitored by stripping voltammetry-attenuated internal reflectance spectroscopy. The voltammetric or optical responses of both metals were examined with respect to solution conditions such as pH and presence of dissolved oxygen. The morphologies of these films were also examined using environmental scanning electron microscopy, and the microscopic electrodeposition patterns were found to influence the optical response. The wavelength dependence of the optical response of deposited copper was determined and compared with calculations; optimal performance was at 400 nm for copper. A linear calibration curve was obtained over a range of 1 x 10(-7)-1 x 10(-4) M for copper and compared with that of cadmium. The simultaneous determination of cadmium and copper was demonstrated, and the mechanism of co-deposition is discussed.

Journal Article↗

PubMed2004

Spectroelectrochemical sensing based on attenuated total internal reflectance stripping voltammetry. 2. Determination of mercury and lead.

Detection of lead and mercury by attenuated total internal reflectance spectroscopy coupled to stripping voltammetry is demonstrated. Changes in attenuation of light passing through an indium tin oxide optically transparent electrode (ITO-OTE) accompany the electrodeposition and stripping of lead and mercury on the electrode surface. The change in absorbance during stripping of electrodeposited metal constitutes the analytical response that enables detection over a range of 2.5 x 10(-7)-5 x 10(-5) and 5 x 10(-8)-5 x 10(-5) M for mercury and lead, respectively. The spectroelectrochemical responses of mercury and lead on the ITO surface are characterized and optimized with respect to solution conditions, the potential excitation signals used for deposition and stripping, and wavelength for detection. The deposited metals were examined by environmental scanning electron miscroscopy, and the electrodeposition pattern of lead and mercury was found to influence the optical response.

Electrochemistry↗

PubMed2004

Further investigations on a poly(vinyl alcohol)- polyelectrolyte chemically selective optical film.

The ionomer poly(vinylbenzyltrimethylammonium chloride) has been blended with cross-linked poly(vinyl alcohol) to form optically clear composites that can be covalently linked as thin films to oxide surfaces. Films are characterized using spectroscopic ellipsometry with refractive index (n) and extinction coefficient (k) data presented for wavelengths 300 to 1100 nm. A refractive index of 1.54 and average thickness of 709 nm are typical of an air dry film prepared by spin-coating. Dynamic in situ ellipsometry results for films exposed to 0.1 M KNO(3) and 1.0 mM Fe(CN)(6)(-3) are presented. Upon initial exposure to 0.1 M KNO(3), an air dry film expands by about 160% and stabilizes in size and refractive index at about 18 hours. Ion exchange of film cationic groups with ferricyanide is marked by slight film contraction, presumably due to electrostatic cross-linking by the multivalent anion. These films are useful in the spectroelectrochemical sensor with our newly developed fluorescence detection mode, as demonstrated by results of the reversible incorporation of the fluorescent anion fluorescein.

Journal Article↗

PubMed2003

Spectroelectrochemical sensing based on multimode selectivity simultaneously achievable in a single device. 16. Sensing by fluorescence.

A fluorescence spectroelectrochemical sensor capable of detecting very low concentrations of metal complexes is described. The sensor is based on a novel spectroelectrochemical sensor that incorporates multiple internal reflection spectroscopy at an optically transparent electrode (OTE) coated with a selective film to enhance detection limits by preconcentrating the analyte at the OTE surface. Nafion was used as the selective cation exchange film for detecting Ru(bpy)(3)(2+), the model analyte, which fluoresces at 605 nm when excited with a 441.6-nm HeCd laser. The unoptimized linear dynamic range of the sensor for Ru(bpy)(3)(2+) is between 1 x 10(-)(11) and 1 x 10(-)(7) M with a calculated 2 x 10(-)(13) M detection limit. The sensor employs extremely thin films ( approximately 12 nm) without significantly sacrificing its sensitivity. The sensor response is demonstrated with varying film thicknesses. A state-of-the-art flow cell design allows variable cell volumes as low as approximately 4 microL. Fluorescence of the sample can be controlled by electromodulation between 0.7 and 1.3 V. Sensor operation is not reversible for the chosen model film (Nafion) and sample (Ru(bpy)(3)(2+)) but it can be regenerated with ethanol for multiple uses.

Journal Article↗

PubMed2003

Spectroelectrochemical sensing based on attenuated total internal reflectance stripping voltammetry. 1. Determination of lead and cadmium.

The electrodeposition and subsequent stripping of lead and cadmium on an indium tin oxide (ITO) optically transparent electrode (OTE) were monitored by attenuated total internal reflectance. Light passing through the ITO-OTE is attenuated proportionally to the concentration of metal ion and deposition time. The wavelength dependence of the optical responses of deposited Pb and Cd was determined; optimal performance based on maximum sensor absorbance was at 750 nm for Pb and at 400 nm for Cd. Calibration curves were obtained over a range of 5 x 10(-8) to 5 x 10(-5) M and 1 x 10(-9) to 1 x 10(-5) M for Pb and Cd, respectively, using change in absorbance that accompanied deposition and subsequent stripping of the electrodeposited metal from the ITO.

Journal Article↗

PubMed2003

Determination of endogenous extracellular signal-regulated protein kinase by microchip capillary electrophoresis.

The application of microchip capillary electrophoresis (CE) to the assay of extracellular signal-regulated protein kinase (ERK) is presented. In this assay, ERK catalyzes the transfer of gamma-phosphate from adenosine 5(')-triphosphate to the threonine residue of a fluorescently labeled nonapeptide (APRTPGGRR), and the phosphorylated and nonphosphorylated peptides were detected by fluorescence. The phosphorylated and nonphosphorylated peptides and the internal standard were separated within 20s, and the increase in magnitude of the phosphorylated peptide peak was monitored to assess ERK activity. ERK reactions were prepared off-chip and analyzed on a single-lane glass microchip fabricated by standard methods. It was demonstrated that microchip CE could be used to measure endogenous amounts of ERK by spiking known concentrations of recombinant ERK2 into the lysates of serum-starved human umbilical vein endothelial cells (HUVEC) and recovering between 90 and 100% for all samples. Endogenous ERK activity was determined by microchip where HUVEC were stimulated with 500pM vascular endothelial growth factor (VEGF) at different times before cell lysis. The results showed a transient VEGF-mediated ERK activation that peaked at 10min, which was consistent with previous reports using conventional techniques. The microchip assay provided a rapid, accurate, and precise alternative to conventional methods of determining endogenous ERK activity.

Cells, Cultured↗

PubMed2003

Development and characterization of simulant pancreatic islets.

Insulin is stored in pancreatic islets as a zinc-insulin complex, and stimulating the islets results in the release of insulin and zinc. Simulant pancreatic islet beads have been developed using agarose beads (50-250 micro m diameter) derivatized with iminodiacetic acid that have been loaded with zinc. A qualitative comparison of the simulant beads with pancreatic islets has been made by staining with dithizone and a zinc-binding fluorescent dye, TSQ. The binding capacity of simulant beads was determined to be 34 micro mol Zn(2+)/g of dried beads using anodic stripping voltammetry. Hydrochloric acid was used to release zinc from beads to mimic the secretion of insulin from pancreatic islets and a release profile was established. The simulant beads can be used to optimize the islet isolation process and reduce the use of real islets in method development.

Animals↗

PubMed2003

Spectroelectrochemical sensing based on multimode selectivity simultaneously achievable in a single device. 11. Design and evaluation of a small portable sensor for the determination of ferrocyanide in Hanford waste samples.

A portable spectroelectrochemical sensor has been designed, evaluated, and demonstrated on a complex sample of radioactive waste. The sensor consisted of a black delrin sample compartment with a total internal sample volume of 800 microL, attached to an indium tin oxide coated glass multiple internal reflection optical element. Detection was by total internal reflection of light from a blue light emitting diode source. After a 10 min uptake for each standard, the sensor showed a linear response in absorbance change for 5 x 10(-5) to 5 x 10(-3) M ferrocyanide with electrochemical modulation by scanning at 20 mV/s from -0.30 V to +0.55 V vs a Ag/AgCl reference electrode. Due to the complex nature of Hanford radioactive tank waste samples containing ferrocyanide, a standard addition method was developed for analysis. The spectroelectrochemical sensor determined a concentration of 9.2 mM ferrocyanide for U-Plant-2 simulant solution containing 9.38 mM ferrocyanide that was prepared according to Hanford process flowsheets. A radioactive tank waste sample from Hanford Tank 241-C-112 was determined to be 1.0 mM in ferrocyanide using the spectroelectrochemical sensor. A value for the ferrocyanide concentration in the sample of 0.61 mM was determined by FTIR spectroscopy.

Electrochemistry↗

PubMed2002

Spectroelectrochemical sensing based on multimode selectivity simultaneously achievable in a single device. 13. Detection of aqueous iron by in situ complexation with 2,2'-bipyridine.

A spectroelectrochemical sensor with attenuated total reflectance at an indium-doped tin oxide (ITO) optically transparent electrode coated with a thin film of Nafion has been demonstrated for the determination of aqueous iron ion. The novelty of this sensor stems from its ability to take up colorless iron ion (Fe2+) from solution and complex it with an organic ligand, 2,2'-bipyridine (bipy), that has been previously loaded in the optically transparent charge-selective Nafion film coating the electrode. The resulting complex ion, tris(2,2'-bipyridyl)iron(II), Fe(bipy)3(2+), absorbs strongly, making it easily detectable via optical spectroscopy. Fe(bipy)3(2+) loaded into the selective film is oxidized to colorless Fe(bipy)3(3+), which gives rise to an absorbance change for quantifying iron. This paper maps the development of this sensor, from the spectroelectrochemical characterization of the complex ion at an ITO optically transparent electrode to an analysis of the uptake, retention, and optical response of the complex ion in the Nafion film. Finally, an evaluation of the uptake of aqueous Fe2+ by the bipy-loaded Nafion film is reported. These data include preliminary results illustrating the dependence of the sensor response on differing concentrations of Fe2+ in solution.

Journal Article↗

PubMed2001

An integrated microfluidic biochemical detection system for protein analysis with magnetic bead-based sampling capabilities.

This paper presents the development and characterization of an integrated microfluidic biochemical detection system for fast and low-volume immunoassays using magnetic beads, which are used as both immobilization surfaces and bio-molecule carriers. Microfluidic components have been developed and integrated to construct a microfluidic biochemical detection system. Magnetic bead-based immunoassay, as a typical example of biochemical detection and analysis, has been successfully performed on the integrated microfluidic biochemical analysis system that includes a surface-mounted biofilter and electrochemical sensor on a glass microfluidic motherboard. Total time required for an immunoassay was less than 20 min including sample incubation time, and sample volume wasted was less than 50 microl during five repeated assays. Fast and low-volume biochemical analysis has been successfully achieved with the developed biofilter and immunosensor, which is integrated to the microfluidic system. Such a magnetic bead-based biochemical detection system, described in this paper, can be applied to protein analysis systems.

Electrochemistry↗