Vincent B. Pizziconi

Characterization of Chlorobium tepidum Chlorosomes: A Calculation of Bacteriochlorophyll c per Chlorosome and Oligomer Modeling

The bacteriochlorophyll (Bchl) c content and organization was determined for Chlorobium (Cb.) tepidum chlorosomes, the light-harvesting complexes from green photosynthetic bacteria, using fluorescence correlation spectroscopy and atomic force microscopy. Single-chlorosome fluorescence data was analyzed in terms of the correlation of the fluorescence intensity with time. Using this technique, known as fluorescence correlation spectroscopy, chlorosomes were shown to have a hydrodynamic radius (Rh) of 25 +/- 3.2 nm. This technique was also used to determine the concentration of chlorosomes in a sample, and pigment extraction and quantitation was used to determine the molar concentration of Bchl c present. From these data, a number of approximately 215,000 +/- 80,000 Bchl c per chlorosome was determined. Homogeneity of the sample was further characterized by dynamic light scattering, giving a single population of particles with a hydrodynamic radius of 26.8 +/- 3.7 nm in the sample. Tapping-mode atomic force microscopy (TMAFM) was used to determine the x,y,z dimensions of chlorosomes present in the sample. The results of the TMAFM studies indicated that the average chlorosome dimensions for Cb. tepidum was 174 +/- 8.3 x 91.4 +/- 7.7 x 10.9 +/- 2.71 nm and an overall average volume 90,800 nm(3) for the chlorosomes was determined. The data collected from these experiments as well as a model for Bchl c aggregate dimensions was used to determine possible arrangements of Bchl c oligomers in the chlorosomes. The results obtained in this study have significant implications on chlorosome structure and architecture, and will allow a more thorough investigation of the energetics of photosynthetic light harvesting in green bacteria.

Transplacental transport of N-acetylcysteine in an ovine model

STUDY OBJECTIVE Acetaminophen freely crosses the placenta, and acetaminophen ingestion is the most frequent intentional overdose in pregnancy. Although most patients do well after maternal treatment with the antidote N-acetylcysteine (NAC), fetal death with massive hepatic necrosis has occurred. It has never been shown whether NAC crosses the placenta to yield fetal plasma levels equal to those associated with hepatoprotective effects in human beings. Our study objective was to evaluate this in a widely accepted large animal model for maternal-fetal research. DESIGN AND TYPE OF PARTICIPANTS: A nonblinded experiment was performed using four domestic sheep at near-term gestation. INTERVENTIONS NAC 150 mg/kg IV was administered to the ewe over 15 minutes. After induction of anesthesia, the fetal head was delivered surgically and a neck vein cannulated for blood sampling. Maternal and fetal blood samples were obtained at the end of NAC infusion, at 30- and then at 60-minute intervals for four hours. Plasma NAC levels were determined by gas chromatography/mass spectroscopy (detection limit, 2 micrograms/mL; quantification limit, 5 micrograms/mL). RESULTS Maternal peak plasma NAC levels were 619, 631, 1,757, and 2,512, micrograms/mL, respectively, within 30 minutes of infusion. However, NAC was only minimally detectable in plasma of two fetal animals and transiently reached quantifiable levels in two others. None of the fetal animals attained serial plasma NAC levels that equalled those associated with therapeutic dosing or hepatoprotective effects in human beings. CONCLUSION Transplacental transport of NAC is clinically insignificant in a mammalian model resembling the human being. These findings suggest that the human fetal liver is not protected from acetaminophen toxicity by maternal NAC therapy.

A microflow amperometric glucose biosensor

We investigate a small glucose sensor that uses a flow-through enzyme bed and reaction endpoint approach that seems particularly suited to microdialysis-type subcutaneous or intravascular glucose sensors. The particular configuration has the advantage of relative insensitivity to blood oxygen changes and also to factors which affect enzyme activity compared to conventional polarographic type glucose sensors. We evaluate the placement of a microdialysis fiber into a near-surface blood vessel in the dog model as a means of blood glucose sampling and to determine the effects of protein deposition. We observe a progressive decline in intravascular membrane fiber transport that must be considered in sensor design.

A cell-based immunobiosensor with engineered molecular recognition— Part III: engineering molecular recognition

We have been studying the feasibility of exploiting the recognition and amplification abilities of living immune cells for the development of hybrid immunosensors. Our group has previously reported that cell metabolic activation responses, induced by calcium ionophore A23187, can be directly transduced using calorimetric transducers, and that enzyme systems can be integrated to enhance sensing response time and output. In this study our goal was to determine the feasibility of transducing the thermal activation responses of mast cells molecularly engineered to a specific antigen. Rat peritoneal mast cells were sensitized to the model antigenic analyte dinitrophenylated-albumin (DNP-A), with monoclonal anti-DNP-A IgE, and challenged with antigen at final concentrations of 10 or 100 ng/ml. The addition of antigen resulted in the molecular triggering of cell activation, yielding thermal responses similar to those obtained previously with the ionophore model. A peak thermal response of 1.7 microW/5 x 10(5) cells was obtained within approximately 7 min of addition of antigen. The incorporation of selected amplification enzyme systems increased peak thermal outputs approximately three-fold, and reduced peak thermal response times to less than 3 min. A preliminary regression analysis of these data suggests a quantitative relationship exists between analyte concentration and peak thermal response (R = 0.988). These results support the feasibility and potential versatility of cell-based immunobiosensors for the selective detection and quantification of immunological analytes of interest.

Immobilization of functional light antenna structures derived from the filamentous green bacterium Chloroflexus aurantiacus.

The integration of highly efficient, natural photosynthetic light antenna structures into engineered systems while their biophotonic capabilities are maintained has been an elusive goal in the design of biohybrid photonic devices. In this study, we report a novel technique to covalently immobilize nanoscaled bacterial light antenna structures known as chlorosomes from Chloroflexus aurantiacus on both conductive and nonconductive glass while their energy transducing functionality was maintained. Chlorosomes without their reaction centers (RCs) were covalently immobilized on 3-aminoproyltriethoxysilane (APTES) treated surfaces using a glutaraldehyde linker. AFM techniques verified that the chlorosomes maintained their native ellipsoidal ultrastructure upon immobilization. Results from absorbance and fluorescence spectral analysis (where the Stokes shift to 808/810 nm was observed upon 470 nm blue light excitation) in conjunction with confocal microscopy confirm that the functional integrity of immobilized chlorosomes was also preserved. In addition, experiments with electrochemical impedance spectroscopy (EIS) suggested that the presence of chlorosomes in the electrical double layer of the electrode enhanced the electron transfer capacity of the electrochemical cell. Further, chronoamperometric studies suggested that the reduced form of the Bchl- c pigments found within the chlorosome modulate the conduction properties of the electrochemical cell, where the oxidized form of Bchl- c pigments impeded any current transduction at a bias of 0.4 V within the electrochemical cell. The results therefore demonstrate that the intact chlorosomes can be successfully immobilized while their biophotonic transduction capabilities are preserved through the immobilization process. These findings indicate that it is feasible to design biophotonic devices incorporating fully functional light antenna structures, which may offer significant performance enhancements to current silicon-based photonic devices for diverse technological applications ranging from CCD devices used in retinal implants to terrestrial and space fuel cell applications.

Interactive nano-visualization for science and engineering education

A novel, web-based Interactive Nano-Visualization for Science and Engineering Education (IN-VSEE) initiative is currently under development at Arizona State University and collaborating institutions that offers asynchronous distance learning of material systems remotely over the WWW. IN-VSEE exploits advanced telecommunications to facilitate remote operation of scanning probe instruments over the web directly from the classroom. The IN-VSEE multidisciplinary initiative can be used to reinforce biomaterials science and engineering concepts, as well as for training bioengineering students interested in understanding and exploiting man-made and naturally derived biomaterials at the micro- and nanoscale.

A cell-based immunobiosensor with engineered molecular recognition— Part II: enzyme amplification systems

Immune cells in vivo routinely perform highly selective immunosensing in blood and tissues as part of their normal immune surveillance functions. We have been investigating the potential of exploiting the immunosensing detection abilities of excitable immune cells (i.e. the mast cell) for the development of whole cell immunobiosensors. A key feature is that these immune cells can be selectively engineered to recognize specific antigens in vitro. In the presence of antigen, these cells undergo excitable activation responses which result in increased metabolism and the exocytosis of stored intracellular mediators. We have previously determined that mast cell metabolic responses can be thermally transduced in real time, thus indicating the possibility of whole cell thermoelectric immunobiosensing. In this work we investigated the use of enzyme amplification systems to enhance the direct transduction of immune cell responses to analyte. It was found that with appropriate enzymes, peak outputs occurred within approximately 5 min (4-20 times faster than without enzymes) and peak response magnitudes were up to nine-fold greater than without enzymes.

Regulatory and Reimbursement Innovation

Coverage with evidence development and parallel review for molecular diagnostics aid regulation and reimbursement. Coverage with evidence development and parallel review for molecular diagnostics aid regulation and reimbursement.

Optoelectronic Energy Transfer at Novel Biohybrid Interfaces Using Light Harvesting Complexes from Chloroflexus aurantiacus

In nature, nanoscale supramolecular light harvesting complexes initiate the photosynthetic energy collection process at high quantum efficiencies. In this study, the distinctive antenna structure from Chloroflexus aurantiacusthe chlorosomeis assessed for potential exploitation in novel biohybrid optoelectronic devices. Electrochemical characterization of bacterial fragments containing intact chlorosomes with the photosynthetic apparatus show an increase in the charge storage density near the working electrode upon light stimulation and suggest that chlorosomes contribute approximately one-third of the overall photocurrent. Further, isolated chlorosomes (without additional photosynthetic components, e.g., reaction centers, biochemical mediators) produce a photocurrent (approximately 8-10 nA) under light saturation conditions. Correlative experiments indicate that the main chlorosome pigment, bacteriochlorophyll-c, contributes to the photocurrent via an oxidative mechanism. The results reported herein are the first to demonstrate that isolated chlorosomes (lipid-enclosed sacs of pigments) directly transduce light energy in an electrochemical manner, laying an alternative, biomimetic approach for designing photosensitized interfaces in biofuel cells and biomedical devices, such as bioenhanced retinal prosthetics.

The application of ion beam analysis to calcium phosphate-based biomaterials.

Ion beam technology may be applied in a straightforward fashion to the analysis and modification of biomaterials. For analytical purposes, characterization using megaelectron-volt He2+ ions provides a standardless, nondestructive means for accurately quantifying the composition of material surfaces and the thickness of thin films. In this study, three complementary ion backscattering techniques were utilized to characterize hydroxyapatite (HA) films: Rutherford backscattering spectrometry (RBS) can determine composition and amounts of elements heavier than He; forward recoil elastic spectrometry (FRES) can determine hydrogen content; resonance-enhanced RBS can quantify small amounts of light elements, e.g. carbon, by choosing a particular incident beam energy resulting in excitation of the light element nucleus. At this resonance energy, the scattering cross section greatly increases, improving elemental sensitivity. Sol-gel chemistry was used to synthesize HA films by spin coating and annealing in a rapid thermal processor. Using these techniques, the chemical composition of unfired films was Ca1.63O5.4H1.8C0.24P with a thickness of 3.01 x 10(18) atoms/cm2 and after firing at 800 degrees C as Ca1.66O4.0H0.26C0.09P with a thickness of 2.11 x 10(18) atoms/cm2. This compares favorably to stoichiometric HA, which has a composition of Ca1.67O4.33H0.33P.