David Benjamin Jaroch

A self referencing platinum nanoparticle decorated enzyme-based microbiosensor for real time measurement of physiological glucose transport

Glucose is the central molecule in many biochemical pathways, and numerous approaches have been developed for fabricating micro biosensors designed to measure glucose concentration in/near cells and/or tissues. An inherent problem for microsensors used in physiological studies is a low signal-to-noise ratio, which is further complicated by concentration drift due to the metabolic activity of cells. A microsensor technique designed to filter extraneous electrical noise and provide direct quantification of active membrane transport is known as self-referencing. Self-referencing involves oscillation of a single microsensor via computer-controlled stepper motors within a stable gradient formed near cells/tissues (i.e., within the concentration boundary layer). The non-invasive technique provides direct measurement of trans-membrane (or trans-tissue) analyte flux. A glucose micro biosensor was fabricated using deposition of nanomaterials (platinum black, multiwalled carbon nanotubes, Nafion) and glucose oxidase on a platinum/iridium microelectrode. The highly sensitive/selective biosensor was used in the self-referencing modality for cell/tissue physiological transport studies. Detailed analysis of signal drift/noise filtering via phase sensitive detection (including a post-measurement analytical technique) are provided. Using this highly sensitive technique, physiological glucose uptake is demonstrated in a wide range of metabolic and pharmacological studies. Use of this technique is demonstrated for cancer cell physiology, bioenergetics, diabetes, and microbial biofilm physiology. This robust and versatile biosensor technique will provide much insight into biological transport in biomedical, environmental, and agricultural research applications.

Fabrication and characterization of tunable polysaccharide hydrogel blends for neural repair.

Hydrogels are an important class of biomaterials that have the potential to be used as three-dimensional tissue engineering scaffolds for regenerative medicine. This is especially true in the central nervous system, where neurons do not have the ability to regenerate due to the prohibitory local environment following injury. Hydrogels can fill an injury site, replacing the growth-prohibiting environment with a more growth-permissive one. In this study, dextran and chitosan were incorporated into a methylcellulose and agarose hydrogel blend. This created several thermally sensitive polysaccharide hydrogel blends that had tunable mechanical and surface charge properties. Cortical neurons were cultured on the hydrogels to determine the blend that had the greatest neuron compatibility. Our results show that softer, more positively charged polysaccharide hydrogel blends allow for greater neuron attachment and neurite extension, showing their promise as CNS regeneration scaffolds.

Optical Nanosensor Architecture for Cell Signaling Molecules Using DNA Aptamer-Coated Carbon Nanotubes

We report a novel optical biosensor platform using near-infrared fluorescent single-walled carbon nanotubes (SWNTs) functionalized with target-recognizing aptamer DNA for noninvasively detecting cell-signaling molecules in real time. Photoluminescence (PL) emission of aptamer-coated SWNTs is modulated upon selectively binding to target molecules, which is exploited to detect insulin using an insulin-binding aptamer (IBA) as a molecular recognition element. We find that nanotube PL quenches upon insulin recognition via a photoinduced charge transfer mechanism with a quenching rate of k(q) = 5.85 × 10(14) M(-1) s(-1) and a diffusion-reaction rate of k(r) = 0.129 s(-1). Circular dichroism spectra reveal for the first time that IBA strands retain a four-stranded, parallel guanine quadruplex conformation on the nanotubes, ensuring target selectivity. We demonstrate that these IBA-functionalized SWNT sensors incorporated in a collagen extracellular matrix (ECM) can be regenerated by removing bound analytes through enzymatic proteolysis. As proof-of-concept, we show that the SWNT sensors embedded in the ECM promptly detect insulin secreted by cultured pancreatic INS-1 cells stimulated by glucose influx and report a gradient contour of insulin secretion profile. This novel design enables new types of label-free assays and noninvasive, in situ, real-time detection schemes for cell-signaling molecules.

A comparative study of enzyme immobilization strategies for multi-walled carbon nanotube glucose biosensors.

This work addresses the comparison of different strategies for improving biosensor performance using nanomaterials. Glucose biosensors based on commonly applied enzyme immobilization approaches, including sol-gel encapsulation approaches and glutaraldehyde cross-linking strategies, were studied in the presence and absence of multi-walled carbon nanotubes (MWNTs). Although direct comparison of design parameters such as linear range and sensitivity is intuitive, this comparison alone is not an accurate indicator of biosensor efficacy, due to the wide range of electrodes and nanomaterials available for use in current biosensor designs. We proposed a comparative protocol which considers both the active area available for transduction following nanomaterial deposition and the sensitivity. Based on the protocol, when no nanomaterials were involved, TEOS/GOx biosensors exhibited the highest efficacy, followed by BSA/GA/GOx and TMOS/GOx biosensors. A novel biosensor containing carboxylated MWNTs modified with glucose oxidase and an overlying TMOS layer demonstrated optimum efficacy in terms of enhanced current density (18.3 ± 0.5 µA mM(-1) cm(-2)), linear range (0.0037-12 mM), detection limit (3.7 µM), coefficient of variation (2%), response time (less than 8 s), and stability/selectivity/reproducibility. H(2)O(2) response tests demonstrated that the most possible reason for the performance enhancement was an increased enzyme loading. This design is an excellent platform for versatile biosensing applications.

Self-referencing optrodes for measuring spatially resolved, real-time metabolic oxygen flux in plant systems

The ability to non-invasively measure metabolic oxygen flux is a very important tool for physiologists interested in a variety of questions ranging from basic metabolism, growth/development, and stress adaptation. Technologies for measuring oxygen concentration near the surface of cells/tissues include electrochemical and optical techniques. A wealth of knowledge was gained using these tools for quantifying real-time physiology. Fiber-optic microprobes (optrodes) have recently been developed for measuring oxygen in a variety of biomedical and environmental applications. We have adopted the use of these optical microsensors for plant physiology applications, and used the microsensors in an advanced sensing modality known as self-referencing. Self-referencing is a non-invasive microsensor technique used for measuring real-time flux of analytes. This paper demonstrates the use of optical microsensors for non-invasively measuring rhizosphere oxygen flux associated with respiration in plant roots, as well as boundary layer oxygen flux in phytoplankton mats. Highly sensitive/selective optrodes had little to no hysteresis/calibration drift during experimentation, and an extremely high signal-to-noise ratio. We have used this new tool to compare various aspects of rhizosphere oxygen flux for roots of Glycine max, Zea mays, and Phaseolus vulgaris, and also mapped developmentally relevant profiles and distinct temporal patterns. We also characterized real-time respiratory patterns during inhibition of cytochrome and alternative oxidase pathways via pharmacology. Boundary layer oxygen flux was also measured for a phytoplankton mat during dark:light cycling and exposure to pharamacological inhibitors. This highly sensitive technology enables non-invasive study of oxygen transport in plant systems under physiologically relevant conditions.

Oscillatory glucose flux in INS 1 pancreatic β cells: A self-referencing microbiosensor study

Signaling and insulin secretion in β cells have been reported to demonstrate oscillatory modes, with abnormal oscillations associated with type 2 diabetes. We investigated cellular glucose influx in β cells with a self-referencing (SR) microbiosensor based on nanomaterials with enhanced performance. Dose-response analyses with glucose and metabolic inhibition studies were used to study oscillatory patterns and transporter kinetics. For the first time, we report a stable and regular oscillatory uptake of glucose (averaged period 2.9±0.6 min), which corresponds well with an oscillator model. This oscillatory behavior is part of the feedback control pathway involving oxygen, cytosolic Ca(2+)/ATP, and insulin secretion (periodicity approximately 3 min). Glucose stimulation experiments show that the net Michaelis-Menten constant (6.1±1.5 mM) is in between GLUT2 and GLUT9. Phloretin inhibition experiments show an EC(50) value of 28±1.6 μM phloretin for class I GLUT proteins and a concentration of 40±0.6 μM phloretin caused maximum inhibition with residual nonoscillating flux, suggesting that the transporters not inhibited by phloretin are likely responsible for the remaining nonoscillatory uptake, and that impaired uptake via GLUT2 may be the cause of the oscillation loss in type 2 diabetes. Transporter studies using the SR microbiosensor will contribute to diabetes research and therapy development by exploring the nature of oscillatory transport mechanisms.

Magnetic insertion system for flexible electrode implantation

Chronic recording electrodes are a vital tool for brain research and neural prostheses. Despite decades of advances in recording technology, probe structures and implantation methods have changed little over time. Then as now, compressive insertion methods require probes to be constructed from hard, stiff materials, such as silicon, and contain a large diameter shank to penetrate the brain, particularly for deeper structures. The chronic presence of these probes results in an electrically isolating glial scar, degrading signal quality over time. This work demonstrates a new magnetic tension-based insertion mechanism that allows for the use of soft, flexible, and thinner probe materials, overcoming the materials limitations of modern electrodes. Probes are constructed from a sharp magnetic tip attached to a flexible tether. A pulsed magnetic field is generated in a coil surrounding a glass pipette containing the electrode. The applied field pulls the electrode tip forward, accelerating the probe into the neural tissue with a penetration depth that is calibrated against the charge voltage. Mathematical modeling and agar gel insertion testing demonstrate that the electrode can be implanted to a predictable depth given system specific parameters. Trial rodent implantations resulted in discernible single-unit activity on one of the probes. The current prototype demonstrates the feasibility of a tension based, magnetically driven implantation system and opens the door to a wide variety of new minimally invasive probe materials and configurations.

Cell-mediated deposition of porous silica on bacterial biofilms

Living hybrid materials that respond dynamically to their surrounding environment have important applications in bioreactors. Silica based sol-gels represent appealing matrix materials as they form a mesoporous biocompatible glass lattice that allows for nutrient diffusion while firmly encapsulating living cells. Despite progress in sol-gel cellular encapsulation technologies, current techniques typically form bulk materials and are unable to generate regular silica membranes over complex geometries for large-scale applications. We have developed a novel biomimetic encapsulation technique whereby endogenous extracellular matrix molecules facilitate formation of a cell surface specific biomineral layer. In this study, monoculture Pseudomonas aeruginosa and Nitrosomonas europaea biofilms are exposed to silica precursors under different acid conditions. Scanning electron microscopy (SEM) imaging and electron dispersive X-ray (EDX) elemental analysis revealed the presence of a thin silica layer covering the biofilm surface. Cell survival was confirmed 30 min, 30 days, and 90 days after encapsulation using confocal imaging with a membrane integrity assay and physiological flux measurements of oxygen, glucose, and NH 4⁺. No statistical difference in viability, oxygen flux, or substrate flux was observed after encapsulation in silica glass. Shear induced biofilm detachment was assessed using a particle counter. Encapsulation significantly reduced detachment rate of the biofilms for over 30 days. The results of this study indicate that the thin regular silica membrane permits the diffusion of nutrients and cellular products, supporting continued cellular viability after biomineralization. This technique offers a means of controllably encapsulating biofilms over large surfaces and complex geometries. The generic deposition mechanism employed to form the silica matrix can be translated to a wide range of biological material and represents a platform encapsulation technology.