[show abstract][hide abstract] ABSTRACT: Inorganic materials have properties that can be advantageous in bioencapsulation for cell transplantation. Our aim was to engineer a hybrid inorganic / soft tissue construct by inducing pancreatic islets to grow an inorganic shell. We created pancreatic islets surrounded by porous silica, which has potential application in the immuno-protection of islets in transplantation therapies for type 1 diabetes. The new method takes advantage of the islet capsule surface as a template for silica formation. Mouse and human islets were exposed to media containing saturating silicic acid levels for 9 to 15 minutes. The resulting tissue constructs were then cultured for up to four weeks under normal conditions. Scanning electron microscopy and energy dispersive X-ray spectroscopy was used to monitor the morphology and elemental composition of the material at the islet surface. Cytokine assay was used to assess biocompatibility with macrophages. Islet survival and function was assessed by confocal microscopy, glucose-stimulated insulin release assays, oxygen flux at the islet surface, expression of key genes by RT-PCR, and syngeneic transplant into diabetic mice.
AJP Endocrinology and Metabolism 09/2013; · 4.51 Impact Factor
[show abstract][hide abstract] ABSTRACT: Abstract Silver nanoparticles (Ag NPs) are gaining popularity as bactericidal agents in commercial products; however, the mechanisms of toxicity (MOT) of Ag NPs to other organisms are not fully understood. It is the goal of this research to determine differences in MOT induced by ionic Ag+ and Ag NPs in Daphnia magna, by incorporating a battery of traditional and novel methods. Daphnia embryos were exposed to sublethal concentrations of AgNO3 and Ag NPs (130-650 ng/L), with uptake of the latter confirmed by confocal reflectance microscopy. Mitochondrial function was non-invasively monitored by measuring proton flux using self-referencing microsensors. Proton flux measurements revealed that while both forms of silver significantly affected proton efflux, the change induced by Ag NPs was greater than that of Ag+. This could be correlated with the effects of Ag NPs on mitochondrial dysfunction, as determined by confocal fluorescence microscopy and JC-1, an indicator of mitochondrial permeability. However, Ag+ was more efficient than Ag NPs at displacing Na+ within embryonic Daphnia, based on inductively coupled plasma-mass spectroscopy (ICP-MS) analysis. The abnormalities in mitochondrial activity for Ag NP-exposed organisms suggest a nanoparticle-specific MOT, distinct from that induced by Ag ions. We propose that the MOT of each form of silver are complementary, and can act in synergy to produce a greater toxic response overall.
[show abstract][hide abstract] ABSTRACT: Type 1 diabetes is preceded by islet β-cell dysfunction, but the mechanisms leading to β-cell dysfunction have not been rigorously studied. Because immune cell infiltration occurs prior to overt diabetes, we hypothesized that activation of inflammatory cascades and appearance of endoplasmic reticulum (ER) stress in β-cells contributes to insulin secretory defects. Prediabetic nonobese diabetic (NOD) mice and control diabetes-resistant NOD-SCID and CD1 strains were studied for metabolic control and islet function and gene regulation. Prediabetic NOD mice were relatively glucose intolerant and had defective insulin secretion with elevated proinsulin:insulin ratios compared with control strains. Isolated islets from NOD mice displayed age-dependent increases in parameters of ER stress, morphologic alterations in ER structure by electron microscopy, and activation of nuclear factor-κB (NF-κB) target genes. Upon exposure to a mixture of proinflammatory cytokines that mimics the microenvironment of type 1 diabetes, MIN6 β-cells displayed evidence for polyribosomal runoff, a finding consistent with the translational initiation blockade characteristic of ER stress. We conclude that β-cells of prediabetic NOD mice display dysfunction and overt ER stress that may be driven by NF-κB signaling, and strategies that attenuate pathways leading to ER stress may preserve β-cell function in type 1 diabetes.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: We describe two hybrid nanomaterial biosensor platforms, based on networks of single-walled carbon nanotubes (SWCNTs) enhanced with Pd nanocubes and Pt nanospheres and grown in situ from a porous anodic alumina (PAA) template. These nanocube and nanosphere SWCNT networks are converted into glutamate biosensors by immobilizing the enzyme glutamate oxidase (cross-linked with gluteraldehyde) onto the electrode surface. The Pt nanosphere/SWCNT biosensor outperformed the Pd nanocube/SWCNT biosensor and previously reported similar nanomaterial-based biosensors by amperometrically monitoring glutamate concentrations with a wide linear sensing range (50 nM to 1.6 mM) and a small detection limit (4.6 nM, 3s). These results combined with the biosensor fabrication scheme (in situ growth of SWCNTs, electrodeposition of metal nanoparticles, and facile enzyme immobilization protocol) create a biosensor that can potentially be scaled for integration into a wide range of applications including the treatment of neurological disorders.
[show abstract][hide abstract] ABSTRACT: We describe two hybrid nanomaterial biosensor platforms, based on networks of single-walled carbon nanotubes (SWCNTs) enhanced with Pd nanocubes and Pt nanospheres and grown in situ from a porous anodic alumina (PAA) template. These nanocube and nanosphere SWCNT networks are converted into glutamate biosensors by immobilizing the enzyme glutamate oxidase (cross-linked with gluteraldehyde) onto the electrode surface. The Pt nanosphere/SWCNT biosensor outperformed the Pd nanocube/SWCNT biosensor and previously reported similar nanomaterial-based biosensors by amperometrically monitoring glutamate concentrations with a wide linear sensing range (50 nM to 1.6 mM) and a small detection limit (4.6 nM, 3 sigma). These results combined with the biosensor fabrication scheme (in situ growth of SWCNTs, electrodeposition of metal nanoparticles, and facile enzyme immobilization protocol) create a biosensor that can potentially be scaled for integration into a wide range of applications including the treatment of neurological disorders.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Quantification of neurotransmitter transport dynamics is hindered by a lack of sufficient tools to directly monitor bioactive flux under physiological conditions. Traditional techniques for studying neurotransmitter release/uptake require inferences from non-selective electrical recordings, are invasive/destructive, and/or suffer from poor temporal resolution. Recent advances in electrochemical biosensors have enhanced in vitro and in vivo detection of neurotransmitter concentration under physiological/pathophysiological conditions. The use of enzymatic biosensors with performance enhancing materials (e.g., carbon nanotubes) has been a major focus for many of these advances. However, these techniques are not used as mainstream neuroscience research tools, due to relatively low sensitivity, excessive drift/noise, low signal-to-noise ratio, and inability to quantify rapid neurochemical kinetics during synaptic transmission. A sensing technique known as self-referencing overcomes many of these problems, and allows non-invasive quantification of biophysical transport. This work presents a self-referencing CNT modified glutamate oxidase biosensor for monitoring glutamate flux near neural/neuronal cells. Concentration of basal glutamate was similar to other in vivo and in vitro measurements. The biosensor was used in self-referencing (oscillating) mode to measure net glutamate flux near neural cells during electrical stimulation. Prior to stimulation, the average influx was 33.9 ± 6.4 fmol cm−2 s−1). Glutamate efflux took place immediately following stimulation, and was always followed by uptake in the 50–150 fmol cm−2 s−1 range. Uptake was inhibited using threo-β-benzyloxyaspartate, and average surface flux in replicate cells (1.1 ± 7.4 fmol cm−2 s−1) was significantly lower than uninhibited cells. The technique is extremely valuable for studying neuropathological conditions related to neurotransmission under dynamic physiological conditions.
Journal of neuroscience methods 03/2010; · 2.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mammalian cell culture platforms often require biomolecular modification to enhance cell adhesion and proliferation, Often, these modifications are performed using self-assembled monolayers or whole protein coatings, Such its collagen. These protocols are inherently useful but generally suffer from repeatability. Undesirable conditions during self-assembly can lead to complications in the surface presentation of the biological ligands. Whole proteins are often unstable and derived from animal sources, making them less attractive for tissue engineering applications. As the biological effect of the material often depends strongly on the concentration of the integrated ligand(s), any complication due to synthesis or stability can lead to unexpected biological results. In this research, we expand upon previous work in peptide-silane modifications to sol-gel derived silica matrixes, demonstrating that the surface density of the peptide can be calibrated by simply modifying the starting liquid precursor concentration. The potential for calibration of peptide surface presentation allows for well-defined cell culture platforms that have the potential to mimic natural proteins in a stable, repeatable manner.
[show abstract][hide abstract] ABSTRACT: Approximately 30% of individuals with epilepsy have refractory seizures that cannot be controlled by current pharmacological treatment measures. For such patients, responsive neurostimulation prior to a seizure may lead to greater efficacy when compared with current treatments. In this paper, we present a real-time adaptive Wiener prediction algorithm implemented on a digital signal processor to be used with local field potential (LFP) recordings. The hardware implementation of the algorithm enables it to be a miniaturized portable system that could be used in a hand-held device. The adaptive nature of the algorithm allows the seizure data to be compared with baseline data occurring in the recent past rather than a preset value. This enhances the sensitivity of the algorithm by accounting for the time-varying dynamics of baseline, inter-ictal and ictal activity. The Wiener algorithm was compared to two statistical-based naïve prediction algorithms. ROC curves, area over ROC curves, predictive power, and time under false positives are computed to characterize the algorithm. Testing of the algorithm via offline Matlab analysis on kainate-treated rats results in prediction of seizures about 27 s before clinical onset, with 94% sensitivity and a false positive rate of 0.009 min(-1). When implemented on a real-time TI C6713 signal processor, the algorithm predicts seizures about 6.7s before their clinical onset, with 92% sensitivity and a false positive rate of 0.08 min(-1). These results compare favorably with those obtained in similar studies in terms of sensitivity and false positive rate.
Computers in biology and medicine 12/2009; 40(1):97-108. · 1.27 Impact Factor
[show abstract][hide abstract] ABSTRACT: Quaternized copolymers of 4-vinylpyridine and poly(ethylene glycol) methyl ether methacrylate are known to have antibacterial properties and have displayed biocompatibility in red blood cell hemolysis assays. The results from hemolysis assays have shown substantial promise, but the technique is rudimentary and only a first step toward the determination of biocompatibility. The present paper further explores the biocompatibility of these copolymers through comprehensive cell viability assays performed on Caco-2 human epithelial cells cultivated in vitro. We have shown that these copolymers are biocompatible at concentrations above their minimum bactericidal concentrations, leading to selectivity values that compare well with other microbicidal products.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of neuroscience methods 08/2009; 183(2):213-22. · 2.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this work we quantified the in vitro calibration relationships between high frequency electrical stimulation and GABA and glutamate release in both mature retinoic acid differentiated P19 neurons and immortalized embryonic cortical cells engineered to express glutamic acid decarboxylase, GAD65. Extracellular glutamate and GABA was quantified by 2D gas chromatography and time of flight mass spectrometry after stimulation at varying amplitudes and frequencies. Amplitude sweeps resulted in a linear calibration for P19 neurons; the level of neurotransmitter varied over one order of magnitude from ~ 200 pg/neuron to ~ 1.2 ng/neuron for glutamate and ~ 1 ng/neuron to ~ 2 ng/neuron for GABA, depending on the stimulation amplitude. Frequency sweeps resulted in a peak release at 250 Hz for glutamate and 400 Hz for GABA in P19 cells. The GABA transporter inhibitor, nipecotic acid, increased extracellular GABA levels and decrease glutamate. In contrast the embryonic cortical cells had a strongly nonlinear dependency of release on stimulation amplitude, and a weak dependence on frequency. These cells had roughly equal extracellular glutamate and GABA levels after stimulation despite the expression of GAD65. In addition glutamate and GABA levels were insensitive to nipecotic acid. These results demonstrate an ability to calibrate and tune neurotransmitter release from neural cells using high frequency stimulation parameters.
International Journal of Neural Systems 07/2009; 19(3):197-212. · 5.05 Impact Factor
[show abstract][hide abstract] ABSTRACT: The reactive tissue response of the brain to chronically implanted materials remains a formidable obstacle to stable recording from implanted microelectrodes. One approach to mitigate this response is to apply a bioactive coating in the form of an ultra-porous silica sol-gel, which can be engineered to improve biocompatibility and to enable local drug delivery. The first step in establishing the feasibility of such a coating is to investigate the effects of the coating on electrode properties. In this paper, we describe a method to apply a thin-film silica sol-gel coating to silicon-based microelectrodes, and discuss the resultant changes in the electrode properties. Fluorescently labeled coatings were used to confirm coating adherence to the electrode. Cyclic voltammetry and impedance spectroscopy were used to evaluate electrical property changes. The silica sol-gel was found to successfully adhere to the electrodes as a thin coating. The voltammograms revealed a slight increase in charge carrying capacity of the electrodes following coating. Impedance spectrograms showed a mild increase in impedance at high frequencies but a more pronounced decrease in impedance at mid to low frequencies. These results demonstrate the feasibility of applying silica sol-gel coatings to silicon-based microelectrodes and are encouraging for the continued investigation of their use in mitigating the reactive tissue response.
Journal of neuroscience methods 06/2009; 180(1):106-10. · 2.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: The successful use of implantable neural microelectrodes as neuroprosthetic devices depends on the mitigation of the reactive tissue response of the brain. One of the factors affecting the ultimate severity of the reactive tissue response and the in vivo electrical properties of the microelectrodes is the initial adsorption of proteins onto the surface of the implanted microelectrodes. In this study we quantify the increase in microelectrode impedance magnitude at physiological frequencies following electrode immersion in a 10% bovine serum albumin (BSA) solution. We also demonstrate the efficacy of a common antifouling molecule, poly(ethylene glycol) (PEG), in preventing a significant increase in microelectrode impedance. In addition, we show the feasibility of using long-duration DC voltage pulses to remove adsorbed proteins from the microelectrode surface.
Conference proceedings: ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference 01/2009; 2009:7139-42.
[show abstract][hide abstract] ABSTRACT: Nitric oxide (NO) is an important cell-signaling molecule whose role in a variety of cellular processes such as differentiation and apoptosis depends strongly on its concentration and flux levels. This work describes and characterizes a novel nitric oxide releasing nanocomposite, capable of photostimulated NO flux that can by dynamically modulated in within a range of biological levels. This material mimics the common compartmentalization strategies used by living cells to achieve its novel features. The material is constructed by encapsulating a photosensitive nitric oxide donor within lipid vesicles with an average diameter of 150 nm. The vesicles are then doped into the interstitial liquid phase of a solid porous silica matrix, which has previously demonstrated biological compatibility and capabilities as a growth surface for mammalian cells. Stimulation by a light source produces a step increase in NO concentration within seconds. The NO flux at the surface of the material is measured to be 14 pmol-cm(-2) sec(-1) using a NO selective self-referencing amperometric microsensor. The NO concentration profile decreases with distance perpendicular to the surface as expected for diffusion from a surface through an aqueous environment. A pattern of one minute light pulses produced uniform pulses of increased NO concentration of one minute duration. A linear relationship exists between NO surface concentration and photon flux, and this relationship can be used to tune the material response.
The Journal of Physical Chemistry B 12/2008; 112(47):15086-93. · 3.61 Impact Factor