Danielle R. Rand

Endothelial cell responses to micropillar substrates of varying dimensions and stiffness

In the vascular niche, the extracellular matrix (ECM) provides a structural scaffold with a rich ligand landscape of essential matrix proteins that supports the organization and stabilization of endothelial cells (ECs) into functional blood vessels. Many of the physical interactions between ECs and macromolecular components of the ECM occur at both the micron and submicron scale. In addition, the elasticity of the ECM has been shown to be a critical factor in the progress of the angiogenic cascade. Here, we sought to determine the effect of substrate topography and elasticity (stiffness) on EC behavior. Utilizing a unique SiO(2) substrate with an array of micropillars, we first demonstrate that micropillars with heights >3 μm significantly decrease EC adhesion and spreading. Fibronectin (Fn) patterning of 1 μm high micropillars enabled EC adhesion onto the micropillars and promoted alignment in a single-cell chain manner. We then developed a robust method to generate a soft micropillar substrate array made of polydimethylsiloxane (PDMS), similar to the SiO(2) substrate. Finally, we examined the kinetics of EC adhesion and spreading on the soft PDMS substrates compared to the stiff SiO(2) substrates. Culturing cells on the PDMS substrates demonstrated an enhanced EC elongation and alignment when compared to stiff SiO(2) with similar topographical features. We conclude that the elongation and alignment of ECs is coregulated by substrate topography and stiffness and can be harnessed to guide vascular organization.

Superimposed topographic and chemical cues synergistically guide neurite outgrowth

Guidance of neuronal extensions is a complex process essential for linking neurons into complex functional networks underlying the workings of the neural system. Decades of research have suggested the ability of neuronal growth cones to integrate multiple types of cues during the extension process, but also have raised numerous still unanswered questions about synergy or antagonism between the superimposed chemical and mechanical signaling inputs. In this study, using a novel microfabricated analysis platform, we investigate the response of primary mouse embryonic hippocampal neurons to superimposed topographic and soluble chemical cues. We find that an optimal spatial frequency of topographic cues exists, maximizing the precision of the neurite extension. This optimal frequency can help the extending neurites navigate a topographically complex environment, providing pronounced directional selectivity. We also demonstrate that this cue can synergistically enhance attractive and suppress repulsive guidance by the bi-functional soluble cue Netrin-1, and eliminate the repulsive guidance by a chemorepellent Semaphorin3A (Sema3A). These results suggest that topographic cues can provide optimal periodic input into the guidance signaling processes involved in growth cone chemoattraction and can synergistically interact with chemical gradients of soluble guidance cues, shedding light on complex events accompanying the development of the functional nervous system.

Bottom-Up SiO2 Embedded Carbon Nanotube Electrodes with Superior Performance for Integration in Implantable Neural Microsystems

The reliable integration of carbon nanotube (CNT) electrodes in future neural probes requires a proper embedding of the CNTs to prevent damage and toxic contamination during fabrication and also to preserve their mechanical integrity during implantation. Here we describe a novel bottom-up embedding approach where the CNT microelectrodes are encased in SiO(2) and Parylene C with lithographically defined electrode openings. Vertically aligned CNTs are grown on microelectrode arrays using low-temperature plasma-enhanced chemical vapor deposition compatible with wafer-scale CMOS processing. Electrodes with 5, 10, and 25 μm diameter are realized. The CNT electrodes are characterized by electrochemical impedance spectroscopy and cyclic voltammetry and compared against cofabricated Pt and TiN electrodes. The superior performance of the CNTs in terms of impedance (≤4.8 ± 0.3 kΩ at 1 kHz) and charge-storage capacity (≥513.9 ± 61.6 mC/cm(2)) is attributed to an increased wettability caused by the removal of the SiO(2) embedding in buffered hydrofluoric acid. Infrared spectroscopy reveals an unaltered chemical fingerprint of the CNTs after fabrication. Impedance monitoring during biphasic current pulsing with increasing amplitudes provides clear evidence of the onset of gas evolution at CNT electrodes. Stimulation is accordingly considered safe for charge densities ≤40.7 mC/cm(2). In addition, prolonged stimulation with 5000 biphasic current pulses at 8.1, 40.7, and 81.5 mC/cm(2) increases the CNT electrode impedance at 1 kHz only by 5.5, 1.2, and 12.1%, respectively. Finally, insertion of CNT electrodes with and without embedding into rat brains demonstrates that embedded CNTs are mechanically more stable than non-embedded CNTs.

Glutamate sensing with enzyme-modified floating-gate field effect transistors

Neurotransmitter release is the key factor of chemical messaging in the brain. Fast, sensitive and in situ detection of single cell neurotransmitter release is essential for the investigation of synaptic transmission under physiological or pathophysiological conditions. Although various techniques have been developed for detecting neurotransmitter release both in vitro and in vivo, the sensing of such events still remains challenging. First of all, the amount of neurotransmitter released during synaptic transmission is unknown because of the limited number of molecules released and the fast diffusion and reuptake of these molecules after release. On the other hand, advances in microelectronic biosensor devices have made possible the fast detection of various analytes with high sensitivity and selectivity. Specifically, enzyme-modified field-effect (ENFET) devices are attractive for such applications due to their fast response, small dimensions and the possibility to integrate a large number of sensors on the same chip. In this paper, we present a floating-gate FET device coated with glutamate oxidase (GLOD) layer. The surface chemistry was optimized for maximal enzyme loading and long-term stability, and characterized by quartz crystal microbalance and colorimetric assays. Enzyme loading was largest on poly-L-lysin-based surfaces combined with glutaraldehyde. The surface chemistry showed excellent stability for at least one month in Tris buffers stored at 4 degrees C. A glutamate detection limit of 10(-7) M has been obtained with the GLOD-coated FET and our sensor proved to be selective to glutamate only. We show that this biosensor is a promising tool for the in vitro detection of glutamate and can be extended to other neurotransmitters.

Coulometric detection of irreversible electrochemical reactions occurring at Pt microelectrodes used for neural stimulation.

The electrochemistry of 50 μm diameter Pt electrodes used for neural stimulation was studied in vitro by reciprocal derivative chronopotentiometry. This differential method provides well-defined electrochemical signatures of the various polarization phenomena that occur at Pt microelectrodes and are generally obscured in voltage transients. In combination with a novel in situ coulometric approach, irreversible H(2) and O(2) evolution, Pt dissolution and reduction of dissolved O(2) were detected. Measurements were performed with biphasic, charge-balanced, cathodic-first and anodic-first current pulses at charge densities ranging from 0.07 to 1.41 mC/cm(2) (real surface area) in phosphate buffered saline (PBS) with and without bovine serum albumin (BSA). The extent to which O(2) reduction occurs under the different stimulation conditions was compared in O(2)-saturated and deoxygenated PBS. Adsorption of BSA inhibited Pt dissolution as well as Pt oxidation and oxide reduction by blocking reactive sites on the electrode surface. This inhibitory effect promoted the onset of irreversible H(2) and O(2) evolution, which occurred at lower charge densities than those in PBS. Reduction of dissolved O(2) on Pt electrodes accounted for 19-34% of the total injected charge in O(2)-saturated PBS, while a contribution of 0.4-12% was estimated for in vivo stimulation. These result may prove important for the interpretation of histological damage induced by neural stimulation and therefore help define safer operational limits.

Surface Amplification of L-Glutamate Using a Patterned Bienzymatic System for Biosensing Applications

Electronic sensing of neurotransmitter molecules is based on a diffusion-limited process, which requires the immobilization of biological recognition elements as close as possible to the active area of the sensor. Moreover, in many applications, the analyte concentration is very low. In particular, the in situ detection of neurotransmitter release from neurons is challenging due to the limited number of molecules secreted and their fast diffusion and reuptake immediately after release. In this paper, we present a method that allows for the local amplification of L-glutamate directly on the chip surface. Our approach is based on the surface patterning of a bienzymatic system consisting of glutamate oxidase (GLOD) and glutamic-pyruvate transaminase (GPT) that amplifies L-glutamate via a recycling process. The surface chemistry was optimized for maximal enzyme loading. The level of amplification was determined using a colorimetric assay. Coimmobilization of GLOD and GPT yielded at least a doubling of the signal, and increasing the surface concentrations of each enzyme led to amplification levels that approached those obtained in solution. We show that these enzymes can be patterned on substrates using a flip chip bonder for aligned microcontact printing. Furthermore, primary mouse hippocampal neurons were successfully cultured on these patterned surfaces and remained viable for at least five days. The enzymatic pattern was preserved on the substrate surface for the same time period. Lastly, amplification of L-glutamate released from neurons seven days in vitro was detected. Thus, we conclude that this bienzymatic system can ultimately be applied to biosensor surfaces for the in vitro detection of L-glutamate.

Local electrical stimulation of cultured embryonic cardiomyocytes with sub-micrometer nail structures

In this paper, we demonstrate the feasibility of selective extracellular electrical stimulation at the (sub)cellular level in dissociated cultured cells. Using a CMOS-compatible process, we have fabricated an electrode array with sub-micrometer nail probes. Due to their particular configuration, the nails are strongly engulfed by the cellular membrane. By measuring the calcium signals, we found that electrical stimulation via the micronails activates the cell locally, in a dose-dependent manner, with very low applied currents. The results suggest the applicability of the device in pharmacological or signal propagation studies.

Selective Protein Immobilization onto Gold Nanoparticles Deposited under Vacuum on a Protein-Repellent Self-Assembled Monolayer

The immobilization of proteins on flat substrates plays an important role for a wide spectrum of applications in the fields of biology, medicine, and biochemistry, among others. An essential prerequisite for the use of proteins (e.g., in biosensors) is the conservation of their biological activity. Losses in activity upon protein immobilization can largely be attributed to a random attachment of the proteins to the surface. In this study, we present an approach for the immobilization of proteins onto a chemically heterogeneous surface, namely a surface consisting of protein-permissive and protein-repellent areas, which allows for significant reduction of random protein attachment. As protein-permissive, i.e., as protein-binding sites, ultra pure metallic nanoparticles are deposited under vacuum onto a protein-repellent PEG-silane polymer layer. Using complementary surface characterization techniques (atomic force microscopy, quartz crystal microbalance, and X-ray photoelectron spectroscopy) we demonstrate that the Au nanoparticles remain accessible for protein attachment without compromising the protein-repellency of the PEG-silane background. Moreover, we show that the amount of immobilized protein can be controlled by tuning the Au nanoparticle coverage. This method shows potential for applications requiring the control of protein immobilization down to the single molecule level.

Site-specific bio-functionalization of surfaces by means of metal nanostructures

We present an approach to control the immobilization process of biomolecules by functionalization of SiO2 substrates decorated with nanostructures. The creation of specific protein permissive and protein resistant areas leads to a directed and site-specific immobilization of proteins. We make use of molecular beam epitaxy and laser assisted cluster deposition to create Au nanostructures on PEG-silanized SiO2 substrates. The chemical contrast provided by the two materials, namely Au and PEG on the substrate surface supports the specificity of thiol-based Au functionalization for biomolecule attachment.

On-Surface Amplification of L-Glutamate Using a Patterned Bi-enzymatic System

We present a method for the amplification of L-glutamate directly on a surface. Our strategy is based on the surface patterning of a bi-enzymatic system consisting of glutamate oxidase (GLOD) and glutamic-pyruvate transaminase (GPT). This bi-enzymatic system amplifies L-glutamate via a recycling process. The surface chemistry on Ta2O5 was optimized for maximal enzyme loading. Enzyme activity was determined using a colormetric assay for the presence of GLOD. Co-immobilization of GLOD and GPT results in at least a doubling of the signal. Furthermore, increasing the surface concentrations of each enzyme leads to amplification levels that approach those obtained in solution. These enzymes can be patterned on substrates using a flip chip bonder for aligned microcontact printing. This bi-enzymatic system can be applied to biosensor surfaces for the in vitro detection of L-glutamate.