Carbon nanotube multi-electrode array chips for noninvasive real-time measurement of dopamine, action potentials, and postsynaptic potentials.

Multi-electrode arrays (MEAs) can be used for noninvasive, real-time, and long-term recording of electrophysiological activity and changes in the extracellular chemical microenvironment. Neural network organization, neuronal excitability, synaptic and phenotypic plasticity, and drug responses may be monitored by MEAs, but it is still difficult to measure presynaptic activity, such as neurotransmitter release, from the presynaptic bouton. In this study, we describe the development of planar carbon nanotube (CNT)-MEA chips that can measure both the release of the neurotransmitter dopamine as well as electrophysiological responses such as field postsynaptic potentials (fPSPs) and action potentials (APs). These CNT-MEA chips were fabricated by electroplating the indium-tin oxide (ITO) microelectrode surfaces. The CNT-plated ITO electrode exhibited electrochemical response, having much higher current density compared with the bare ITO electrode. Chronoamperometric measurements using these CNT-MEA chips detected dopamine at nanomolar concentrations. By placing mouse striatal brain slices on the CNT-MEA chip, we successfully measured synaptic dopamine release from spontaneous firings with a high S/N ratio of 62. Furthermore, APs and fPSPs were measured from cultured hippocampal neurons and slices with high temporal resolution and a 100-fold greater S/N ratio. Our CNT-MEA chips made it possible to measure neurotransmitter dopamine (presynaptic activities), postsynaptic potentials, and action potentials, which have a central role in information processing in the neuronal network. CNT-MEA chips could prove useful for in vitro studies of stem cell differentiation, drug screening and toxicity, synaptic plasticity, and pathogenic processes involved in epilepsy, stroke, and neurodegenerative diseases.

Control of neural network patterning using collagen gel photothermal etching.

Two-dimensional (2D) micropatterning techniques have been developed to guide dissociated neurons into predefined distributions on solid substrates, such as glass and plastic. Micropatterning methods using three-dimensional (3D) substrates or scaffolds that reproduce aspects of the in vivo microenvironment could facilitate the engineering of functional tissues for transplantation or more robust experimental models. We developed a 3D collagen gel photothermal etching method using an infrared laser that precisely controls the area of cell adhesion and neurite projection by etching a small targeted section of the collage gel. It was then possible to guide neural network formation under microscopic observation. After conventional cell seeding, we succeeded in creating isolated 3D networks, while controlling (1) the number of each neural subtype (neurons, glia, and fluorescently-labeled neurons) and (2) the direction of neurite elongation. Neurons seeded on a 10-µm-thick collagen gel survived longer and projected greater numbers of neurites than neurons growing on 2D culture substrates. Intracellular Ca(2+) imaging revealed both synchronous and discordant oscillations in different neuronal populations that suggested the pattern and strength of synaptic connectivity. This photothermal etching technique allows for the creation of designed 3D neural networks during cultivation for use in studies of synaptic transmission, neuron-glial signaling, pathogenesis, and drug responses.

A simplified micropatterning method for straight-line neurite extension of cultured hippocampal neurons.

We report a simplified micropatterning method for the straight-line extension of the neurites of cultured neurons. We prepared a poly-D-lysine (PDL)-patterned surface using a polydimethylsiloxane microfluidic stamp. Hippocampal neurons were cultured on the PDL-bound substrate with the stamp removed, allowing for conventional cell seeding and detailed optical observation without fluorescent label. Cultured neurons elongated neurites along straight lines at the single-cell level and displayed spontaneous firing as detected by time-lapse imaging and Ca(2+) imaging.

Development of a package-free transparent disposable biosensor chip for simultaneous measurements of blood constituents and investigation of its storage stability.

A package-free transparent disposable biosensor chip was developed by a screen-printing technique. The biosensor chip was fabricated by stacking a substrate with two carbon electrodes on its surface, a spacer consisting of a resist layer and an adhesive layer, and a cover. The structure of the chip keeps the interior of the reaction-detecting section airtight until use. The chip is equipped with double electrochemical measuring elements for the simultaneous measurement of multiple items, and the reagent layer was developed in sample-feeding path. The sample-inlet port and air-discharge port are simultaneously opened by longitudinally folding in two biosensor units with a notch as a boundary. Then the shape of the chip is changed to a V-shape. The reaction-detecting section of the chip has a 1.0 microl sample volume for one biosensor unit. Excellent results were obtained with the chip in initial simultaneous chronoamperometric measurements of both glucose (r=1.00) and lactate (r=0.998) in the same samples. The stability of the enzyme sensor signals of the chip was estimated at ambient atmosphere on 8 testing days during a 6-month period. The results were compared with those obtained for an unpackaged chip used as a control. The package-free chip proved to be twice as good as the control chip in terms of the reproducibility of slopes from 16 calibration curves (one calibration curve: 0, 100, 300, 500 mg dl(-1) glucose; n=3) and 4.6 times better in terms of the reproducibility of correlation coefficients from the 16 calibration curves.

A new BOD estimation method employing a double-mediator system by ferricyanide and menadione using the eukaryote Saccharomyces cerevisiae.

A new biochemical oxygen demand (BOD) sensing method employing a double-mediator (DM) system coupled with ferricyanide and a lipophilic mediator, menadione and the eukaryote Saccharomyces cerevisiae has been developed. In this study, a stirred micro-batch-type microbial sensor with a 560muL volume and a two-electrode system was used. The chronamperometric response of this sensor had a linear response between 1muM and 10mM hexacyanoferrate(II) (r(2)=0.9995, 14 points, n=3, average of relative standard deviation and R.S.D.(av)=1.3%). Next, the optimum conditions for BOD estimation by the DM system (BOD(DM)) were investigated and the findings revealed that the concentration of ethanol, used to dissolve menadione, influenced the sensor response and a relationship between the sensor output and glucose glutamic acid concentration was obtained over a range of 6.6-220mgO(2)L(-1) (five points, n=3, R.S.D.(av) 6.6%) when using a reaction mixture incubated for 15min. Subsequently, the characterization of this sensor was studied. The sensor responses to 14 pure organic substances were compared with the conventional BOD(5) method and other biosensor methods. Similar results with the BOD biosensor system using Trichosporon cutaneum were obtained. In addition, the influence of chloride ion, artificial seawater and heavy metal ions on the sensor response was investigated. A slight influence of 20.0gL(-1) chloride ion and artificial seawater (18.4gL(-1) Cl(-)) was observed. Thus, the possibility of BOD determination for seawater was suggested in this study. In addition, no influence of the heavy metal ions (1.0mgL(-1) Fe(3+), Cu(2+), Mn(2+), Cr(3+) and Zn(2+)) was observed. Real sample measurements using both river water and seawater were performed and compared with those obtained from the BOD(5) method. Finally, stable responses were obtained for 14 days when the yeast suspension was stored at 4 degrees C (response reduction, 93%; R.S.D. for 6 testing days, 9.1%).

Development of a self-sterilizing lancet coated with a titanium dioxide photocatalytic nano-layer for self-monitoring of blood glucose.

A photocatalyst was applied to a lancet for pricking the finger to obtain an antibacterial property. A photocatalytic and uniform nano-layer of titanium dioxide (TiO2) on the surface of the lancet (0.36 mm x 24.5 mm) was formed by sputtering and annealed for crystallization of the TiO2 layer. By elementary analysis of the TiO2 layer, titanium and oxygen were detected. Next, for the estimation of the antibacterial properties resulting from the photocatalytic effect, the lancet was packed into a capillary tube filled with a suspension of Escherichia coli K-12 (non-spore-forming bacterium), and was continuously rolled in a continuous UV-irradiation system under black-light irradiation. Distinct antibacterial effects after irradiation at 0.5 mW cm(-2) for 45 min were observed in the crystallized TiO2 layer on the lancet. Finally, lancing resistances obtained by pricking an artificial skin sheet were examined using control lancets, and lancets with an unannealed TiO2 layer or an annealed TiO2 layer. The results showed almost the same lancing resistances for the control (0.53+/-0 N, n=3) and the lancet with an annealed TiO2 layer (0.51+/-0.018 N), while the lancet with an unannealed TiO2 layer showed a high lancing resistance compared with the other lancets (0.62+/-0.05 N). In conclusion, the lancet coated with a crystallized, velvety nano-layer of TiO2 obtained by annealing had antibacterial properties and a similar lancing resistance compared with the bare lancet, and showed potential for application in monitoring blood glucose in diabetes.

Structural development of a minimally invasive sensor chip for blood glucose monitoring.

Our newly developed glucose sensor chip has the world smallest blood sample volume, as small as 200 nL, which makes lancing with less pain possible. Forming the inside walls of the cavity, where the blood is collected, by screen printing of the adhesive ink instead of the conventional adhesive sheet placing drastically reduced the cavity thickness, to less than 50 microm, and the blood sample volume. On this thin-cavity type sensor, the cavity thickness was proven to have the strong influence on the sensor response. This means that thinner cavity requires more accurate printing, in terms of thickness control. The printing conditions were adjusted to minimize the dispersion in the cavity geometry. One of the challenges was how to print patterns without saddles, which was achieved by selecting proper ink. After the adjustment, the contribution of the dispersion in the cavity thickness and the electrode area to the dispersion in sensor responses was estimated, respectively, which indicated the contribution of the cavity thickness still existed. Finally, the sensor was evaluated using whole sheep blood containing glucose of from 60 to 493 mg dL(-1). Irrespective of the influence relating to the cavity thickness, the coefficients of variation of the sensor responses were from 4.4 to 7.6. In addition, the correlation curve showed linearity in this blood glucose range, and the coefficient of determination r2 was 0.98. That is, the sensor was verified to have sufficient performance for practical use.