Yen-Chung Chang

A flexible hydrophilic-modified graphene microprobe for neural and cardiac recording.

UNLABELLED A graphene-based flexible microprobe developed by microelectromechanical system technology shows high resolution for the detection of electrophysiological signals from various bio-objects. The hydrophilization post-treatment using steam plasma was performed on the graphene surface to decrease the interfacial impedance between graphene and electrolyte, and thus improve the signal-to-noise ratio during neural and cardiac recording. The signal-to-noise ratio of the action potentials from axons of a crayfish measured by hydrophilic-modified graphene microprobe (27.8±4.0dB) is higher than that of untreated device (20.3±3.3dB). Also, the form of the QRS complex and T wave in the electrocardiogram of the zebrafish heart can be clearly distinguished using the modified device. The total measured noise levels of the overall stability of the system were 4.2μVrms (hydrophilic graphene) and 7.64μVrms (hydrophobic graphene). The graphene-based implant can be further used for in vivo, long-term recording and retina prosthesis. FROM THE CLINICAL EDITOR In this study a graphene-based flexible microprobe developed using microelectromechanical system technology was demonstrated to enable high resolution detection of electrophysiological signals, including EKG in zebrafish models. Both hydrophilic and hydrophobic graphene were studied, paving the way to potential future clinical applications of this new technology.

A three-dimensional flexible microprobe array for neural recording assembled through electrostatic actuation

We designed, fabricated and tested a novel three-dimensional flexible microprobe to record neural signals of a lateral giant nerve fiber of the escape circuit of an American crayfish. An electrostatic actuation folded planar probes into three-dimensional neural probes with arbitrary orientations for neuroscientific applications. A batch assembly based on electrostatic forces simplified the fabrication and was non-toxic. A novel fabrication for these three-dimensional flexible probes used SU-8 and Parylene technology. The mechanical strength of the neural probe was great enough to penetrate into a bio-gel. A flexible probe both decreased the micromotion and alleviated tissue encapsulation of the implant caused by chronic inflammation of tissue when an animal breathes or moves. The cortex consisted of six horizontal layers, and the neurons of the cortex were arranged in vertical structures; the three-dimensional microelectrode arrays were suitable to investigate the cooperative activity for neurons in horizontal separate layers and in vertical cortical columns. With this flexible probe we recorded neural signals of a lateral giant cell from an American crayfish. The response amplitude of action potentials was about 343 µV during 1 ms period; the average recorded data had a ratio of signal to noise as great as 30.22 ± 3.58 dB. The improved performance of this electrode made feasible the separation of neural signals according to their distinct shapes. The cytotoxicity indicated a satisfactory biocompatibility and non-toxicity of the flexible device fabricated in this work.

A cone-shaped 3D carbon nanotube probe for neural recording

A novel cone-shaped 3D carbon nanotube (CNT) probe is proposed as an electrode for applications in neural recording. The electrode consists of CNTs synthesized on the cone-shaped Si (cs-Si) tip by catalytic thermal chemical vapor deposition (CVD). This probe exhibits a larger CNT surface area with the same footprint area and higher spatial resolution of neural recording compared to planar-type CNT electrodes. An approach to improve CNT characteristics by O(2) plasma treatment to modify the CNT surface will be also presented. Electrochemical characterization of O(2) plasma-treated 3D CNT (OT-CNT) probes revealed low impedance per unit area (∼64.5 Ω mm(-2)) at 1 kHz and high specific capacitance per unit area (∼2.5 mF cm(-2)). Furthermore, the OT-CNT probes were employed to record the neural signals of a crayfish nerve cord. Our findings suggest that OT-CNT probes have potential advantages as high spatial resolution and superb electrochemical properties which are suitable for neural recording applications.

An active, flexible carbon nanotube microelectrode array for recording electrocorticograms

A variety of microelectrode arrays (MEAs) has been developed for monitoring intra-cortical neural activity at a high spatio-temporal resolution, opening a promising future for brain research and neural prostheses. However, most MEAs are based on metal electrodes on rigid substrates, and the intra-cortical implantation normally causes neural damage and immune responses that impede long-term recordings. This communication presents a flexible, carbon-nanotube MEA (CMEA) with integrated circuitry. The flexibility allows the electrodes to fit on the irregular surface of the brain to record electrocorticograms in a less invasive way. Carbon nanotubes (CNTs) further improve both the electrode impedance and the charge-transfer capacity by more than six times. Moreover, the CNTs are grown on the polyimide substrate directly to improve the adhesion to the substrate. With the integrated recording circuitry, the flexible CMEA is proved capable of recording the neural activity of crayfish in vitro, as well as the electrocorticogram of a rat cortex in vivo, with an improved signal-to-noise ratio. Therefore, the proposed CMEA can be employed as a less-invasive, biocompatible and reliable neuro-electronic interface for long-term usage.

Efficient precipitation and accurate quantitation of detergent-solubilized membrane proteins

The protein assay method of I. Polacheck and E. Cabib (Anal. Biochem. 117, 311-314, 1981) has been modified to provide a general method for quantitating protein samples in the presence of detergents. Dilute detergent-solubilized membrane proteins, by using ribonucleic acid as a carrier, have been efficiently precipitated here by trichloroacetic acid (TCA) in the presence of sodium dodecyl sulfate (SDS). Washing the pellets once with TCA solution has removed most of the reagents present in the original sample. The washed sample could then be quantitated by the Lowry method (O. J. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265-275, 1951). This procedure could be used to assay protein solutions of a concentration as low as 5 micrograms/ml in the presence of the following reagents: Triton X-100, Triton X-114, Tween 20, N-octylglucoside, deoxycholate, cholate, Thesit, octanoyl-N-methylglucamide, isotridecylpoly (ethyleneglycoether)n, Nonidet P-40, glucose, methyl-D-glucopyranoside, methyl-D-mannopyranoside, N-acetyl-glucosamine, Mn2+, Ca2+, Mg2+, and many buffer reagents. Proteins solubilized from porcine brain myelin sheath and synaptic plasma membranes were quantitated by amino acid analysis and by the TCA/SDS precipitation method described here. The resultant protein concentrations were almost identical. The results have suggested this TCA/SDS precipitation method to be useful for quantitating dilute protein samples containing high concentrations of detergents and other reagents commonly employed in studying membrane proteins.

Structural role of zinc ions bound to postsynaptic densities

Postsynaptic densities (PSDs) isolated from porcine cerebral cortices are large aggregates consisting of more than 30 different proteins. Inductively coupled plasma–mass spectrometric analyses revealed that isolated PSDs contained zinc at a concentration of 4.1 nmol per mg protein. Treatment with 8 m urea lead to dissociation of the PSDs into small components and, concomitantly, depletion of most of their bound zinc. After removal of the urea by dialysis, urea‐dissociated PSD proteins did not reassemble into aggregates by themselves. Adding ZnCl2 to urea‐treated PSD samples resulted in the assembly of urea‐dissociated proteins into large aggregates with morphology and protein composition closely resembling those of the original PSDs. Mg2+, Ca2+, Co2+, Cd2+, Cu2+, Mn2+, Fe3+, K+ and Na+ ions at higher concentrations also induced the aggregation of urea‐dissociated PSD protein. The structures of the K+‐, Na+‐, Mg2+‐ and Ca2+‐induced aggregates were distinct from that of the original PSDs. Our results indicate that the structure of the PSD could be disassembled and reassembled under in vitro conditions. They further suggest that Zn2+ ions, by binding to certain zinc‐binding proteins, play an important role in the formation and maintenance of the structure of the PSD.

Syntheses of (2S, 4S)- and (2S, 4R)-4-substituted glutamic acid analogues for neuroexcitatory activity studies

The stereospecific syntheses of various length of 4(R)- and 4(S)-substituted 2(S)-glutamic acid were described

A novel NMDA receptor antagonist protects against N-methyl-d-aspartate- and glutamate-induced neurotoxicity in the goldfish retina

4(R)-(3-Phenylpropyl)-2(S)-glutamic acid, C(3), is a synthetic analogue of L-glutamate. This analogue reversibly inhibits the membrane depolarization of neurons in the CA1 region of rat hippocampal slices evoked by N-methyl-D-aspartate (NMDA), with an EC50 value of 3.6 microM, whereas the depolarization of these neurons evoked by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid is not inhibited by C(3). Analyses of the inhibitory effect of C(3) on NMDA-evoked currents of dissociated rat hippocampal neurons further revealed that C(3) acts as a competitive antagonist of NMDA receptors and that the inhibitory action of C(3) is not use-dependent. Using goldfish retina as a model, we found that the neuronal damage produced by glutamate or by NMDA was effectively prevented by C(3). Incubation of retinas with high concentrations of C(3), up to 1 mM, did not induce pathomorphological changes in retinal neurons. These results suggest that C(3) is a useful neuroprotectant against excitotoxic damage of neurons.

A Battery-Less, Implantable Neuro-Electronic Interface for Studying the Mechanisms of Deep Brain Stimulation in Rat Models

Although deep brain stimulation (DBS) has been a promising alternative for treating several neural disorders, the mechanisms underlying the DBS remain not fully understood. As rat models provide the advantage of recording and stimulating different disease-related regions simultaneously, this paper proposes a battery-less, implantable neuro-electronic interface suitable for studying DBS mechanisms with a freely-moving rat. The neuro-electronic interface mainly consists of a microsystem able to interact with eight different brain regions bi-directionally and simultaneously. To minimize the size of the implant, the microsystem receives power and transmits data through a single coil. In addition, particular attention is paid to the capability of recording neural activities right after each stimulation, so as to acquire information on how stimulations modulate neural activities. The microsystem has been fabricated with the standard 0.18 μm CMOS technology. The chip area is 7.74 mm 2, and the microsystem is able to operate with a single supply voltage of 1 V. The wireless interface allows a maximum power of 10 mW to be transmitted together with either uplink or downlink data at a rate of 2 Mbps or 100 kbps, respectively. The input referred noise of recording amplifiers is 1.16 μVrms, and the stimulation voltage is tunable from 1.5 V to 4.5 V with 5-bit resolution. After the electrical functionality of the microsystem is tested, the capability of the microsystem to interface with rat brain is further examined and compared with conventional instruments. All experimental results are presented and discussed in this paper.

Identification of an enzymatic activity that hydrolyzes protein-bound ADP-ribose in skeletal muscle

An enzymatic activity present in high-speed supernatant fluids of rat skeletal muscle was found that catalyzes the release of ADP-ribose from ADP-ribosylated-modified lysozyme. The nature of the product was proved by chromatographic studies and proton nuclear magnetic resonance spectroscopy. The enzyme activity is stimulated by Mg2+, dithioerythritol, and flouride. These results and those published earlier (Soman, G., Mickelson, J.R., Louis, C.F., and Graves, D.J. (1984) Biochem. Biophys. Res. Commun. 120, 973-980) show that ADP-ribosylation is a reversible process in skeletal muscle.