Single-cell recording and stimulation with a 16k micro-nail electrode array integrated on a 0.18 μm CMOS chip

To cope with the growing needs in research towards the understanding of cellular function and network dynamics, advanced micro-electrode arrays (MEAs) based on integrated complementary metal oxide semiconductor (CMOS) circuits have been increasingly reported. Although such arrays contain a large number of sensors for recording and/or stimulation, the size of the electrodes on these chips are often larger than a typical mammalian cell. Therefore, true single-cell recording and stimulation remains challenging. Single-cell resolution can be obtained by decreasing the size of the electrodes, which inherently increases the characteristic impedance and noise. Here, we present an array of 16,384 active sensors monolithically integrated on chip, realized in 0.18 μm CMOS technology for recording and stimulation of individual cells. Successful recording of electrical activity of cardiac cells with the chip, validated with intracellular whole-cell patch clamp recordings are presented, illustrating single-cell readout capability. Further, by applying a single-electrode stimulation protocol, we could pace individual cardiac cells, demonstrating single-cell addressability. This novel electrode array could help pave the way towards solving complex interactions of mammalian cellular networks.     

Characterization of a microfluidic dispensing system for localised stimulation of cellular networks

We present a 3-D microfluidic device designed for localized drug delivery to cellular networks. The device features a flow cell comprising a main channel for nutrient delivery as well as multiple channels for drug delivery. This device is one key component of a larger, fully integrated system now under development, based upon a microelectrode array (MEA) with on-chip CMOS circuitry for recording and stimulation of electrogenic cells (e.g. neurons, cardiomyocytes). As a critical system unit, the microfluidics must be carefully designed and characterized to ensure that candidate drugs are delivered to specific regions of the culture at known concentrations. Furthermore, microfluidic design and functionality is dictated by the size, geometry, and material/electrical characteristics of the CMOS MEA. Therefore, this paper reports on the design considerations and fabrication of the flow cell, including theoretical and experimental analysis of the mass transfer properties of the nutrient and drug flows, which are in good agreement with one another. To demonstrate proof of concept, the flow cell was mounted on a dummy CMOS chip, which had been plated with HL-1 cardiomyocytes. A test chemical compound was delivered to the cell culture in a spatially resolved manner. Envisioned applications of this stand-alone system include simultaneous toxicological testing of multiple compounds and chemical stimulation of natural neural networks for neuroscience investigations.     

The potential of microelectrode arrays and microelectronics for biomedical research and diagnostics

Planar microelectrode arrays (MEAs) are devices that can be used in biomedical and basic in vitro research to provide extracellular electrophysiological information about biological systems at high spatial and temporal resolution. Complementary metal oxide semiconductor (CMOS) is a technology with which MEAs can be produced on a microscale featuring high spatial resolution and excellent signal-to-noise characteristics. CMOS MEAs are specialized for the analysis of complete electrogenic cellular networks at the cellular or subcellular level in dissociated cultures, organotypic cultures, and acute tissue slices; they can also function as biosensors to detect biochemical events. Models of disease or the response of cellular networks to pharmacological compounds can be studied in vitro, allowing one to investigate pathologies, such as cardiac arrhythmias, memory impairment due to Alzheimer’s disease, or vision impairment caused by ganglion cell degeneration in the retina.     

Biological application of microelectrode arrays in drug discovery and basic research

Electrical activity of electrogenic cells in neuronal and cardiac tissue can be recorded by means of microelectrode arrays (MEAs) that offer the unique possibility for non-invasive extracellular recording from as many as 60 sites simultaneously. Since its introduction 30 years ago, the technology and the related culture methods for electrophysiological cell and tissue assays have been continually improved and have found their way into many academic and industrial laboratories. Currently, this technology is attracting increased interest owing to the industrial need to screen selected compounds against ion channel targets in their native environment at organic, cellular, and sub-cellular level.As the MEA technology can be applied to any electrogenic tissue (i.e., central and peripheral neurons, heart cells, and muscle cells), the MEA biosensor is an ideal in vitro system to monitor both acute and chronic effects of drugs and toxins and to perform functional studies under physiological or induced pathophysiological conditions that mimic in vivo damages. By recording the electrical response of various locations on a tissue, a spatial map of drug effects at different sites can be generated, providing important clues about a drug's specificity.In this survey, examples of MEA biosensor applications are described that have been developed for drug screening and discovery and safety pharmacology in the field of cardiac and neural research. Additionally, biophysical basics of recording and concepts for analysis of extracellular electrical signals are presented.Abbreviations AP action potential - DG dentate gyrus - EC entorhinal cortex - ECG electrocardiogram - ERG electroretinogram - LFP local field potentials - MEA microelectrode array - PSTH peri-stimulus–time histogram - SNR signal-to-noise ratio     

Cell adhesion promotion strategies for signal transduction enhancement in microelectrode array in vitro electrophysiology: An introductory overview and critical discussion

Microelectrode arrays (MEAs) find application both in vitro and in vivo to record and stimulate electrical activity in electrogenic cells such as neurons, cardiomyocytes, pancreatic beta cells or immortalized cell lines derived therefrom (e.g., PC12, HL-1). In MEA electrophysiology, the quality of the predominantly extracellularly recorded or elicited electrical signals strongly depends on the distance, strength and stability of the interfacial contact between the electrogenic cells and an electrode. Decorating the substrate or electrode with biochemical adhesion factors and physical guidance cues does not only determine the tightness of that junction, but it also modulates substrate biocompatibility, its biostability, cell differentiation as well as cell fate. If an interface is furthermore topologically, chemically or physically patterned or constrained, neural interconnectivity may be steered towards directional organization. In this introductory and selective overview, we briefly discuss adhesion events at the chemical and biological level, review the general role and mechanisms of cell adhesion in (neuro)biology, then explore how cells adhere to artificial substrates. This will lead to the discussion of popular strategies for enhancing and steering interfacial interactions at the bio-hardware boundary with particular focus on MEA substrates. It will include a critical treatment of open issues with respect to the origin and shape of extracellularly recorded signals and their modulation by cell-culture-inherent events.     

A vertically aligned carbon nanotube-based impedance sensing biosensor for rapid and high sensitive detection of cancer cells

A novel vertically aligned carbon nanotube based electrical cell impedance sensing biosensor (CNT-ECIS) was demonstrated for the first time as a more rapid, sensitive and specific device for the detection of cancer cells. This biosensor is based on the fast entrapment of cancer cells on vertically aligned carbon nanotube arrays and leads to mechanical and electrical interactions between CNT tips and entrapped cell membranes, changing the impedance of the biosensor. CNT-ECIS was fabricated through a photolithography process on Ni/SiO(2)/Si layers. Carbon nanotube arrays have been grown on 9 nm thick patterned Ni microelectrodes by DC-PECVD. SW48 colon cancer cells were passed over the surface of CNT covered electrodes to be specifically entrapped on elastic nanotube beams. CNT arrays act as both adhesive and conductive agents and impedance changes occurred as fast as 30 s (for whole entrapment and signaling processes). CNT-ECIS detected the cancer cells with the concentration as low as 4000 cells cm(-2) on its surface and a sensitivity of 1.7 × 10(-3)Ω cm(2). Time and cell efficiency factor (TEF and CEF) parameters were defined which describe the sensor's rapidness and resolution, respectively. TEF and CEF of CNT-ECIS were much higher than other cell based electrical biosensors which are compared in this paper.     

Well-ordered porous conductive polypyrrole as a new platform for neural interfaces

We present the preparation of electrically conductive, porous polypyrrole surfaces and demonstrate their use as an interactive substrate for neuronal growth. Nerve growth factor (NGF)-loaded porous conducting polymers were initially prepared by electrochemical deposition of a mixture of pyrrole monomers and NGF into two- or three-dimensional particle arrays followed by subsequent removal of a sacrificial template. Morphological observation by scanning electron microscopy (SEM) revealed these to possess high regularity and porosity with well-defined topographical features. A four-point probe study demonstrated remarkable electrical activities despite the presence of voids. In addition, we investigated the effects of these surfaces on cellular behaviors using PC 12 cells in the presence and absence of electrical stimulation. Our results suggest that the surface topography as well as an applied electrical field can play a crucial role in determining further cell responses. Indeed, surface-induced preferential regulation leads to enhanced cellular viability and neurite extension. Establishing the underlying cellular mechanisms in response to various external stimuli is essential in that one can elicit positive neuronal guidance and modulate their activities by engineering a series of electrical, chemical, and topographical cues.     

Controlled on-chip stimulation of quantal catecholamine release from chromaffin cells using photolysis of caged Ca2+ on transparent indium-tin-oxide microchip electrodes

Photorelease of caged Ca(2+) is a uniquely powerful tool to study the dynamics of Ca(2+)-triggered exocytosis from individual cells. Using photolithography and other microfabrication techniques, we have developed transparent microchip devices to enable photorelease of caged Ca(2+), together with electrochemical detection of quantal catecholamine secretion from individual cells or cell arrays as a step towards developing high-throughput experimental devices. A 100 nm thick transparent indium-tin-oxide (ITO) film was sputter-deposited onto glass coverslips, which were then patterned into 24 cell-sized working electrodes (approximately 20 microm by 20 microm). We loaded bovine chromaffin cells with acetoxymethyl (AM) ester derivatives of the Ca(2+) cage NP-EGTA and Ca(2+) indicator dye fura-4F, then transferred these cells onto the working ITO electrodes for amperometric recordings. Upon flash photorelease of caged Ca(2+), a uniform rise of [Ca(2+)](i) within the target cell leads to quantal release of oxidizable catecholamines measured amperometrically by the underlying ITO electrode. We observed a burst of amperometric spikes upon rapid elevation of [Ca(2+)](i) and a "priming" effect of sub-stimulatory [Ca(2+)](i) on the response of cells to subsequent [Ca(2+)](i) elevation, similar to previous reports using different techniques. We conclude that UV photolysis of caged Ca(2+) is a suitable stimulation technique for higher-throughput studies of Ca(2+)-dependent exocytosis on transparent electrochemical microelectrode arrays.     

Optimization of processing parameters on the controlled growth of ZnO nanorod arrays for the performance improvement of solid-state dye-sensitized solar cells

High-transparency and high quality ZnO nanorod arrays were grown on the ITO substrates by a two-step chemical bath deposition (CBD) method. The effects of processing parameters including reaction temperature (25-95 °C) and solution concentration (0.01-0.1 M) on the crystal growth, alignment, optical and electrical properties were systematically investigated. It has been found that these process parameters are critical for the growth, orientation and aspect ratio of the nanorod arrays, showing different structural and optical properties. Experimental results reveal that the hexagonal ZnO nanorod arrays prepared under reaction temperature of 95 °C and solution concentration of 0.03 M possess highest aspect ratio of ∼21, and show the well-aligned orientation and optimum optical properties. Moreover the ZnO nanorod arrays based heterojunction electrodes and the solid-state dye-sensitized solar cells (SS-DSSCs) were fabricated with an improved optoelectrical performance.     

Synthesis and characterization of axial heterojunction inorganic-organic semiconductor nanowire arrays

The end-to-end P-N heterojunction nanowire arrays combined organic (poly[1,4-bis(pyrrol-2-yl)benzene], BPB) and inorganic (CdS) molecules have been successfully designed and fabricated. The electrical properties of P-N heterojunctions of organic-inorganic nanowire arrays were investigated. The diode nature and rectifying feature of P-N heterojunction nanowire arrays were observed. The rectification ratio of the diode increased from 29.9 to 129.7 as the illumination intensity increased. The material exhibits a new property, which is an improvement in the integration of the physical and chemical properties of the two independent components.     

Synthesis, structures, and magnetic properties of transition metal compounds with 2,2'-dinitrobiphenyl-4,4'-dicarboxylate and N,N'-chelating ligands

Solvothermal reactions of Co(II), Ni(II), Zn(II) salts with 2,2'-dinitrobiphenyl-4,4'-dicarboxylate (dnpdc) and 2,2'-bipyridyl-like chelating ligands yielded five compounds formulated as [Co(dnpdc)(bipy)](n)·nH(2)O (1), [M(dnpdc)(phen)](n) (2, M = Co; 3, M = Ni; 4, M = Zn) and [Co(dnpdc)(biql)](n)·2nH(2)O (5) (bipy = 2,2'-bipyridine, phen = 1,10-phenanthroline and biql = 2,2'-biquinoline). With bipy or phen as coligands, compounds 1-4 exhibit isomorphous 3D M(dnpdc) metal-organic frameworks in which double carboxylate bridged chains are interlinked by the backbones of the dicarboxylate ligands. The bipy or phen ligands are involved in interchain hydrogen bonding or π-π interactions to form 1D zipper-like arrays in the rhombic channels of the frameworks, playing a templating role and determining the channel dimensions. The biql coligand is too bulky for the 1D double carboxylate bridged chain and the rhombic channel. Instead, in compound 5, the dnpdc ligands link metal ions into 1D zigzag metal-organic chains and the biql ligands are arranged into 2D (6,3) arrays through extensive π-π stacking interactions. In compounds 1-3, the double carboxylate bridges in the nonplanar syn-skew conformation mediate ferromagnetic interactions along the chains, while the chelating ligands provide supramolecular pathways for interchain antiferromagnetic interactions. The π-π interactions in 5 also evoke weak antiferromagnetic interactions.     

Fabrication and Optical and Electrical Properties of Poly(2,5-di-n-butoxyphenylene) Nanowire Arrays

Regular patterned arrays of nanomaterials have been widely fabricated and studied for their benefits in construction of novel type of optical, electron and magnetic device1-2, these kinds of devices center on the inorganic materials. With the development of synthesis and application of new type of polymer material, the design and construction of organic nanopolymer have become a great interest. Poly(p-phenylene)(PPP) and some derivatives have been widely investigated as a candidate for high strength, high temperature and conducting polymers, and can be used as electrode materials in electrochemical cells, blue emitting diodes: The polymers obtained by oxidative coupling polymerization of benzene nuclei with aluminum chloride and copper(Ⅱ) chloride is insoluble in all solvent and inflexible, which hinders revealing their basic properties. Introduction of flexible side chains into the aromatic rings can not only render solubility and processibility, but also improve or modify optical and electrical properties of the polymers. As a further step in assembling method and optoelectronic studies, it is attractive to investigate the properties of photoluminescence and electroluminescene of regular patterned arrays of poly(p-phenylene) deriva-tives nanowires.     

Charge induced closing of Dionaea muscipula Ellis trap

In terms of bioelectrochemistry, Venus flytrap responses can be considered in three stages: stimulus perception, electrical signal transmission, and induction of mechanical and biochemical responses. When an insect touches the trigger hairs, these mechanosensors generate receptor potentials, which induce solitary waves activating the motor cells. We found that the electrical charge injected between a midrib and a lobe closes the Venus flytrap leaf by activating motor cells without mechanical stimulation of trigger hairs. The mean electrical charge required for the closure of the Venus flytrap leaf is 13.6 muC. To close the trap, electrical charge can be submitted as a single charge or applied cumulatively by small portions during a short period of time. Ion channel blocker such as Zn(2+) as well as an uncoupler CCCP, dramatically decreases the speed of the trap closing a few hours after treatment of the soil. This effect is reversible. After soil washing by distilled water, the closing time of Venus flytrap treated by CCCP or ZnCl(2) decreases back from 2-5 s to 0.3 s, but higher electrical charge is needed for trap closure. The mechanism behind closing the upper leaf of Venus flytrap is discussed.     

Rapid activation of the melibiose permease MelB immobilized on a solid-supported membrane

Rapid solution exchange on a solid-supported membrane (SSM) is investigated using fluidic structures and a solid-supported membrane of 1 mm diameter in wall jet geometry. The flow is analyzed with a new technique based on specific ion interactions with the surface combined with an electrical measurement. The critical parameters affecting the time course of the solution exchange and the transfer function describing the time resolution of the SSM system are determined. The experimental data indicate that solution transport represents an intermediate situation between the plug flow and the Hagen-Poiseuille laminar flow regime. However, to a good approximation the rise of the surface concentration can be described by Hagen-Poiseuille flow with ideal mixing at the surface of the SSM. Using an improved cuvette design, solution exchange as fast as 2 ms was achieved at the surface of a solid-supported membrane. As an application of the technique, the rate constant of a fast electrogenic reaction in the melibiose permease MelB, a bacterial ( Escherichia coli) sugar transporter, is determined. For comparison, the kinetics of a conformational transition of the same transporter was measured using stopped-flow tryptophan fluorescence spectroscopy. The relaxation time constant obtained for the charge displacement agrees with that determined in the stopped-flow experiments. This demonstrates that upon sugar binding MelB undergoes an electrogenic conformational transition with a rate constant of k approximately 250 s (-1).     

Monitoring Electropermeabilization of Adherent Mammalian Cells Through Electrochemical Impedance Spectroscopy

In this work, cell adhesion and electroporation effects have been studied through impedance measurements. Chinese Hamster Ovary (CHO-K1) cells have been plated and grown adherent to multi-electrode arrays and then stimulated to obtain the electroporation. We used Electrochemical Impedance Spectroscopy (EIS) measurements to analyze the impedance variation that occurs with cell adhesion and, consequently, to obtain the intrinsic electrical parameters of the electrode-cell interface through a lumped parameter model. The whole model allows to estimate the correction factor β and the sealing resistance R_seal, both dependent on the cell coverage and adhesion. The electroporation has been performed after cell adhesion, using a custom stimulation bench, able to evaluate the cell-electrode coupling and to stimulate an individually addressable site. By performing EIS measurements, before and after the electroporation, cell status, adhesion and membrane resistance can be detected. From measurements, an impedance decrease as a function of pulse amplitude can be noted. The proposed equivalent electrical model allows to evaluate the variation in membrane conductance due to pores formation. Moreover, average pore radius between 0.5 nm and 2.2 nm, increasing with pulse amplitude, was estimated.     

Electrical conducting bis(oxalato)platinate complex with direct connection of CuII ions

Reactions of K1.62[Pt(ox)2].2H2O and [Cu(bpy)(H2O)3](NO3)2 yielded partially oxidized one-dimensional (1D) bis(oxalato)platinates of [Cu(bpy)(H2O)n]6[Pt(ox)2]7.7H2O (n = 2, 3, or 4) (1) and [Cu(bpy)(H2O)n]8[Pt(ox)2]10.8H2O (n = 3 or 4) (2). The average oxidation numbers of the platinum ions in 1 and 2 are +2.29 and +2.40, respectively. Complexes 1 and 2 crystallize in the triclinic P and monoclinic C2/c space groups, respectively, and the [Pt(ox)2]n- anions are stacked along the crystallographic b axis with 7-fold periodicity for 1 and 10-fold periodicity for 2. In 1, an oxalato ligand in the platinum chain directly coordinates to a paramagnetic [Cu(bpy)(H2O)3]2+ ion, whereas no such direct coordination was observed for 2. The electrical conductivity of 2 at room temperature along the platinum chain is approximately 3 orders of magnitude smaller (sigma||= 1.3 x 10(-3) S cm(-1)) than that of 1 (sigma|| = 0.9-0.5 S cm(-1)), and the activation energies of 1 and 2 are 29 and 67 meV, respectively. The longest inter-platinum distances in 1 and 2 are 2.762 and 3.0082 A, respectively, and this is responsible for the lower electrical conductivity of 2. An X-ray oscillation photograph taken along the b axis of 1 reveals the 7-fold periodicity in the 1D chain, consistent with the period of the Peierls distortion estimated from the degree of partial oxidation. The semiconducting state of 1 can therefore be regarded as a commensurate Peierls state. The magnetoresistance of 1 at ambient pressure indicates no interaction between conduction electrons in the platinum chain and local spins of the paramagnetic CuII ions. Application of hydrostatic pressures of up to 3 GPa enhances electrical conduction, as is often seen as the usual pressure effect on the electrical conductivity, which is due to enhanced orbital (Pt-5dz2) overlap by pressure application.     

Advances in Large Gap Peripheral Nerve Injury Repair and Regeneration with Bridging Nerve Guidance Conduits

Peripheral nerve injury is a common complication of accidents and diseases. The traditional autologous nerve graft approach remains the gold standard for the treatment of nerve injuries. While sources of autologous nerve grafts are very limited and difficult to obtain. Nerve guidance conduits are widely used in the treatment of peripheral nerve injuries as an alternative to nerve autografts and allografts. However, the development of nerve conduits does not meet the needs of large gap peripheral nerve injury. Functional nerve conduits can provide a good microenvironment for axon elongation and myelin regeneration. Herein, the manufacturing methods and different design types of functional bridging nerve conduits for nerve conduits combined with electrical or magnetic stimulation and loaded with Schwann cells, etc., are summarized. It summarizes the literature and finds that the technical solutions of functional nerve conduits with electrical stimulation, magnetic stimulation and nerve conduits combined with Schwann cells can be used as effective strategies for bridging large gap nerve injury and provide an effective way for the study of large gap nerve injury repair. In addition, functional nerve conduits provide a new way to construct delivery systems for drugs and growth factors in vivo.     

TRANSFORMATION OF A BOP-HOP-SOP-I-SOP-II-Halobacterium halobium MUTANT TO BOP+: EFFECTS OF BACTERIORHODOPSIN PHOTOACTIVATION ON CELLULAR PROTON FLUXES AND SWIMMING BEHAVIOR

We have transformed Pho81, a Halobacterium halobium mutant strain which does not contain any of the four retinylidene proteins known in this species, with the bop gene cluster to create Pho81BR, a BR+HR-SR-I-SR-II-strain. The absorption spectrum, pigment reconstitution process, light-dark adaptation and photochemical reaction cycle of the expressed protein are indistinguishable from those of native bacteriorhodopsin (BR) in purple membrane of wild type strains. Strain Pho81BR permits for the first time characterization of effects of BR photoactivation alone on cell swimming behavior and energetics in the absence of the spectrally similar phototaxis receptor sensory rhodopsin I (SR-I) and electrogenic chloride pump halorhodopsin (HR). A non-adaptive upward shift in spontaneous swimming reversal frequency occurs following 3 s of continuous illumination of Pho81BR cells with green light (550 +/- 20 nm). This effect is abolished by low concentrations of the proton ionophore carbonylcyanide m-chlorophenylhydrazone. Although BR does not mediate phototaxis responses in energized Pho81BR cells under our culture conditions, proton pumping by BR in Pho81BR cells partially deenergized by inhibitors of respiration and adenosine triphosphate synthesis results in a small attractant response. Based on our measurements, we attribute the observed effects of BR photoactivation on swimming behavior to secondary consequences of electrogenic proton pumping on metabolic or signal transduction pathways, rather than to primary sensory signaling such as that mediated by SR-I. Proton extrusion by BR activates gated proton influx ports resulting in net proton uptake in wild-type cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution processable diketopyrrolopyrrole semiconductor: towards bio-electronic applications

In this paper, the possibility to use diketopyrrolopyrrole (DPP) for the construction of electrical devices designed to interact with animal cells was studied. For this purpose, the biocompatibility and electrical properties of the selected DPP derivative (3,6-bis(5-(benzofuran-2-yl)thiophen-2-yl)-2,5-bis(2-ethyl-hexyl)pyrrolo[3,4-c]pyrrole-1,4-dione) [referred as DPP(TBFu)₂] were researched. The electrical properties were studied using model organic field-effect transistors. Mainly investigated was under what conditions maximum charge carrier mobility can be achieved. Using the cumulative effect of self-assembled monolayers on dielectrics and electrodes and detailed thermal analysis of the DPP, a higher charge carrier mobility was achieved than has been previously reported (5.5?×?10^?3 cm² V^?1 s^?1). The biocompatibility was studied based on a culture of 3T3 fibroblasts. This research revealed that DPP(TBFu)₂ can be used in applications involving direct contact with living animal cells. The conclusions found with these model devices can be applied to components suitable for biosensing applications, e.g., water- or electrolyte-gated organic field-effect transistors.