Enzyme-based choline and L-glutamate biosensor electrodes on silicon microprobe arrays.

Brain-implantable microprobe arrays, 6.5 mm shaft-length, incorporating several recessed Pt microelectrodes (50 µm×150 µm) and an integrated Ag/AgCl reference electrode fabricated by silicon micromachining dry etching techniques (DRIE) are described. The microelectrodes are coated by an enzyme membrane and a semi-permeable m-phenylenediamine layer for the selective detection of the neurotransmitters choline and L-glutamate at physiologically relevant concentrations. The functionalisation is based on electrochemically aided adsorption (EAA) combined with chemical co-cross-linking using glutaraldehyde and electrochemical polymerisation, respectively. These deposition methods are fully compatible with the fabricated microprobe arrays for the simultaneous detection of several analytes in different brain target areas. They are spatially controlled and allow fabricating biosensors on several microelectrodes in parallel or providing a cross-talk-free coating of closely spaced microelectrodes with different enzyme membranes. A sensitivity of 132±20 µA mM(-1) cm(-2) for choline and 95±20 µA mM(-1) cm(-2) for L-glutamate with limits of detections below 0.5 µM was obtained. The results of in vitro and in vivo experiments confirm the functional viability of the choline and l-glutamate biosensors.

Extracellular recordings from locally dense microelectrode arrays coupled to dissociated cortical cultures.

High-density microelectrode arrays (MEAs) enabled by recent developments of microelectronic circuits (CMOS-MEA) and providing spatial resolutions down to the cellular level open the perspective to access simultaneously local and overall neuronal network activities expressed by in vitro preparations. The short inter-electrode separation results in a gain of information on the micro-circuit neuronal dynamics and signal propagation, but requires the careful evaluation of the time resolution as well as the assessment of possible cross-talk artifacts. In this respect, we have realized and tested Pt high-density (HD)-MEAs featuring four local areas with 10microm inter-electrode spacing and providing a suitable noise level for the assessment of the high-density approach. First, simulated results show how possible artifacts (duplicated spikes) can be theoretically observed on nearby microelectrodes only for very high-shunt resistance values (e.g. R(sh)=50 kOmega generates up to 60% of false positives). This limiting condition is not compatible with typical experimental conditions (i.e. dense but not confluent cultures). Experiments performed on spontaneously active cortical neuronal networks show that spike synchronicity decreases by increasing the time resolution and analysis results show that the detected synchronous spikes on nearby electrodes are likely to be unresolved (in time) fast local propagations. Finally, functional connectivity analysis results show stronger local connections than long connections spread homogeneously over the whole network demonstrating the expected gain in detail provided by the spatial resolution.

High-resolution MEA platform for in-vitro electrogenic cell networks imaging.

A platform based on an active-pixel-sensor electrode array (APS-MEA) for high-resolution imaging of in-vitro electrogenic cell cultures is presented, characterized and validated under culture conditions. The system enables full frame acquisition at 8 kHz from 4096 microelectrodes integrated with separations of 21 microm and zoomed area acquisition with temporal resolutions down to 8 micros. This bi-modal acquisition feature opens new perspectives in particular for neuronal activity analysis and for the correlation of micro-scale and macro-scale behaviors. The low-noise performances of the integrated amplifier (11 microVRMS) combined with a hardware implementation reflecting image-/video-concepts enable high-resolution acquisitions with real-time processing capabilities adapted to the handling of the large amount of acquired data.

High-density electrode array for imaging in vitro electrophysiological activity.

The development of a high-density active microelectrode array for in vitro electrophysiology is reported. Based on the Active Pixel Sensor (APS) concept, the array integrates 4096 gold microelectrodes (electrode separation 20 microm) on a surface of 2.5 mmx2.5 mm as well as a high-speed random addressing logic allowing the sequential selection of the measuring pixels. Following the electrical characterization in a phosphate solution, the functional evaluation has been carried out by recording the spontaneous electrical activity of neonatal rat cardiomyocytes. Signals with amplitudes from 130 microVp-p to 300 microVp-p could be recorded from different pixels. The results demonstrate the suitability of the APS concept for developing a new generation of high-resolution extracellular recording devices for in vitro electrophysiology.

Development of a multifunctional platform for extra- and intracellular ionic activities recording.

We present the development of a multifunctional platform equipped with an array of silicon nitride micropipettes with dimensions allowing the implementation of extra- and intracellular operations. Micropipettes with outer diameter that ranges from 6 mum down to 300 nm and with walls thicknesses of 500 down to 150 nm are presented. The generic technology developed to fabricate these micropipettes has a number of advantages, including the ability to be implemented as ion-selective electrodes for (A) intracellular and (B) extracellular recordings and as (C) local drug microdispensers.

Enzyme modified microband electrodes: cross-talk effects and their elimination.

One microband of an array of four microband electrodes (1 mm long and 25 microm wide with a 25 microm gap) was modified with glucose oxidase by direct electrochemically assisted immobilisation, giving a stable microbiosensor with an apparent Michaelis-Menten constant of 12 mM and an i(max) of 80 nA. Cross-talk effects on the adjacent microbands were studied and three different methods for their elimination were tested: the most efficient one involved catalase deposition on the adjacent microband. Under these conditions, the maximum response at the unmodified microbands was in the worst case about 3% compared with the response of the modified microband. This approach has the potential to fabricate a multianalyte microbiosensor.

Individually addressable gel-integrated voltammetric microelectrode array for high-resolution measurement of concentration profiles at interfaces.

The application of a novel voltammetric probe, based on an individually addressable gel-integrated microelectrode array (IA-GIME), for real-time, high-spatial resolution concentration profile measurements at interfaces is described. Reliability and validity of steep metal concentration gradients obtained with this novel system have been demonstrated by performing systematic tests at well-controlled liquid-liquid and liquid-solid interfaces. The liquid-liquid interface was formed by two layers of aqueous solutions with different components; only one layer contained trace metal ions (Pb(II) and Cd(II)); the individually addressable microelectrode array was placed at the interface of the liquid-liquid system; the concentration profiles were recorded as function of time; and the effective diffusion coefficients were calculated. The liquid-"solid" interface was formed from an aqueous solution layer overlying a bed of silica particles saturated with an aqueous solution. The sensor array has been used to monitor the diffusion processes of Tl(I) or Pb(II) from the liquid phase to the "solid" phase. The influences of porosity, geometry of the porous media, and complexation between metal ion and silica, on the diffusion processes, have been studied. All these results show that correct diffusion profiles of metal ions at interfaces can be obtained with 200-microm resolution with the IA-GIME. They also demonstrate that, for measurements in "solid" phase, the aforementioned factors must be considered carefully for correct calibration of any electrodes and the gel-integrated microelectrodes are unique tools to enable calibration of the sensors with synthetic solutions.

Biocompatibility of silicon-based arrays of electrodes coupled to organotypic hippocampal brain slice cultures.

In this study we examined the passive biocompatibility of a three-dimensional microelectrode array (MEA), designed to be coupled to organotypic brain slice cultures for multisite recording of electrophysiological signals. Hippocampal (and corticostriatal) brain slices from 1-week-old (and newborn) rats were grown for 4-8 weeks on the perforated silicon chips with silicon nitride surfaces and 40 microm sized holes and compared with corresponding tissue slices grown on conventional semiporous membranes. In terms of preservation of the basic cellular and connective organization, as visualized by Nissl staining, Timm sulphide silver-staining, microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) immunostaining, the slice cultures grown on chips did not differ from conventionally grown slice cultures. Neither were there any signs of astrogliosis or neurodegeneration around the upper recording part of the 47-microm-high platinum-tip electrodes. Slice cultures grown on a separate set of chips with platinum instead of silicon nitride surfaces also displayed normal MAP2 and GFAP immunostaining. The width of the GFAP-rich zone (glia limitans) at the bottom surface of the slice cultures was the same ( approximately 20 microm) in cultures grown on chips with silicon nitride and platinum surfaces and on conventional insert membranes. The slice cultures grown on chips maintained a normal, subfield differentiated susceptibility to the glutamate receptor agonist N-methyl-D-aspartate (NMDA) and the neurotoxin trimethyltin (TMT), as demonstrated by the cellular uptake of propidium iodide (PI), which was used as a reproducible and quantifiable marker for neuronal degeneration. We conclude that organotypic brain slice cultures can grow on silicon-based three-dimensional microelectrode arrays and develop normally with display of normal subfield differentiated susceptibilities to known excito- and neurotoxins. From this it is anticipated that the set-up, designed for recording of electrophysiological parameters, can be used for long-term studies of defined neuronal networks and provide valuable information on both normal, neurotoxicological and neuropathological conditions.

A new calcium-sensor based on ion-selective conductometric microsensors--membranes and features.

Based on the concept of ion-selective conductometric microsensors (ISCOM) a new calcium sensor was developed and characterized. ISCOM have a single probe, all-solid-state construction and do not need a reference electrode. These sensors are amenable to miniaturization and integration in the true sense of integrated circuit and microsystem technologies. The detection is accomplished by measurement of the bulk conductance Gm of a thin polymeric membrane containing an ion-complexing agent, where the magnitude of Gm can be related to the content of the primary ion in the analyzed solution. Thin-film platinum electrodes forming an interdigitated electrode are used as the transducer to detect the conductivity of the polymeric membrane. Optimization of the membrane composition was carried out by testing different types of calcium-ionophores, polymers, and plasticizers. The sensor characteristics have been investigated. The limit of detection is about 10(-7) mol L(-1). The dynamic range is 10(-6)-10(-1) mol L(-1) with a response time of less than 5 s. These parameters are comparable to those of corresponding potentiometric calcium selective electrodes (ISE). The Ca(2+)-ISCOM demonstrates good practical relevant selectivities against typical interfering ions for biomedical and environmental applications.

Coupling of organotypic brain slice cultures to silicon-based arrays of electrodes.

Fetal or early postnatal brain tissue can be cultured in viable and healthy condition for several weeks with development and preservation of the basic cellular and connective organization as so-called organotypic brain slice cultures. Here we demonstrate and describe how it is possible to establish such hippocampal rat brain slice cultures on biocompatible silicon-based chips with arrays of electrodes with a histological organization comparable to that of conventional brain slice cultures grown by the roller drum technique and on semiporous membranes. Intracellular and extracellular recordings from neurons in the slice cultures show that the electroresponsive properties of the neurons and synaptic circuitry are in accordance with those described for cells in acutely prepared slices of the adult rat hippocampus. Based on the recordings and the possibilities of stimulating the cultured cells through the electrode arrays it is anticipated that the setup eventually will allow long-term studies of defined neuronal networks and provide valuable information on both normal and neurotoxicological and neuropathological conditions.

An array of Pt-tip microelectrodes for extracellular monitoring of activity of brain slices.

A microelectrode array (MEA) consisting of 34 silicon nitride passivated Pt-tip microelectrodes embedded on a perforated silicon substrate (porosity 35%) has been realized. The electrodes are 47 microns high, of which only the top 15 microns are exposed Pt-tips having a curvature of 0.5 micron. The MEA is intended for extracellular recordings of brain slices in vitro. Here we report the fabrication, characterization and initial electrophysiological evaluation of the first generation of Pt-tip MEAs.

Electrochemiluminescence of Tris(2,2'-bipyridine)ruthenium in Water at Carbon Microelectrodes.

Electrochemiluminescence (ECL) of Ru(bpy)(3)(2+) in water only, without any added electrolyte or reducing agents, has been obtained at carbon interdigitated microelectrode arrays (C-IDAs) of 2 µm width and spacing. In a generation/collection biasing mode, ECL can be clearly seen with the naked eye in normal room lighting at concentrations greater than 1 mM. Using a conventional photomultiplier tube (PMT), a detection limit of 10(-)(7) M Ru(bpy)(3)(2+) has been achieved for an electrode area of 0.25 mm(2). In comparison, the ECL intensity produced at Pt-IDA of the same geometry, under identical experimental conditions, was more than 300 times less. The ECL obtained at C-IDAs is attributed to the annihilation reaction of the reduced and oxidized forms of the Ru(bpy)(3)(2+) made possible due to the small electrode spacing.

Microelectrode arrays for electrophysiological monitoring of hippocampal organotypic slice cultures.

A three-dimensional platinum (Pt) microelectrode array embedded on a micromachined silicon (Si) substrate (porosity of 13%, via hole diameter of 40 microns) has been developed. Electrodes are 35-micron wide and 20-microns high, spaced 200 microns apart and arranged in an elliptic geometry. Integrated within a microperfusion chamber, the devices were used for stimulation and recording experiments of hippocampal slice cultures over a period of several days.

Micromachined analyzers on a silicon chip.

For an example of a silicon-based micromachined analyzer, we describe a combined PO2, PCO2, and pH sensor designed for extracorporeal blood gas monitoring. The clinically well-accepted amperometric (PO2) and potentiometric (PCO2, pH) sensing principles are used, realized in a planar and miniaturized form on a single silicon chip (6 x 22 mm). The transducer part of the chip is fabricated by standard silicon technology. Polyacrylamide and polysiloxane polymeric layers, which are used as internal electrolyte and gas-permeable membrane, respectively, are deposited and patterned by photopolymerization. The entire sensor is fabricated on the wafer level by using integrated-circuit-compatible processes, thus allowing mass production. By integrating a flow-through channel directly on the chip, the sample size and the reagent consumption are substantially reduced. The device was characterized in aqueous solutions and in blood intended for transfusion. The sensor has a typical sensitivity of 0.36 nA/mmHg (PO2), -39 mV/decade (PCO2), and 51 mV/pH (pH); low drift; and a functional lifetime of > 2 months. The analytical precision in the physiologically expected range is better than 2 mmHg for the PO2 and PCO2 sensor, and 0.02 pH unit for the pH sensor.

Glucose-sensitive enzyme field effect transistor using potassium ferricyanide as an oxidizing substrate.

A glucose-sensitive field effect transistor was fabricated by immobilizing glucose oxidase on the gate of a pH-sensitive field effect transistor. Calibration curves of the biosensor were measured in phosphate and TRIS buffers in the presence of potassium ferricyanide. The use of the latter as an oxidizing substrate in the biocatalytic oxidation of glucose leads to an increase of the acidification rate of the solution inside the enzymatic layer, because three protons are now generated per one molecule of glucose instead of only one when the natural oxidizing cosubstrate, oxygen, is used. Depending on the concentration of ferricyanide we observe a 10-100 times increase of the biosensor response in concentrated buffer solutions and a substantial extension of its dynamic range. At sufficiently high concentrations of ferricyanide, the calibration curves in both buffers have a sigmoidal shape in linear coordinates with local pH changes on the surface of the field effect transistor reaching about two pH units in the saturation range. The resulting saturation of the curves at higher glucose concentrations is due to the inhibition of the activity of glucose oxidase at acidic pH by Cl- ions present in the solution. The proposed approach may be extended to allow the detection of a wide range of analytes using enzyme field effect transistors based on the enzymes for which reoxidation of the cofactor (coenzyme) leads to a liberation of H+ ions.

Polymeric membranes for silicon based (bio)sensors.

During the last decade, chemical and biochemical sensor research has benefited from the availability of new technologies and materials. New embodiments of classical devices have resulted from the use of e.g., solid state technology for the realization of the transducers. In this paper we describe several examples of membrane deposition techniques used in connection with planar, silicon based electrochemical transducers. Casting and electrochemical deposition of glucose oxidase containing membranes are described for the fabrication of glucose enzyme electrodes. Photolithographic patterning of polyacrylamide hydrogel and of siloxane based gas permeable membrane is used for the realization of an amperometric oxygen sensor and an ISFET-based pCO2 device. The last example is that of a free-chlorine sensor for which the photolithographic patterning of the polyHEMA hydrogel layer is described.