A CMOS-based microelectrode array for interaction with neuronal cultures.

We report on the system integration of a CMOS chip that is capable of bidirectionally communicating (stimulation and recording) with electrogenic cells such as neurons or cardiomyocytes and that is targeted at investigating electrical signal propagation within cellular networks in vitro. The overall system consists of three major subunits: first, the core component is a 6.5 mm x 6.5 mm CMOS chip, on top of which the cells are cultured. It features 128 bidirectional electrodes, each equipped with dedicated analog filters and amplification stages and a stimulation buffer. The electrodes are sampled at 20 kHz with 8-bit resolution. The measured input-referred circuitry noise is 5.9 microV root mean square (10 Hz to 100 kHz), which allows to reliably detect the cell signals ranging from 1 mVpp down to 40 microVpp. Additionally, temperature sensors, a digital-to-analog converter for stimulation, and a digital interface for data transmission are integrated. Second, there is a reconfigurable logic device, which provides chip control, event detection, data buffering and an USB interface, capable of processing the 2.56 million samples per second. The third element includes software that is running on a standard PC performing data capturing, processing, and visualization. Experiments involving the stimulation of neurons with two different spatio-temporal patterns and the recording of the triggered spiking activity have been carried out. The response patterns have been successfully classified (83% correct) with respect to the different stimulation patterns. The advantages over current microelectrode arrays, as has been demonstrated in the experiments, include the capability to stimulate (voltage stimulation, 8 bit, 60 kHz) spatio-temporal patterns on arbitrary sets of electrodes and the fast stimulation reset mechanism that allows to record neuronal signals on a stimulating electrode 5 ms after stimulation (instantaneously on all other electrodes). Other advantages of the overall system include the small number of needed electrical connections due to the digital interface and the short latency time that allows to initiate a stimulation less than 2 ms after the detection of an action potential in closed-loop configurations.

Patterned cell adhesion by self-assembled structures for use with a CMOS cell-based biosensor.

A strategy for patterned cell adhesion based on chemical surface modification is presented. To confine cell adhesion to specific locations, an engineered surface for high-contrast protein adsorption and, hence, cell attachment has been developed. Surface functionalization is based on selective molecular-assembly patterning (SMAP). An amine-terminated self-assembled monolayer is used to define areas of cell adhesion. A protein-repellent grafted copolymer, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG), is used to render the surrounding silicon dioxide resistant to protein adsorption. X-ray photoelectron spectroscopy, scanning ellipsometry and fluorescence microscopy techniques were used to monitor the individual steps of the patterning process. Successful guided growth using these layers is demonstrated with primary neonatal rat cardiomyocytes, up to 4 days in vitro, and with the HL-1 cardiomyocyte cell line, up to 7 days in vitro. The advantage of the presented method is that high-resolution engineered surfaces can be realized using a simple, cost-effective, dip-and-rinse process. The technique has been developed for application on a CMOS cell-based biosensor, which comprises an array of microelectrodes to extracellularly record electrical activity from cardiomyocytes.

Single-chip microelectronic system to interface with living cells.

A high degree of connectivity and the coordinated electrical activity of neural cells or networks are believed to be the reason that the brain is capable of highly sophisticated information processing. Likewise, the effectiveness of an animal heart largely depends on such coordinated cell activity. To advance our understanding of these complex biological systems, high spatiotemporal-resolution techniques to monitor the cell electrical activity and an ideally seamless interaction between cells and recording devices are desired. Here we present a monolithic microsystem in complementary metal oxide semiconductor (CMOS) technology that provides bidirectional communication (stimulation and recording) between standard electronics technology and cultured electrogenic cells. The microchip can be directly used as a substrate for cell culturing, it features circuitry units per electrode for stimulation and immediate cell signal treatment, and it provides on-chip signal transformation as well as a digital interface so that a very fast, almost real-time interaction (2 ms loop time from event recognition to, e.g., a defined stimulation) is possible at remarkable signal quality. The corresponding spontaneous and stimulated electrical activity recordings with neuronal and cardiac cell cultures will be presented. The system can be used to, e.g., study the development of neural networks, reveal the effects of neuronal plasticity and study cellular or network activity in response to pharmacological treatments.

Long-Term, High-Spatiotemporal Resolution Recording From Cultured Organotypic Slices with High-Density Microelectrode Arrays.

A novel system to cultivate and record brain slices directly on high-density microelectrode arrays (HD-MEA) was developed. This system allows to continuously record electrical activity of selected individual neurons at high spatial resolution, while monitoring neuronal network activity at the same time. For the first time, properties of single neurons and the corresponding neuronal network in an organotypic hippocampal slice culture were studied over four consecutive weeks at daily intervals.

On-chip lysis of mammalian cells through a handheld corona device.

On-chip lysis is required in many lab-on-chip applications involving cell studies. In these applications, the complete disruption of the cellular membrane and a high lysis yield is essential. Here, we present a novel approach to lyse cells on-chip through the application of electric discharges from a corona handheld device. The method only requires a microfluidic chip and a low-cost corona device. We demonstrate the effective lysis of BHK and eGFP HCT 116 cells in the sub-second time range using an embedded microelectrode. We also show cell lysis of non-adherent K562 leukemia cells without the use of an electrode in the chip. Cell lysis has been assessed through the use of bright-field microscopy, high-speed imaging and cell-viability fluorescence probes. The experimental results show effective cell lysis without any bubble formation or significant heating. Due to the simplicity of both the components involved and the lysis procedure, this technique offers an inexpensive lysis option with the potential for integration into lab-on-a-chip devices.

Conferring selectivity to chemical sensors via polymer side-chain selection: thermodynamics of vapor sorption by a set of polysiloxanes on thickness-shear mode resonators

Entropy of mixing is shown to be the driving interaction for the endothermic physisorption process of organic vapor partitioning into seven systematically side-chain-modified (polar, acidic, basic, polarizable side groups and groups interacting via H-bridges) polysiloxanes on thickness-shear mode resonators. Each sensor was exposed to seven analytes, selected for their diversity of functional groups. This systematic investigation of sorption yields benchmarking data on physisorption selectivity: response data and modeling reveal a direct correlation of partition coefficients with interactions between specific polymer side chains and analyte functional groups. Partition coefficients were determined for every polymer/analyte pairing over the 273-343 K range at 10 K intervals; from partition coefficient temperature dependence, overall absorption enthalpies and entropies were calculated. By subtracting the enthalpy and entropy of condensation for a given pure analyte, its mixing entropy (primarily combinatorial) and mixing enthalpy (associated with intermolecular interactions) with each polymer matrix were determined. These two crucial thermodynamic parameters determine the chemical selectivity patterns of the polymers for the analytes. Simple molecular modeling based on the polymer contact surface share of the modified side group or the introduced functional group reveals a direct correlation between the partition coefficients and the side-group variation.

CMOS microelectrode array for the monitoring of electrogenic cells.

Signal degradation and an array size dictated by the number of available interconnects are the two main limitations inherent to standalone microelectrode arrays (MEAs). A new biochip consisting of an array of microelectrodes with fully-integrated analog and digital circuitry realized in an industrial CMOS process addresses these issues. The device is capable of on-chip signal filtering for improved signal-to-noise ratio (SNR), on-chip analog and digital conversion, and multiplexing, thereby facilitating simultaneous stimulation and recording of electrogenic cell activity. The designed electrode pitch of 250 microm significantly limits the space available for circuitry: a repeated unit of circuitry associated with each electrode comprises a stimulation buffer and a bandpass filter for readout. The bandpass filter has corner frequencies of 100 Hz and 50 kHz, and a gain of 1000. Stimulation voltages are generated from an 8-bit digital signal and converted to an analog signal at a frequency of 120 kHz. Functionality of the read-out circuitry is demonstrated by the measurement of cardiomyocyte activity. The microelectrode is realized in a shifted design for flexibility and biocompatibility. Several microelectrode materials (platinum, platinum black and titanium nitride) have been electrically characterized. An equivalent circuit model, where each parameter represents a macroscopic physical quantity contributing to the interface impedance, has been successfully fitted to experimental results.

Nanochemical surface analyzer in CMOS technology.

We have developed an atomic force microscopy (AFM) cantilever system, fabricated using a standard CMOS process and a few post-processing steps, capable of detecting the difference between hydrophilic and hydrophobic samples for the purpose of nanochemical surface analysis. The fully integrated cantilever comprises a thermal actuator for cantilever deflection and a Wheatstone bridge to sense cantilever bending, thus obviating the need for cumbersome laser detection and external piezoelectric drives. Glass microspheres have been affixed to the cantilevers and, were either modified with a self-assembled monolayer to form hydrophobic tips, or left unmodified for hydrophilic tips. Force-distance curves have been used to measure the force between the functionalized/unfunctionalized tips and hydrophobic/hydrophilic sample surfaces. In an optimization step three different Wheatstone bridge sensors have been designed and characterized; best Wheatstone bridge sensitivity is 8.0 microV/nm with a 713 nm/mW actuator efficiency.

Compact voltage and current stimulation buffer for high-density microelectrode arrays.

We report on a compact (0.02 mm(2) ) buffer for both voltage and current stimulation of electrogenic cells on a complementary metal-oxide semiconductor microelectrode array. In voltage mode, the circuit is a high-current class-AB voltage follower, based on a local common-mode feedback (LCMFB) amplifier. In current mode, the circuit is a current conveyor of type II, using the same LCMFB amplifier with cascode stages to increase the gain. The circuit shows good linearity in the 0.5-3.5 V input range and has extensively been used for stimulation of neuronal cultures.

Microelectronic system for high-resolution mapping of extracellular electric fields applied to brain slices.

There is an enduring quest for technologies that provide - temporally and spatially - highly resolved information on electric neuronal or cardiac activity in functional tissues or cell cultures. Here, we present a planar high-density, low-noise microelectrode system realized in microelectronics technology that features 11,011 microelectrodes (3,150 electrodes per mm(2)), 126 of which can be arbitrarily selected and can, via a reconfigurable routing scheme, be connected to on-chip recording and stimulation circuits. This device enables long-term extracellular electrical-activity recordings at subcellular spatial resolution and microsecond temporal resolution to capture the entire dynamics of the cellular electrical signals. To illustrate the device performance, extracellular potentials of Purkinje cells (PCs) in acute slices of the cerebellum have been analyzed. A detailed and comprehensive picture of the distribution and dynamics of action potentials (APs) in the somatic and dendritic regions of a single cell was obtained from the recordings by applying spike sorting and spike-triggered averaging methods to the collected data. An analysis of the measured local current densities revealed a reproducible sink/source pattern within a single cell during an AP. The experimental data substantiated compartmental models and can be used to extend those models to better understand extracellular single-cell potential patterns and their contributions to the population activity. The presented devices can be conveniently applied to a broad variety of biological preparations, i.e., neural or cardiac tissues, slices, or cell cultures can be grown or placed directly atop of the chips for fundamental mechanistic or pharmacological studies.

Using microelectronics technology to communicate with living cells.

A monolithic microsystem in CMOS (complementary metal oxide semiconductor) technology is presented that provides bidirectional communication (stimulation and recording) between standard microelectronics and cultured electrogenic cells. The 128-electrode chip can be directly used as a substrate for cell culturing. It features circuitry units for stimulation and immediate cell signal treatment near each electrode. In addition, it provides on-chip A/D conversion as well as a digital interface so that a fast interaction is possible at good signal quality. Spontaneous and stimulated electrical activity recordings with neuronal and cardiac cell cultures will be presented. The system can be used to, e.g., study the behavior and development of neural networks in vitro, to reveal the effects of neuronal plasticity and to study network activity in response to pharmacological treatments.

Cell recordings with a CMOS high-density microelectrode array.

Recordings have been performed with a CMOS-based microelectrode array (MEA) featuring 11'016 metal electrodes and 126 channels, each of which comprises recording and stimulation electronics for extracellular, bidirectional communication with electrogenic cells. The important features of the device include (i) high spatial resolution at (sub) cellular level with 3'200 electrodes per mm(2) (diameter 7 microm, pitch 18 microm), (ii) a reconfigurable routing of the electrodes to the 126 channels, and (iii) low noise levels. Recordings from neonatal rat cardiomyocytes forming confluent layers and microtissues are shown. Moreover, signals from dissociated rat hippocampal neurons and from neurons in an acute cerebellar slice preparation are presented.

Micro hot plate-based sensor array system for the detection of environmentally relevant gases.

A monolithic stand-alone gas sensor system is presented, which includes on a single chip an array of three metal oxide-coated micro hot plates with integrated MOS-transistor heaters, as well as a specifically designed digital system architecture. An octagonal-shaped micro hot plate design with MOS-transistor heaters has been adopted for the three gas sensors. The integrated circuitry includes a programmable digital temperature regulation, digital sensor readout units, and a standard serial interface. The programmable digital temperature controllers enable individual regulation of the micro hot plate temperatures in constant or dynamic mode. Nanocrystalline tin oxide thick films with different Pd dopings (undoped, 0.2 and 3 wt %) were used. Gas test measurements for environmentally relevant gases were carried out and evidenced detection limits of less than 1 ppm for carbon monoxide, or 100 ppm for methane, both at 40% relative humidity. Temperature modulation techniques were successfully applied for improved analyte discrimination.

Magnetically actuated complementary metal oxide semiconductor resonant cantilever gas sensor systems.

In the present paper, an electromagnetically actuated resonant cantilever gas sensor system is presented that features piezoresistive readout by means of stress-sensitive MOS transistors. The monolithic gas sensor system includes a polymer-coated resonant cantilever and the necessary oscillation feedback circuitry, both monolithically integrated on the same chip. The fully differential feedback circuit allows for operating the device in self-oscillation with the cantilever constituting the frequency-determining element of the feedback loop. The combination of magnetic actuation and transistor-based readout entails little power dissipation on the cantilever and reduces the temperature increase in the sensitive polymer layer to less than 1 degrees C, whereas previous designs with thermally actuated cantilevers showed a temperature increase of up to 19 degrees C. The lower temperature of the sensitive polymer layer on the cantilever directly improves the sensitivity of the sensor system as the extent of analyte physisorption decreases with increasing temperature. The electromagnetic sensor design shows an almost 2 times larger gas sensitivity than the earlier design, which is thermally actuated and read out using p-diffused resistors. The gas sensor is fabricated using an industrial complementary metal oxide semiconductor (CMOS) process and post-CMOS micromachining.

Search for extraterrestrial enantioenrichment by using chemical microsensors.

The use of enantioselective chemical microsensors is proposed for the search of extraterrestrial homochirality in space. The already established enantiomer-discrimination-capability of chemical sensors and the feasibility of quantitatively determining the enantiomeric composition of a target analyte are demonstrated. The benefits of applying modern microsensor technology are presented followed by some concepts and scenarios including how chemical microsensors could be used in space.

Effective use of molecular recognition in gas sensing: results from acoustic wave and in situ FT-IR measurements.

To probe directly the analyte/film interactions that characterize molecular recognition in gas sensors, we recorded changes to the in situ surface vibrational spectra of specifically functionalized surface acoustic wave (SAW) devices concurrently with analyte exposure and SAW measurement of the extent of sorption. Fourier transform infrared external-reflectance spectra (FT-IR-ERS) were collected from operating 97-MHz SAW delay lines during exposure to a range of analytes as they interacted with thin-film coatings previously shown to be selective: cyclodextrins for chiral recognition, nickel camphorates for Lewis bases such as pyridine or organophosphonates, and phthalocyanines for aromatic compounds. In most cases where specific chemical interactions--metal coordination, "cage" compound inclusion, or pi-stacking--were expected, analyte dosing caused distinctive changes in the IR spectra, together with anomalously large SAW sensor responses. In contrast, control experiments involving the physisorption of the same analytes by conventional organic polymers did not cause similar changes in the IR spectra, and the SAW responses were smaller. For a given conventional polymer, the partition coefficients (or SAW sensor signals) roughly followed the analyte fraction of saturation vapor pressure. These SAW/FT-IR results support earlier conclusions derived from thickness-shear mode resonator data.

Use of linear solvation energy relationships for modeling responses from polymer-coated acoustic-wave vapor sensors.

The applicability and performance of linear solvation energy relationships (LSERs) as models of responses from polymer-coated acoustic-wave vapor sensors are critically examined. Criteria for the use of these thermodynamic models with thickness-shear-mode resonator (TSMR) and surface-acoustic-wave (SAW) vapor sensors are clarified. Published partition coefficient values derived from gas-liquid chromatography (GLC) are found to be consistently lower than those obtained gravimetrically, in accordance with previous reports, suggesting that LSERs based on GLC-derived partition coefficients will not provide accurate estimates of acoustic-wave sensor responses. The development of LSER models directly from polymer-coated TSMR vapor sensor response data is demonstrated and a revised model developed from SAW vapor sensor response data, which takes account of viscoelastic changes in polymeric coating films, is presented and compared to those developed by other methods.

Chiral discrimination using piezoelectric and optical gas sensors.

Odour perception in humans can sometimes discriminate different enantiomers of a chiral compound, such as limonene. Chiral discrimination represents one of the greatest challenges in attempts to devise selective and sensitive gas sensors. The importance of such discrimination for pharmacology is dear, as the physiological effect of enantiomers of drugs and other biologically active molecules may differ significantly. Here we describe two different sensor systems that are capable of recognizing different enantiomers and of qualitatively monitoring the enantiomeric composition of amino-acid derivatives and lactates in the gas phase. One sensor detects changes in mass, owing to binding of the compound being analysed (the 'analyte'), by thickness shear-mode resonance; the other detects changes in the thickness of a surface layer by reflectometric interference spectroscopys. Both devices use the two enantiomers of a chiral polymeric receptor, and offer rapid on-line detection of chiral species with high selectivity.

Chiral discrimination in the gas phase using different transducers: thickness shear mode resonators and reflectometric interference spectroscopy.

The discrimination of optical isomers (enantiomers) in the gas phase has been performed using two different analytical tools: thickness shear mode resonators (TSMRs) and reflectometric interference spectroscopy (RIFS). The selective coatings included both enantiomers ((S)- and (R)-receptor) of a Chirasil-Val derivative (stationary phase material in GC) with octyl side chains. Successful discrimination of the enantiomers of different types of analytes (amino acids and lactates) was achieved. The results of both transduction methods were consistent and in good agreement with GC measurements. In addition, different mixtures of both enantiomers of the respective analyte were measured, and the enantiomeric composition could be quantitatively determined with excellent reliability. Since the sensors allow on-line monitoring (not possible with GC) of enantiomeric purity, an application in industrial synthesis (process control) of such compounds represents an interesting feature, especially with regard to the tested derivatives of lactic acid.

Performances of mass-sensitive devices for gas sensing: thickness shear mode and surface acoustic wave transducers.

In this work we investigated different thickness shear mode resonators (TSMRs) with fundamental frequencies of 10 and 30 MHz and surface acoustic wave devices with fundamental frequencies of 80 and 433 MHz. Four aspects were of primary interest in this comparison: noise levels and signal-to-noise ratios (S/N), influence of the polymer film thickness, influence of temperature on the transducer signal before and after coating, and minimum threshold values for monitoring different volatile organic compounds in the environment. We limited our investigations to a temperature range between 298 and 308 K, with 303 K the routine measuring temperature. Analyte concentrations (n-octane, tetrachloroethene) were chosen from the minimum detection limit up to 5000 µg/L. The temperature was found to strongly affect the performance of all the devices. The sorption of the analyte vapors into the polymeric films was demonstrated to be transducer-independent (identical partition coefficients for all the devices). The 30 MHz TSMRs showed very satisfying results in terms of S/N and limits of detection.