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Genetically Engineered Cell-Based Biosensors for Specific Agent Classification

Conference
DeBusschere, B. D., Aravanis, A. M., Chruscinski, A. J., Hedgepath, K. R., Borkholder, D. A., Kobilka, B. K., and Kovacs, G. T. A.
Proceedings of Transducers '99, the Tenth Annual Conference on Solid-State Sensors and Actuators, June 7 - 10, 1999, Sendai, Japan, pp. 1348 - 1351.
Publication year: 1999

Cell-based biosensors utilize whole cells as the primary transducer to detect a biologically active agent as a cellular signal. A secondary transducer converts this cellular signal into an electrical signal that can be processed and analyzed. Although these sensors have shown success in detecting the presence of biological agents, efforts to classify the type of agent have proven difficult for several reasons. One reason for ambiguities in output signal interpretation is that multiple biochemical pathways can lead to the same cellular response. However, a new approach described in this paper translates this crosstalk noise into common-mode noise that can be rejected. This approach uses a differential measurement between wild-type cells and cells genetically engineered to lack a specific receptor (knockouts). Any biological agent that targets that receptor will evoke a response in the wild-type cell but not in the knockout. This approach provides the benefits of a functional assay with specificity while simplifying signal analysis. This concept was successfully implemented using genetically engineered mouse myocardial cells that lacked the β1-adrenergic receptor (β1-AR).

 

Genetically-Engineered-Cell-Based-Biosensors-for-Specific-Agent-Classification

Development and Application of Cell-Based Biosensors

Journal
Pancrazio, J.J., Whelan, J. P., Borkholder, D.A., Ma, W. and Stenger, D.A.
Annals of Biomedical Engineering, 27:697–711 (1999).
Publication year: 1999

Biosensors incorporate a biological sensing element that converts a change in an immediate environment to signals conducive for processing. Biosensors have been implemented for a number of applications ranging from environmental pollutant detection to defense monitoring. Biosensors have two intriguing characteristics: (1) they have a naturally evolved selectivity to biological or biologically active analytes; and (2) biosensors have the capacity to respond to analytes in a physiologically relevant manner. In this paper, molecular biosensors, based on antibodies, enzymes, ion channels, or nucleic acids, are briefly reviewed. Moreover, cell-based biosensors are reviewed and discussed. Cell-based biosensors have been implemented using microorganisms, particularly for environmental monitoring of pollutants. Biosensors incorporating mammalian cells have a distinct advantage of responding in a manner that can offer insight into the physiological effect of an analyte. Several approaches for transduction of cellular signals are discussed; these approaches include measures of cell metabolism, impedance, intracellular potentials, and extracellular potentials. Among these approaches, networks of excitable cells cultured on microelectrode arrays are uniquely poised to provide rapid, functional classification of an analyte and ultimately constitute a potentially effective cell-based biosensor technology. Three challenges that constitute barriers to increased cell-based biosensor applications are presented: analytical methods, reproducibility, and cell sources. Possible future solutions to these challenges are discussed.

Development-and-Application-of-Cell-Based-Biosensors

Cell-Based Biosensors Using Microelectrodes

Thesis
Borkholder, D.A.
Doctoral thesis, Stanford University (1999).
Publication year: 1999

Instruments for the study of living cells have historically been of significant importance for such things as basic neuroscience and cell biology. More recent work has included extending the use of cell based sensors to pharmaceutical screening, environmental monitoring, and toxin detection. While there are a variety of different measurement techniques, microelectrode arrays provide a simple interface for monitoring the electrical activity and impedance characteristics of populations of cultured cells over extended periods. The cell / sensor interface is created as cells attach directly to planar electrode structures. With excitable cells, both intrinsic and stimulated electrical activity has been directly monitored in this way. Impedance techniques have also been used to monitor such things as cellular adhesion, motility and proliferation of both electrically active and non-electrically active cell types.

For this work, a 36 element array of platinized platinum electrodes was packaged and used as a substrate for the culture of neurons, cardiac and glial cells. Electrical activity from a spontaneously beating syncytium of cardiac cells was monitored and analyzed in detail. Power spectral density analysis was used as a means of identifying the class of ionic channel affected by pharmacological compounds or toxins. Impedance measurements on neurons and glial cells were targeted towards measuring changes in cellular membrane conductance in addition to cellular adhesion and motility.

This thesis describes the basic cell theory relevant to measurement of electrical activity and cellular impedance. Classical measurement techniques are compared to planar microelectrode array technologies. New packaging technologies and systems developed for both electrical activity and impedance measurements are described along with pharmacological data for neurons, cardiac and glial cells. The practicality of using microelectrode based sensor technologies for monitoring the activity of cultured cells is discussed.

 

Cell-Based-Biosensors-Using-MicroelectrodesCell-Based-Biosensors-Using-Microelectrodes2

System Requirements for a Portable Cell Based Sensor,

Conference
DeBusschere, B. D., Borkholder, D. A. and Kovacs, G.T.A.,
Proceedings of the UTAS '98 Workshop, Banff, Canada, 13-16 October, Kluwer Academic Publishers, 443-446 (1998)
Publication year: 1998

The use of cell based biosensors outside of the laboratory has been limited due to many issues including preparation of the sample, maintenance of the biological environment, incorporation of the appropriate sensors, and integration of the electronics for data collection and analysis. This paper describes these system issues and briefly presents current efforts to address packaging, fluidics, and data interpretation. These include the development of an integrated silicon-PDMS cell cartridge system that provides reliable electrical and fluidic interconnect and a regulated cell environment, the development of low power and low overhead sensors and electronics, and the necessary fluidic sample preparation.

 

System-Requirements-for-a-Portable-Cell-Based-Sensor

Portable Cell-Based Biosensor System for Toxin Detection

Journal
Pancrazio, J.J., Bey, P.P. Jr., Cuttino, D.S., Kusel, J.K., Borkholder, D.A.
Sensors and Actuators B: Chemical, 53(3):179-185 (1998)
Publication year: 1998

A portable cell-based biosensor has been developed and characterized. The prototype system relies on extracellular recording from excitable cells cultured over an array of platinized gold microelectrodes. Extracellular potentials were bandpass filtered between 80 Hz to 2.8 kHz and amplified with a selectable gain of either 1000 or 5000. The input-referred noise level of the system was only 8.7 μVRMS in the laboratory setting, reaching only 10.6 μVRMS in an outdoor environment, more than sufficient for measurement of extracellular potentials from excitable cells. The system also incorporates a feedback control system for temperature regulation and a 36-channel multiplexer for selection of up to four output channels for simultaneous display. Wherever possible, low-cost ‘off-the-shelf’ components were utilized in this prototype biosensor design. Using this system, extracellular recordings from chick myocardiocytes were performed under both laboratory and outdoor conditions.

Portable-Cell-Based-Biosensor-System-for-Toxin-Detection

 

Design of an Integrated Silicon-PDMS Cell Cartridge

Conference
DeBusschere, B. D., Borkholder, D. A. and Kovacs, G.T.A.
Technical digest of the 1998 Solid-State Sensor and Actuator Workshop, Hilton Head Island, SC, 6/7-6/11, Transducer Research Foundation, Cleveland, pp. 358-362 (1998)
Publication year: 1998

The use of cell based biosensors for applications outside of the laboratory has been limited in part due to packaging issues. A design for an integrated cell cartridge that addresses the requirements of sterile fluidic interconnect and environmental regulation is presented. The device consists of a PDMS (polydimethylsiloxane) part, a glass cover, and a silicon sensing die mounted on a printed circuit board. The PDMS part forms the fluidic channels, interconnect ports, septa, and two 10 μl chambers over the active sensing area. The silicon die will include integrated biological sensors and a temperature regulation system, and the glass cover seals the chambers. Electrical and fluidic connections are made simultaneously as needles pierce septa on the cartridge when it is plugged into a zero-insertion force (ZIF) socket. The viability of injecting suspended cells into a 10 μl volume chamber and culturing them for greater than one week using a continuous flow perfusion system has been demonstrated. Initial prototypes of the cell cartridges have been assembled and cells have been cultured in the 10 μl PDMS chambers.

Description and Demonstration of a CMOS Amplifier-Based-System with Measurement and Stimulation Capability for Bioelectrical Signal Transduction

Journal
Pancrazio J.J., Bey, P.P.Jr., Loloee, A., Manne, S., Chao, H.C., Howard, L.L., Gosney, W.M., Borkholder, D.A., Kovacs, G.T.A., Manos, P., Cuttino, D.S. and Stenger, D.A.
Biosensors and Bioelectronics 13:971-979 (1998).
Publication year: 1998

An extracellular recording system incorporating an electrode array and an amplifier/stimulator CMOS chip is described and characterized. Important features of this custom VLSI chip include 16 instrumentation amplifiers with a gain of 50 and the incorporation of a cross-point array allowing designation of an extracellular microelectrode as either a stimulator or sensor. The planar array consisted of 32 microelectrodes, 14 mm in diameter, and four larger reference electrodes. Microelectrodes, interconnecting traces, and bond pads were patterned with a 500-nm layer of gold. The interconnecting traces were passivated with a 1-mm thick layer of silicon nitride to provide chemical and electrical insulation and microelectrode impedance was lowered utilizing electrode position of platinum black. The amplifier exhibited a nearly flat frequency response with high pass and low pass corner frequencies of 0.7 Hz and 50 kHz, respectively. The input referred noise over the 50 kHz bandwidth was 12–16 mVRMS, well below the magnitude of previously reported extracellular potentials. Crosstalk between neighboring channels resulted in an output signal below the amplifier noise level, even for relatively large extracellular potentials. Using this system, extracellular recordings were demonstrated yielding typical peak-to-peak biopotentials of magnitude 0.9–2.1 mV and 100–400 mV for chick cardiac myocytes and rat spinal cord neurons, respectively. The key components of this extracellular recording system can be manufactured using industry standard thin film photolithographic techniques.

Description-and-Demonstration-of-a-CMOS-Amplifier-Based-System-with-Measurement-and-Stimulation-Capability-for-Bioelectrical-Signal-Transduction

Cell-based Sensor Microelectrode Array Characterized by Imaging X-ray Photoelectron Spectroscopy, Scanning Electron Microscopy, Impedance Measurements, and Extracellular Recordings

Journal
Jung, D.R., Cuttino, D.S., Pancrazio, J.J., Manos, P., Cluster, T., Sathanoori, R.S., Aloi, L.E., Coulombe, M.G., Bey, P., Czarnaski, M.A., Borkholder, D.A., Kovacs, G.T.A., Stenger, D.A. and Hickman, J.J.
Journal of Vacuum Science & Technology A, 16(3):1183-1188 (1998).
Publication year: 1998

We are developing a cell-based biosensor consisting of a planar microelectrode array that allows detection of extracellular potentials and their modulation in the presence of toxins or other active agents. To improve cell–electrode coupling, the microelectrodes were electroplated with platinum black. We report on the use of imagingx-ray photoelectron spectroscopy(XPS),scanning electron microscopy(SEM), impedance measurements, and extracellular recordings to assess the effectiveness of this procedure. SEM provided highly detailed images of the shape and structure of well-formed deposits of thickness on the order of 1 μm or more. Because of its inherent high surface sensitivity, imagingXPS could reveal the presence of platinum deposits that were too thin to be detected by SEM. For typical, well-plated microelectrodes, impedance measurements showed reductions in the electrical resistance at 100 Hz from roughly 60 MΩ or more 1 MΩ. The overall electronic coupling of biopotentials to the microelectrodes was demonstrated by recordings obtained from beating rat myocytes and from rat spinal cord cells.

Cell-based-Sensor-Microelectrode-Array-Characterized-by-Imaging-X-ray-Photoelectron-Spectroscopy-Scanning-Electron-Microscopy-Impedance-Measurements-and-Extracellular-Recordings

An Approach to the Classification of Unknown Biological Agents with Cell-based Sensors

Conference
Borkholder, D.A., DeBusschere, B.D., and Kovacs, G.T.A.
1998
Publication year: 1998

The broad-spectrum sensitivity of cell based biosensors offers the capability for detecting previously unknown biological agents. One cellular parameter that is often measured is the action potential of electrically active cells. However, the complexity of this signal makes interpretation of the cellular response to a compound difficult to interpret. By analyzing shifts in the signal’s power spectrum, it may be possible to classify the ionic channels modulated by the agent.

A system is described for the measurement of action potentials from cells cultured on a planar microelectrode array. Experimental results, simulations, and analyses are presented for three pharmaceuticals tested on chick myocardial cells. While the actual agents could readily be distinguished experimentally, the models used for simulation were a partial success, accurately predicting the response to one of the three agents tested.

An-Approach-to-the-Classification-of-Unknown-Biological-Agents-with-Cell-based-SensorsAn-Approach-to-the-Classification-of-Unknown-Biological-Agents-with-Cell-based-Sensors2

Plasma-Etched Neural Probes

Journal
Kewley, D.T., Hills, M.D., Borkholder, D.A., Opris, I.E., Maluf, N.I., Storment, C.W., Bower, J.M. and Kovacs, G.T.A.
Sensors and Actuators: A Physical, 58:27-35, (1997)
Publication year: 1997

A new method is presented for microfabricating silicon-based neural probes that are designed for neurobiology research. Such probes provide unique capabilities to record high-resolution signals simultaneously from multiple, precisely defined locations within neural tissue. The fabrication process utilizes a plasma etch to define the probe outline, resulting in sharp tips and compatibility with standard CMOS processes. A low-noise amplifier array has been fabricated through the MOSIS service to complete a system that has been used in multiple successful physiological experiments.

 

Plasma-Etched-Neural-ProbesPlasma-Etched-Neural-Probes2

Microelectrode Arrays for Stimulation of Neural Slice Preparations

Journal
Borkholder, D., Bao, J., Maluf, N., Perl, E., and Kovacs, G.
Journal of Neuroscience Methods, 77:61-66 (1997)
Publication year: 1997

A planar 6×6 array of iridium electrodes with four reference electrodes has been developed for use with neural tissue preparations. Precise knowledge of the relative locations of the array elements allows for spatial neurophysiological analyses. The 10 μm diameter platinized iridium electrodes on a 100 μm pitch have been used to stimulate acutely prepared slices of spinal cord from free-ranging rodents. An intracellular recording from a single neuron in the substantia gelatinosa (SG) using the whole-cell, tight-seal technique allowed low noise, high resolution studies of excitatory or inhibitory electrical responses of a given neuron to inputs from the primary afferent fibers or from stimulation by individual electrodes of the array. The resulting maps of responses provide an indication of the interconnectivity of neural processes. The pattern emerging is that of limited interconnectivity in the SG from areas surrounding a recorded neuron but with strong excitatory or inhibitory effects from those oriented in a longitudinal (rostral–caudal) direction relative to the neuron. The observations to date suggest the neurons of the SG are arranged in sets of independent networks, possibly related to sensory modality and input from particular body regions.

 

Microelectrode-Arrays-for-Stimulation-of-Neural-Slice-Preparations

Plasma-Etched Neural Probes

Conference
Kewley, D.T., Hills, M.D., Borkholder, D.A., Opris, I.E., Maluf, N.I., Storment, C.W., Bower, J.M. and Kovacs, G.T.A.
Technical digest of the 1996 Solid-State Sensor and Actuator Workshop, Hilton Head Island, SC, pp. 266-271, June 2-6 (1996)
Publication year: 1996

A new method is presented for microfabricating silicon-based neural probes that are designed for neurobiology research. Such probes provide unique capabilities to record high-resolution signals simultaneously from multiple, precisely defined locations within neural tissue. The fabrication process utilizes a plasma etch to define the probe outline, resulting in sharp tips and compatibility with standard CMOS processes. A low-noise amplifier array has been fabricated through the MOSIS service to complete a system that has been used in multiple successful physiological experiments.

Planar Electrode Array Systems for Neural Recording and Impedance Measurements

Conference
Borkholder, D.A., Opris, I.E., Maluf, N.I. and Kovacs, G.T.A.
Conference Proceedings of the 1996 IEEE Engineering in Medicine and Biology, Amsterdam, The Netherlands, 10/31-11/3/96, IEEE (1996)
Publication year: 1996

Systems designed to significantly reduce equipment cost and size for neurophysiological studies and hybrid biosensor applications were developed. Custom integrated circuits, each providing eighteen channels of amplification and filtering were designed, fabricated and tested. Planar arrays of iridium microelectrodes were fabricated and packaged in a standard 40 pin dual-in-line package for cultured cell and neural slice preparation studies. An impedance imaging system was developed to monitor the impedance of the cell / electrode interface across the array, thereby expanding the possible biosensor applications to non-electrically active cell types. Thermal regulation was achieved via a Peltier effect thermoelectric device allowing temperature control both above and below ambient temperature. While designed to work together, the system components presented may be easily applied to existing systems for enhancement of capabilities while reducing size and cost.

 

Planar-Electrode-Array-Systems-for-Neural-Recording-and-Impedance-Measurements

Impedance Imaging for Hybrid Biosensor Applications

Conference
Borkholder, D.A., Maluf, N.I. and Kovacs, G.T.A.
Solid-State Sensor and Actuator Workshop, Hilton Head Island, South Carolina, pp. 156 - 160, June 2-6 (1996)
Publication year: 1996

The impedance characteristics of individual cell / electrode systems are used to monitor cellular viability, position, adhesion, and response to external stimuli in hybrid biosensor applications. A planar microelectrode array consisting of 36 platinized iridium electrodes (10 μm diameter) is used as a substrate for the culture of mammalian cells. Electrode impedance is monitored across the array as different environmental factors are changed. Maps of electrode impedance have been shown to correlate directly to cell positioning over an electrode and general cellular viability. Exposure to a well known voltage-gated Na+ channel blocker (tetrodotoxin) provided significant cellular response as compared to control electrodes without cells. The effective use of small electrodes (10μm diameter) to study single cell / electrode interactions has been demonstrated.

Impedance-Imaging-for-Hybrid-Biosensor-Applications

Quarter-Micron Lithography with a Gapped Markle-Dyson System

Journal
Owen, G., Borkholder, D.A., Knorr, C., Markle, D.A. and Pease, R.F.W.
Journal of Vacuum Science & Technology B, 12(6):3809-3813 (1994)
Publication year: 1994

0.25‐μm lithography has previously been demonstrated using an ungapped prototype Markle–Dyson system, in which the wafer was held in soft contact with the mask. However, such an in‐contact scheme would be inappropriate for semiconductor fabrication, and so a second prototype has been designed and constructed, in which a gap of 25 μm is introduced between the mask and the wafer. The two major technical problems to be overcome in implementing the gap are the measurement and setting of the gap itself, and correction for the spherical aberration which it introduces. In the new prototype, the gap is set by a piezo‐electric actuator and measured using a capacitance gauge, and the spherical aberration is corrected by using a mirror which deviates slightly (by 0.33 μm) from sphericity. The system has been tested lithographically, using chromium reflective masks. It has demonstrated a resolution of 0.25 μm in Shipley XP89131photoresist.

Masks for Markle-Dyson Optics

Conference
Grenville, A., Owen, G., Robinson, J., Borkholder, D., Frank, C., Pease, F. and Markle, D.
Proceedings of the SPIE - The International Society for Optical Engineering, 2087:2-9 (1994).
Publication year: 1994

The optical performance of Markle-Dyson projection optics is now well established. Here we describe options for 1X reflective optical masks that might achieve the desired linewidth control. One option is the use of aluminum as the reflecting material. A film less than 50 nm thick has nearly twice the reflectivity of the silicon used until now, and so it should be possible to develop an etching process (for such a thin film) that is adequately precise. Moreover options exist for repairing both opaque and clear defects. An interesting alternative configuration, that eliminates the need to etch the aluminum, is to use a patterned absorber on the substrate and to deposit the aluminum over the patterned absorber.

Flexible, Dry-Released Process for Aluminum Electrostatic Actuators

Journal
Storment, C.W., Borkholder, D.A., Westerlind, V., Suh, J.W., Maluf, N.I. and Kovacs, G.T.A.
IEEE J. Microelectromechanical Systems, 3(3):90-6 (1994)
Publication year: 1994

We present an all-aluminum MEMS process (Al-MEMS) for the fabrication of large-gap electrostatic actuators with process steps that are compatible with the future use of underlying, pre-fabricated CMOS control circuitry. The process is purely additive above the substrate as opposed to processes that depend on etching pits into the silicon, and thereby permits a high degree of design freedom. Multilayer aluminum metallization is used with organic sacrificial layers to build up the actuator structures. Oxygen-based dry etching is used to remove the sacrificial layers. While this approach has been previously used by other investigators to fabricate optical modulators and displays, the specific process presented herein has been optimized for driving mechanical actuators with relatively large travels. The process is also intended to provide flexibility for design and future enhancements. For example, the gap height between the actuator and the underlying electrode(s) can be set using an adjustable polyimide sacrificial layer and aluminum “post” deposition step. Several Al-MEMS electrostatic structures designed for use as mechanical actuators are presented as well as some measured actuation characteristics

Dry-Released Process for Aluminum Electrostatic Actuators

Conference
Storment, C.W., Borkholder, D.A., Westerlind, V.A., Maluf, N.I. and Kovacs, G.T.A.
Technical Digest of the Solid-State Sensor and Actuator Workshop, Hilton Head Island, SC, pp. 95-98, June 13-16 (1994)
Publication year: 1994

An all-aluminum MEMS process (Al-MEMS) for the fabrication of large-gap electrostatic actuators is presented. The process is purely additive above the substrate and thereby permits a high degree of design freedom. All process steps are compatible with the future addition of underlying CMOS control circuits. Multilayer aluminum metallization is used with organic sacrificial layers to build up the actuator structures. Oxygen-based dry etching is used to remove the sacrificial layers. While this approach has been previously used by other investigators to fabricate optical modulators and displays, the specific process presented herein has been optimized for driving mechanical actuators with relatively large travels and/or out-of-plane attachments such as electroplated “digits”. The gap height between the actuator and the underlying eletrode(s) can be set using an adjustable polyimide sacrificial layer and aluminum “post” deposition step. Several Al-MEMS electrostatic actuators designed for use as mechanical actuators are presented.