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Cell Clinics


Overview | Sensors | Integration and Miniaturization | Cell Culture

Overview

cell_clinics_0 Biological agents as transducers between stimuli and electronic sensors have applications in healthcare, military defense, scientific research, and other arenas. In collaboration with Dr. Elisabeth Smela, we are developing an integrated bioelectronic and biophotonic interface known as cell clinics, for capturing and characterizing small groups of living biological cells. Each of these cell clinics is an integrated micro-electro-mechanical system (MEMS) which consists of a cell-sized well with an actuated lid and circuitry for sensing, signal-processing, and actuation. Cell clinics provide an opportunity to characterize many individual cells in parallel, in contrast with traditional techniques which characterize average properties of an ensemble of cells.

We've demonstrated several technological advances that permit the successful interface of integrated circuits with biological cells: first, the ability to culture cells on-chip and to restrain mobile cells without adhesives or complex structures; next, the ability to characterize cells using sensors tailored to monitor their responses; and finally, the ability to combine many components together into a complex system. Our lab has designed and tested integrated sensors that directly monitor cell presence and viability via capacitance measurement and optical imaging, cell tracking via imaging, and cellular response to environmental stimuli via fluorescence sensing and extracellular electrical potentials (for electrogenic cells). Weve also demonstrated system-level integration for control of MEMS structures using integrated circuits.





Sensors that Directly Monitor Cellular Responses


Cell-Substrate Capacitance Sensors

cell_clinics_1 Living cells growing on a supporting substrate behave capacitively when exposed to weak, low-frequency electric fields. The capacitance results primarily from the insulating nature of cells and the polarization of the surrounding solution under these excitation conditions [J9, C41, C33, C27, C18]. Cell-substrate capacitance sensing relies on the fact that healthy cells possess well-formed plasma membranes and adhere strongly to their substrates. In contrast, unhealthy cells possess compromised membrane structures and are weakly adherent. Consequently, viable cells offer higher electrical capacitance and show more activity compared to cells with compromised viability. This forms the basis for employing on-chip capacitance sensing for cell viability monitoring.

A complementary metal-oxide-semiconductor (CMOS) chip with an array of capacitance sensors has been designed and tested on the bench and in vitro with bovine aortic smooth muscle cells (BAOSMC) cultured on the chip surface [J9, C33, C27, C18]. The sensors employ a charge-based sensing technique for translating the cell-substrate capacitance values to output voltages. Cell monitoring experiments with BAOSMC have demonstrated that on-chip capacitance measurements track the cell-substrate interaction process and reflect changes in cell viability. Capacitance measurements are strongly correlated with results obtained from concurrent cell viability assessments using traditional cell culture techniques [J9, C27].

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Bioamplifiers

cell_clinics_4 Bioamplifiers are amplifiers with gain and bandwidth characteristics appropriate for recording extracellular electrical potentials corresponding to depolarization events in electrogenic cells. A bioamplifier measures field-induced potentials resulting from the flow of ionic currents across the cell membrane. The acquired signal depends on many factors including cell health and age, electrode configuration, cell orientation with respect to sensing electrode, and environmental parameters.

A CMOS chip comprising an array of bioamplifiers connected to on-chip electrodes has been designed, fabricated and tested [C33, C13, C10]. The amplifiers were characterized with bovine aortic smooth muscle cells (BAOSMC) cultured on the chip surface. Spike potentials measuring several 100 V were recorded. Long term measurements monitoring the responses of cells cultured on the chip revealed interesting phenomena such as spike propagation across the network of cells over the electrode array [C33].

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Contact-imaging for Cell Detection and Tracking

Contact imaging is an imaging configuration in which the sample is in close proximity to the sensor surface without the use of an intermediate lens [J10, C30, C19]. In cell clinics, the sample comprises individual cells or small groups of cells which are located on the sensor surface. To detect absorption, standard farfield illumination is applied, and the cells in between the light source and the sensor surface block light; thus, the locations of the cells are represented by dark pixels in the image. To detect fluorescence, the cells are labeled with a fluorescent tag, an excitation source is placed behind the cells, and an excitation filter is placed between the cells and the sensor surface. In this configuration, the only light received by the image sensor is the fluorescent light emitted by the cells, and the location of each cell is represented by a bright spot in the image. We have demonstrated the use of contact imaging to detect the location of cells cultured overtop a sensor surface. We have also begun to extend this work by developing image sensors with on-chip signal processing capable of determining the locations of cells [C30, C26, C24, C20].




Fluorescence Imaging

Integrated fluorescence sensing requires the development of low-noise optical detectors and optical filters capable of rejecting the totality of the excitation light while transmitting the relatively weak fluorescent signal. High performance detectors and filters are important for high quality fluorescence detection, in order to improve the sensitivity to low fluorophore concentrations or to enable the use of weakly emitting fluorophores. We have designed and tested novel photodetectors [C42, C37, C34, C31] with improved noise performance relative to conventional active pixel sensors (APS). We have also developed ultraviolet-blocking microscale polymeric filters capable of achieving the rejection and transmission performance of macroscale filters [J12, C34].

cell_clinics_7 The fluorescence sensor is an extension of APS with a differential readout architecture in order to reduce the overall noise. Experimental results show that the reset noise is reduced by a factor of 1.42 and readout noise is reduced by a factor of 9.20 when the pixel is operated in differential mode versus single-ended mode. Spectral responsivity characteristics show that the photodiodes are responsive over the entire visible and near-infrared spectrum, with highest sensitivity at 480 nm [C37, C34]. Ratiometric measurements using were performed using the differential photodetector in conjunction with an external emission filter for a 5M solution of the calcium indicator Fura-2. The estimated Ca2+ concentration varied linearly with Ca2+ concentration, and the sensor was able to reliably detect a free calcium concentration of 17 nM [C37].

cell_clinics_8 The microscale spectral filters were developed for use in blocking near-UV excitation light from reaching the detector. They are based on absorption of light by a chromophore embedded in an optically clear polymer matrix [J12, C34]. The transmission spectra of the polymer filters achieved rejection levels from 300 nm to 370 nm comparable with that of a commercial macroscale bandpass filter (Chroma 7100a), while a micromachined 39-layer interference filter shows large ripples in the stopband and is very sensitive to variations in the layer thickness. Moreover, the polymer filters only required one deposition step, so they proved to be easier to integrate than micromachined interference filters.





CMOS/MEMS Integration and System Miniaturization

cell_clinics_9 The primary goal of this research is to develop cell clinics: CMOS/MEMS hybrid microsystems for capturing and performing in situ investigation of living cells cultured in vitro within controllable microenvironments [C15, C14, C13, C10, C7]. The technology aims at enabling high-speed, automated and economical cell monitoring for various applications including studies of specific biochemical mechanisms, fast medical diagnosis, pharmaceutical tests, and detection of biochemicals of military or environmental relevance. The project is a collaborative effort with Dr. Elisabeth Smela's group in the Department of Mechanical Engineering. Cell clinics comprise arrays of lidded microvials fabricated on top of CMOS chips. The chips house electrode arrays and circuitry for cell sensing, signal conditioning, and control.

The cell clinics microvial lids are opened and closed by bilayered actuators comprising polypyrrole (PPy) and gold [C15, C14, C13]. PPy is an electroactive polymer that changes volume due to electrochemical oxidation and reduction. At the macro-scale, such reactions are controlled using an electronic instrument known as a potentiostat. In the first generation cell clinics the MEMS structures were controlled using an external potentiostat instrument. In an effort to enable system miniaturization and portability we have developed a CMOS potentiostat for in situ control of PPy/gold microactuators, to be integrated into the next generation cell clinics system [C29]. The integrated potentiostat comprises CMOS control circuitry connected to microelectrodes that constitute the on-chip electrochemical cell. The potentiostat module has been successfully tested and validated for in situ deposition and control of PPy films on the on-chip electrodes. The chip has also been tested for off-chip control of PPy/gold microactuator arrays [C29].

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Cell Culture

cell_clinics_12 In order to test the cell sensors described above, weve developed a lot of experience in culturing cells. To date we have maintained active cultures of:

  • BAOSMC (primary bovine aortic smooth muscle cells)
  • PC-12 (cell line derived from a transplantable rat pheochromocytoma)
  • MDA-MB-231 (mammary gland epithelial cell)
  • RAW 264.7 (mouse macrophage)
  • A549 (lung epithelial cell)
  • CACO-2 (colon cells)

All of these cell types were specifically chosen to be used in the experimental characterization of biosensors designed by the lab. For example, the BAOSMC and PC12 cells (after exposure to nerve growth factor) are electrically active cells and suitable for testing with the bioamplifiers. We perform biocompatibility tests on all materials used to fabricate cell clinics by comparing the numbers of viable cells with controls using various dyes such as trypan blue and neutral red. In addition to the dyes, we monitor the growth of the cells over the course of a two week period by counting the cell population every other day. We also frequently use MTT and Alamar blue assays as a measure of cytotoxicity. While MTT is an endpoint assay that can only be administered at the end of an experiment, Alamar blue can be used throughout an experiment as a measure of the metabolic activity. Our cells have been successfully cultured on fabricated CMOS chips in the presence of chip packaging material such as that used to protect the bond wires. All cell culturing and experimentation occurs in laboratory space of the Bioengineering Department in the new Jeong H. Kim Engineering Building where we have access to Class II bio-safety cabinets, centrifuge, and autoclave in addition to a dedicated CO2 incubator.




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