Integration and Miniaturization |
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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.