Chip Assembly A 4 × 4 microelectrode array comprising of platinum electrodes
(diameter: 80 μm, center-to-center spacing: 200 μm) spanning a surface area
of 0.88 × 0.88 mm2 on a silicon/silicon nitride substrate with electrode leads (6 μm
thick) terminating at electrode pads (100 μm × 120 μm) has been fabricated using standard
microlithography techniques [102]. To achieve a stable local microenvironment for
sensing, the microelectrode array has been integrated to a silicone chamber (16 × 16 ×
2.5mm3) with a microfluidic channel (50 μm, wide); to pump in the testing agent and pump
out the test buffer once the sensing process has been completed. The flow rate of the buffer
was 40 μL/min. The silicone chamber was provided with an opening (8 × 8 × 2.5 mm3)
and covered by a glass cover slip for in-situ monitoring. Simultaneous electrical and optical
monitoring has been achieved by using a MicrozoomTM(Nyoptics Inc, Danville, CA)
68 CENGIZ S. OZKAN ET AL.
http://s271.photobucket.com/albums/jj125/jjames40/bio20-a/?action=view¤t=untitled.jpg
FIGURE 4.5. Schematic representation of the measurement system. It provides simultaneous electrical and optical
monitoring capability [105].
optical probe station under 8 × and 25 × magnification. The electrical stimulation and
measurements were achieved by using micromanipulators (Signatone, Gilroy, CA).
4.3.6.2. Environmental Chamber The optical probe station along with the chip assembly
was enclosed by an acrylic chamber (S&W Plastics, Riverside, CA). The environment
in the chamber is controlled so as to maintain a constant temperature of 37◦C. A heat
gun (McMaster, Santa Fe Springs, CA) inside the chamber heats the air in the chamber and
this is linked to a temperature controller (Cole Parmer, Vernon Hills, Illinois) that stops the
heat gun from functioning above the desired temperature. A 6” fan (McMaster, Santa Fe
Springs, CA) inside the chamber circulates the hot air to maintain temperature uniformity
throughout the chamber and is monitored by a J-type thermocouple probe attached to the
temperature controller. The carbon dioxide concentration inside the chamber is maintained
at 5% and is humidified to prevent excessive evaporation of the medium. This chamber
with all of its components will ensure cell viability over long periods of time and stable cell
physiology in the absence of the chemical agents.
4.3.7. Cell Culture
4.3.7.1. Neuron Culture The H19-7 cell line is derived from hippocampi dissected
from embryonic day 17 (E17) Holtzman rat embryos and immortalized by retroviral transduction
of temperature sensitive tsA58 SV40 large T antigen. H19-7 cells grow at the
permissive temperature (34◦C) in epidermal growth factor or serum. They differentiate
to a neuronal phenotype at the non-permissive temperature (39C) when induced by basic
CELL BASED SENSING TECHNOLOGIES 69
fibroblast growth factor (bFGF) in N2 medium (DMEM-high glucose medium with supplements).
H19-7/IGF-IR cells are established by infecting H19-7 cells with a retroviral vector
expressing the human type I insulin-like growth factor receptor (IGF-IR). The cells are
selected in medium containing puromycin.H19-7/IGF-IR cells express the IGF-IR protein.
IGF-IR is known to send two seemingly contradictory signals inducing either cell proliferation
or cell differentiation, depending on cell type and/or conditions. At 39◦C, expression
of the human IGF-IR in H19-7 cells induces an insulin-like growth factor (IGF) I dependent
differentiation. The cells extend neuritis and show increased expression of NF68. This cell
line does not express detectable levels of the SV40 T antigen. Following spin at 100 × g
for 10 minutes at room temperature; cells were re-suspended in a separation buffer (see
Table 4.1). The density of the re-suspended cells (2500 cell/mL) ensured single cell positioning
over individual electrodes. Separation buffer used for neurons contained 250 mM
sucrose/1640 RPMI (Roswell Park Memorial Institute), with a conductivity of 1.2 mS/cm
and a pH of 7.48. The separation buffer was replaced by a buffer comprising of minimum
essential medium/10% Fetal Bovine Serum (FBS)/5% Phosphate buffer saline (PBS) of
conductivity 2.48 mS/cm and pH of 7.4 suitable for cell viability.
4.3.7.2. Primary Osteoblast Culture Primary rat osteoblast cells were cultured to a
concentration of 10,000 cells in 1 mL for sensing experiments. To achieve the patterning
of a single cell over a single electrode, a 10 μL of cell culture solution was mixed with
500 μL Dulbeco modified eagle medium (DMEM; Gibco, Grand Island NY) supplemented
with 10% fetal bovine serum (FBS; Gibco, Grand Island NY), 100 μg/mL penicillin, and
100 μg/mL streptomycin (P/S; Gibco, Grand Island NY). The cells were centrifuged and
re-suspended in 1 mL of separation buffer consisting of 1:9 dilutions of Phosphate Buffer
Saline 250 mM Sucrose (Sigma, St Louis) and de-ionized water (weight/vol
(diameter: 80 μm, center-to-center spacing: 200 μm) spanning a surface area
of 0.88 × 0.88 mm2 on a silicon/silicon nitride substrate with electrode leads (6 μm
thick) terminating at electrode pads (100 μm × 120 μm) has been fabricated using standard
microlithography techniques [102]. To achieve a stable local microenvironment for
sensing, the microelectrode array has been integrated to a silicone chamber (16 × 16 ×
2.5mm3) with a microfluidic channel (50 μm, wide); to pump in the testing agent and pump
out the test buffer once the sensing process has been completed. The flow rate of the buffer
was 40 μL/min. The silicone chamber was provided with an opening (8 × 8 × 2.5 mm3)
and covered by a glass cover slip for in-situ monitoring. Simultaneous electrical and optical
monitoring has been achieved by using a MicrozoomTM(Nyoptics Inc, Danville, CA)
68 CENGIZ S. OZKAN ET AL.
http://s271.photobucket.com/albums/jj125/jjames40/bio20-a/?action=view¤t=untitled.jpg
FIGURE 4.5. Schematic representation of the measurement system. It provides simultaneous electrical and optical
monitoring capability [105].
optical probe station under 8 × and 25 × magnification. The electrical stimulation and
measurements were achieved by using micromanipulators (Signatone, Gilroy, CA).
4.3.6.2. Environmental Chamber The optical probe station along with the chip assembly
was enclosed by an acrylic chamber (S&W Plastics, Riverside, CA). The environment
in the chamber is controlled so as to maintain a constant temperature of 37◦C. A heat
gun (McMaster, Santa Fe Springs, CA) inside the chamber heats the air in the chamber and
this is linked to a temperature controller (Cole Parmer, Vernon Hills, Illinois) that stops the
heat gun from functioning above the desired temperature. A 6” fan (McMaster, Santa Fe
Springs, CA) inside the chamber circulates the hot air to maintain temperature uniformity
throughout the chamber and is monitored by a J-type thermocouple probe attached to the
temperature controller. The carbon dioxide concentration inside the chamber is maintained
at 5% and is humidified to prevent excessive evaporation of the medium. This chamber
with all of its components will ensure cell viability over long periods of time and stable cell
physiology in the absence of the chemical agents.
4.3.7. Cell Culture
4.3.7.1. Neuron Culture The H19-7 cell line is derived from hippocampi dissected
from embryonic day 17 (E17) Holtzman rat embryos and immortalized by retroviral transduction
of temperature sensitive tsA58 SV40 large T antigen. H19-7 cells grow at the
permissive temperature (34◦C) in epidermal growth factor or serum. They differentiate
to a neuronal phenotype at the non-permissive temperature (39C) when induced by basic
CELL BASED SENSING TECHNOLOGIES 69
fibroblast growth factor (bFGF) in N2 medium (DMEM-high glucose medium with supplements).
H19-7/IGF-IR cells are established by infecting H19-7 cells with a retroviral vector
expressing the human type I insulin-like growth factor receptor (IGF-IR). The cells are
selected in medium containing puromycin.H19-7/IGF-IR cells express the IGF-IR protein.
IGF-IR is known to send two seemingly contradictory signals inducing either cell proliferation
or cell differentiation, depending on cell type and/or conditions. At 39◦C, expression
of the human IGF-IR in H19-7 cells induces an insulin-like growth factor (IGF) I dependent
differentiation. The cells extend neuritis and show increased expression of NF68. This cell
line does not express detectable levels of the SV40 T antigen. Following spin at 100 × g
for 10 minutes at room temperature; cells were re-suspended in a separation buffer (see
Table 4.1). The density of the re-suspended cells (2500 cell/mL) ensured single cell positioning
over individual electrodes. Separation buffer used for neurons contained 250 mM
sucrose/1640 RPMI (Roswell Park Memorial Institute), with a conductivity of 1.2 mS/cm
and a pH of 7.48. The separation buffer was replaced by a buffer comprising of minimum
essential medium/10% Fetal Bovine Serum (FBS)/5% Phosphate buffer saline (PBS) of
conductivity 2.48 mS/cm and pH of 7.4 suitable for cell viability.
4.3.7.2. Primary Osteoblast Culture Primary rat osteoblast cells were cultured to a
concentration of 10,000 cells in 1 mL for sensing experiments. To achieve the patterning
of a single cell over a single electrode, a 10 μL of cell culture solution was mixed with
500 μL Dulbeco modified eagle medium (DMEM; Gibco, Grand Island NY) supplemented
with 10% fetal bovine serum (FBS; Gibco, Grand Island NY), 100 μg/mL penicillin, and
100 μg/mL streptomycin (P/S; Gibco, Grand Island NY). The cells were centrifuged and
re-suspended in 1 mL of separation buffer consisting of 1:9 dilutions of Phosphate Buffer
Saline 250 mM Sucrose (Sigma, St Louis) and de-ionized water (weight/vol