WO2001048144A2

WO2001048144A2 - Mammalian cell culture chamber

Info

Publication number: WO2001048144A2
Application number: PCT/US2000/041800
Authority: WO
Inventors: Hal C. Cantor
Original assignee: Advanced Sensor Technologies, Inc.
Priority date: 1999-11-02
Filing date: 2000-11-02
Publication date: 2001-07-05
Also published as: AU5515501A; WO2001048144A3

Abstract

The present invention relates to a cell culture chamber (10) having a sterile environment (12) for the culture of cells, a cover (14) for allowing for a transfer of gases and a sensor array.

Description

MAMMALIAN CELL CULTURE CHAMBER

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. Section

119(e) of United States Patent Application Number 60/163,470, filed November 2,

1999, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

TECHNICAL FIELD

The present invention relates to a cell culture chamber 10. More

specifically, the present invention relates to a cell culture chamber 10 utilizing a

sensor array.

BACKGROUND ART

Biosensors, sensors including biological materials employed to

detect and/or monitor an environment, offer several advantages. Biosensor

development can utilize the highly sensitive nature of biological materials to

directly detect the presence or absence of analytes by their effect upon cellular

metabolism. For example, utilizing cultured cell systems it is possible to screen for

a broad range of toxins, thereby achieving a fast turn-around time, while

maintaining high sensitivity. Moreover, a degree of selectively can be achieved by

a choice of cell type.

Cellular metabolism refers to the orchestration of the chemical and

enzymatic reactions that constitute the life process of a cell. These reactions

include a vast number of different chemical and enzymatic reactions, relating to

the growth and maintenance of the cell. These chemical and enzymatic reactions occur simultaneously within an active cell. Moreover, these reactions do not take

place in isolation; rather the pace of each reaction is regulated, in turn, by the

product of one or more other reactions. Overall, the organization of cellular

metabolism is embedded in a vast network of inter-related cellular reactions.

Given this interdependency, it is apparent that analytes that affect one or more

aspects of cellular metabolism are likely to manifest their impact on characteristics

of the cell, including impedance; action potential parameters, including action

potential rate, action potential amplitude, and action potential shape, among

others; membrane conductance, membrane capacitance and secretion of cellular

products such as hormones, neurotransmitters, and other cellular metabolites.

Because of the above advantages, biosensors including live, intact

cells (referred to as hybrid biosensors) have several commercially significant

applications. For example, such biosensors are particularly useful in detecting

chemical and biological warfare (CBW) agents. Biosensors may also supplement

existing methods for pharmaceutical screening. It is possible that this type of

biosensor technology will eliminate, or at least greatly reduce, animal testing

employed in pharmaceutical screening.

Some progress in the development of hybrid biosensor technology

has been described within the scientific literature. For example, techniques have

been described by Giaever and Keese (in conjunction with others) to monitor the

impedance characteristics at cell/electrode interfaces. See Kowolenko M., et al.,

1990; Keese C.R., et al., 1990; Ghosh, P.M., et al., 1993; Keese, C.R., et al.,

1994; Giaever, I., et al., 1986; U.S. Pat. Nos. 4,054,646; 4,920,047; and

5,187,096. However, the systems described by Giaever and Keese utilize large area electrodes (e.g., 250 μm diameter) which cannot be completely covered by a

single cell. Therefore, accurate measurement of an individual cell's impedance

characteristics or secretory profiles, is interfered with by the parallel impedance or

secretory profiles of the uncovered electrode area and the impedance or secretory

profiles of cell-cell contact areas (due to the space between adjacent cells).

Moreover, the efforts of Giaever and Keese reveal only motility changes in cells.

Further, cellular membranes are modeled as having a constant capacitance and

conductance; any changes in capacitance and conductance are explained in

terms of varying cellular membrane area.

In Lind et al., 1991 , a system using relatively large area electrodes

similar to that described above was employed, as well as a system utilizing

electrodes smaller than the cell to be monitored. In this system, single cell effects

could be examined without the shunting effects descπbed above, but this system

was only used to monitor cell motility by means of changes in the impedance.

Moreover, in such systems membrane conductance and capacitance was

presumed to be constant. Accordingly, there remains a need for a technique that

utilizes electrodes smaller than a cell's diameter, but which provides for monitoring

changes in cellular membrane capacitance and conductance, as well as being

capable of monitoring secreted cellular products such as hormones,

neurotransmitters and cellular metabolites. Such a technique would also permit

monitoring of activation of voltage-gated ionic channel conductance. In addition,

such a technique would permit detection and monitoring of compounds that affect

the impedance, action potential parameters, membrane conductance, membrane

capacitance of a cell, and cell/substrate seal resistance, as well as being capable of monitoring secreted cellular products such as hormones, neurotransmitters and

cellular metabolites.

For example, voltage-gated Na⁺ channels (among other ion

channels) help make nerve cells electrically excitable and enable them to conduct

action potentials. When the membrane of a cell with many Na⁺ channels is

partially depolarized by a momentary stimulus, some of the channels promptly

open, allowing Na⁺ ions to enter the cell. The influx of positive charge depolarizes

the membrane further, thereby opening more channels, which admit more Na⁺,

causing still further depolarization. This process continues in a self-amplifying

fashion until the membrane potential has shifted from its resting value of about

-70 mV all the way to the Na⁺ equilibrium potential of about +50 mV. At that point,

where the net electrochemical driving force for the flow of Na⁺ is zero, the cell

would come to a new resting state with all its Na⁺ channels permanently open, if

the open channel conformation were stable. The cell is saved from such a

permanent electrical tetanus by the automatic inactivation of the Na⁺ channels,

which now gradually close and stay closed until the membrane potential has

returned to its initial negative resting value. The whole cycle, from initial stimulus

to return to the original resting state, takes a few milliseconds or less.

In many types of neurons, though not all, the recovery is hastened

by the presence of voltage-gated K⁺ channels in the plasma membrane. Like the

Na⁺ channels, these channels open in response to membrane depolarization, but

they do so relatively slowly. By increasing the permeability of the membrane to K⁺

just as the Na⁺ channels are closing through inactivation, the K⁺ channels help to

bring the membrane rapidly back toward the K⁺ equilibrium potential, so returning it to the resting state. The repolarization of the membrane causes the K⁺ channels

to close again and allows the Na⁺ channels to recover from their inactivation. In

this way the cell membrane can be made ready in less than a millisecond to

respond to a second depolarizing stimulus.

Examining the membrane potential relative to time, an action

potential exhibits various characteristics or parameters, including action potential

rate (if cells spontaneously depolarize), action potential amplitude, and action

potential shape, among others. Action potential rate refers to the frequency with

which a cell produces an action potential (rapid depolarization). Action potential

amplitude refers to the height of the peak depolarization that occurs in the course

of the action potential. Action potential shape refers to the time course of the

depolarization and repolarization.

It would therefore be useful to develop a cell chamber 10 which

provides a sterile gas permeable environment containing therein a sensor array

16.

SUMMARY OF THE INVENTION

The present invention relates to a cell culture chamber 10 having a

sterile environment 12 for the culture of cells, a cover 14 for allowing for a transfer

of gases and a sensor array 16.

DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention will be readily appreciated

as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings

wherein:

Figure 1 is a photograph showing the sensor array 16 chip bonded

with the ceramic 100-pin pin grid array (near actual size);

Figures 2 A-C are photomicrographs of the sensor array 16 chip

wire-bonded to the ceramic carriers' insert, Figure A is the test sensor, Figure B is

an 8 μm electrode sensor array 16, and Figure C is a 32 μm electrode sensor

array 16;

Figures 3 A and B are pictures showing the silicone applied to the

top of the sensor chip leaving approximately 2 mm x 2 mm exposed over the

sensor array 16, Figure A shows a 2 μm electrode sensor array 16, and Figure B

shows an 8 μm electrode sensor array 16;

Figure 4 is a picture showing the image of the entire cell culture

chamber 10 with the integrated sensor array 16, cells and medium are maintained

in the cloning cylinder mounted on top of the sensor array 16 in the 35 mm Petri

dish which maintains sterility during transport and minimizes fluid evaporation;

Figure 5 is a side view of the preferred embodiment of the cell

culture chamber of the present invention;

Figure 6 is another view of the preferred embodiment of the cell

culture chamber of the present invention;

Figure 7 is a top view of the preferred embodiment of the cell culture

chamber of the present invention;

Figure 8 is a photomicrograph at hNT neurons growing on the

sensor array; and Figure 9 is a picture of dozens of mammalian cell culture chambers.

DETAILED DESCRIPTION

Generally, the present invention provides a cell culture chamber 10

providing a sterile environment 12 for the culture of cells, a cover 14 for allowing

the free transfer of gases, and a sensor array 16.

By "sterile environment 12" it is meant that the environment or area

in which the cells are being grown is devoid of contaminants and bacteria. The

definition of contaminants differs depending upon the cells being cultured, and the

specific property being measured. For example, the chamber 10 can include a

Petri dish or other container. Any such sterile environment 12 must have a hole

drilled in the bottom of sufficient size to accommodate the sensor array 16.

By "cover 14" it is meant a device sufficient to maintain the sterility of

the environment while enabling the free transfer of required gases. These gases

will differ depending upon the cells being cultured and the property being

measured. In the preferred embodiment, the cover 14 is made of a clear material

which enables a user to observe the culture medium, sensor, and cells. However,

the cover 14 can also include a semi-permeable membrane. This membrane can

be made of a hydrophobic or hydrophilic material.

The present invention permits detection and monitoring of membrane,

chemical, and electrical characteristics of a cell. As noted at the outset, these

characteristics include cell impedance (cell membrane capacitance and

conductance), action potential parameters, cell membrane capacitance, cell

membrane conductance, neuronal action potential, cellular metabolic products (hormones, neurotransmitters, waste products, etc.), and cell/substrate seal

resistance. These electrical characteristics, in turn, correlate well with the

metabolic state of the cell. The present invention, thus, provides an apparatus

and method for monitoring cells and for monitoring the impact that an analyte has

upon the metabolism of a cell. It is to be appreciated that such analytes include

pharmaceutical agents, drugs, environmental factors, toxins, chemical agents,

biological agents, viruses, and cellular adhesion promoters. Poly-D-lysine and

Matrigel matrix (Becton Dickinson Labware, Bedford, MA) were used to provide a

basement membrane for the cells.

The sensor array was first coated with poly-D-lysine to promote bonding of

the Matrigel matrix. Sterile poly-D-lysine, at a concentration of 10μg/ml in distilled

water, was applied to each of the 2mm x 2mm exposed sensor arrays and allowed

to incubate at room temperature for 2 hours. The poly-D-lysine solution was then

aspirated with a sterile pipette. The sensor arrays were placed at an incline with

lids off in a sterile laminar flow hood and allowed to dry for 1.5 hours.

Matrigel matrix was thawed overnight in a refrigerator, and then diluted to

1 :40 in cold DMEM/ F12 (Life Technologies, Rockville, MD). 20μl of the Matrigel

matrix was applied to each sensor array and spread evenly using a Pasture

pipette. The solution was allowed to completely dry at room temperature in a

sterile laminar flow hood. The Matrigel application was then repeated.

The sensor arrays could be stored for at least 2 months with one

coat of poly-D-lysine and one coat of Matrigel matrix. The final coat of Matrigel

matrix must be applied on the day of use. Under the microscope, a dry coated sensor appeared to have a fine, frost-like mesh. Care was taken to avoid opaque

clots indicating the Matrigel matrix concentration was too high.

Thus, the present invention is useful in screening and assaying

broad classes of materials for their impact on cellular metabolism.

Cells are cultured within the medium according to known techniques.

A layer of cells (Figure 8) adhere to the respective surfaces of the microelectrode

array provided on the integrated device. As each of the microelectrodes is driven

with the applied voltage signal, a current flows between the microelectrodes and

the reference electrode. The impedance is determined from this current signal.

Based on the impedance, various electrical characteristics of the cells which

adhere to the surfaces of the microelectrodes can be monitored. Such

characteristics include the impedance of the individual cell (i.e., the combined cell

membrane capacitance and conductance), the action potential parameters of the

individual cell, the cell membrane capacitance, the cell membrane conductance,

and the cell/substrate seal resistance. The electrodes can also monitor the

membrane potential produced by the cell, such as neuronal action potentials. The

action potential parameters include, among others, action potential rate, action

potential amplitude, and action potential shape and action potential power

spectrum.

It is a feature of the invention that each of the microelectrodes is

sufficiently small to enable monitoring of an individual cell, and its cellular

membrane, and any secreted cellular products or metabolites. Conventionally, the

relative size of the reference electrode is large in comparison to the measuring

electrodes so that the measured impedance across each electrode and the reference electrode is dominated by the interface between the microelectrode and

the cell and the cell membrane impedance. In addition, the present invention

utilizes microelectrodes with a small surface such that the diameter of a cell is

larger than the diameter of a microelectrode so that electrical and chemical

characteristics related to individual cells and cell membranes can be resolved.

The specific size of the microelectrodes varies according to the

specific application and size of the cells to be monitored. Preferably, the diameter

of the microelectrodes is less than or equal to one-half the diameter of the cell to

be monitored, thereby permitting a given microelectrode to monitor an individual

cell and cell membrane. For example, in one preferred embodiment,

microelectrodes with diameters of about 4 to 20 μm have been utilized. The

combination of the relatively small size of the microelectrodes, the relatively low

impedance of the microelectrodes (i.e., relative to cell membrane impedance) and

the low noise characteristics of the signal detection components enables the

present invention to monitor changes in the electrical and chemical characteristics

of the cell, including changes in the capacitance and conductance of the cellular

membrane. It also permits a variety of other techniques, described in greater

detail below. These techniques include monitoring the action potential parameters

of the cells; monitoring activation of voltage-gated ion channels; pharmaceutical

screening and toxin detection; identifying particular cell/microelectrode junctions

characterized by a higher degree of cell adherence to the microelectrode; and

testing changes in cell adherence as adhesion promoting agents are employed.

It is understood that the signal monitoring and processing means

referred to includes various devices for calculating the various cellular characteristics referred to above. For example, this element can comprise a

personal computer configured to calculate the impedance between each

microelectrode and the reference electrode based on the signals detected with the

signal detecting means. By monitoring these values over time, the present

invention can determine the various characteristics of individual cells which adhere

to each microelectrode. The signal monitoring and processing means can be

configured to perform a spectral analysis so as to monitor, in real time,

characteristics such as changes in cell action potential parameters and cell

impedance. The sensors can also be operated to perform amperometric and

potentiometric determinations of chemical compositions and concentrations.

The surface of a single microelectrode of the array is exposed

through a via in a passivation layer provided over a substrate. The microelectrode

is driven with a signal source relative to the reference electrode.

The microelectrode has a known impedance Z_elect. As known in the

art, this value is described by a classical model of a metal in an electrolyte, and is

calculated based on the material of the microelectrode or is measured separately

prior to introduction of cells into the system. In series with the impedance of the

microelectrode is the resistance of the solution R_so,_n and the respective

capacitance and conductive values associated with the microelectrode/cell

junction. Parasitics to the substrate and parasitics to the passivation layer, can be

reduced by use of a glass substrate, or can be factored out by normalizing.

Although often modeled as constants in prior art systems, the values

of the capacitance and conductance associated with the cell actually vary in response to added toxins, pharmaceuticals and other substances, applied

voltages, and changes in cellular morphology.

The impedances of the microelectrode, Z_e,_ect, and the reference

electrode, Z_ref, can be determined in a variety of ways. These values, as well as

the resistance of the solution, R_soln, can be factored out, for example, by

normalizing to data obtained prior to introduction of the cells to the system. Thus,

by measuring the total impedance across each microelectrode and the reference

electrode, it is possible to resolve impedances or changes in impedance relating to

cell motility, adhesion to the cell substrate (based on R_seaι)_. and changes in cellular

membrane capacitance and conductance. It is further possible to monitor the

activity of cellular ion channels and the action potential parameters of the cell as a

result of their effects on the impedance measured between each microelectrode

and the reference electrode or by actually monitoring the cell membrane potential.

It is noted that the particular electrical characteristics of the cell, such

as the capacitance and the conductance of the cellular membrane, are determined

from the measured impedance by modeling the cell in accordance with known

techniques. Various cell models can be used. For example, the cell can be

modeled as a flat, circular "pancake" (i.e., as a disk). Other models can include a

square or rectangular "pancake", a sphere, a cube, or a rectangular box.

In an embodiment of the invention, a homodyne detection technique

is used to detect the signals resulting from the application of a signal voltage

between each microelectrode and the reference electrode. A quadrature

synthesizer is used to generate both sine and cosine signals of a programmed

frequency and amplitude. The sine voltage signal is attenuated as needed and selectively applied to individual microelectrodes. The resulting signal is detected

by a transimpedance stage which holds the large reference electrode at a

reference potential (in this case, ground) via negative feedback. Automatic gain

control is used to amplify the voltage output of the transimpedance stage. The

amplified signal is multiplied in quadrature by the source signals and is low pass

filtered to provide the real and imaginary components of the measurement.

Quadrature multiplication allows for signal detection, for example, at the excitation

frequency with high noise immunity. Known resistance values are used to calibrate

the system for electrode impedance measurements.

In another embodiment, a quadrature synthesizer generates both a

sine wave and cosine wave, A sin (ωt) and A cos (ωt). In this particular example,

the generated signals have a frequency of 1 kHz and an amplitude of 10 V peak to

peak (P--P). Of course, it is understood that the present invention is not limited to

these particular values. For example, a system has been constructed capable of

applying signals from 100 Hz to 100 kHz.

The sine wave is attenuated in this example, in a range from 0 to

-80 dB. The attenuated signal is then selectively applied to particular

microelectrodes. In this example, an analog multiplexer is used to apply the

attenuated voltage signal to each of an array of fifty-eight microelectrodes. Again,

the invention is not limited to the number of microelectrodes in the array or to the

components used for selective application of the signal to each of the

microelectrodes.

The resulting current is detected by a transimpedance amplifier

which maintains the large reference electrode at a virtual ground potential. The transimpedance amplifier outputs a signal, B sin (ω+φ). In this example, a known

resistance R_sense_ is applied across the input and output of the transimpedance

amplifier.

The signal from the transimpedance amplifier is then amplified by an

automatic gain control (AGC) amplifier having a variable gain A_v. The signal is

amplified in a range between 0 and 120 dB. The resulting signal, A_v B sin (ω+φ), is

multiplied in quadrature with mixers 38a and 38b and then low pass filtered to

obtain real and imaginary components X and Y.

Thus, the signal from AGC amplifier is mixed with the signal

A sin (ω.t) to obtain the following signal:

(Ay AB/2)[cos φ-cos (2ωt+φ)]

The signal from the AGC amplifier is also mixed with the signal

A cos (ωt) to obtain the following signal:

(Ay AB/2)[sin φ+sin (2ωt+φ)]

Thus, when low pass filtered, the following components remain:

X=(A_V AB cos φ)/2

and

Y=(A_V AB sin φ)/2

In one example, the system is calibrated with a known value

resistance to obtain calibration values X_CAL and Y_CAL. The magnitude |CAL| and

phase φ_CAL of the calibration values are then determined as follows:

φ_CAL =arc tan [Y_CAL /X_CAL ]|CAL|=[X_CAL ² +Y_CAL ² ]^1/2

Once the calibration values are obtained, measurements are taken

with the microelectrodes and measurement values X_MEAS and Y_MEAS are calculated as indicated above. Based on these values, the phase and magnitude for the

respective measurements, φ_MEAS and |MEAS| are calculated as follow:

φ_MEAS =arc tan[X_MEAS /Y_MEAS ]|MEAS|=[X_MEAS ² +Y_MEAS ² ]^1/2

Given the respective values for X_CAL, Y_CAL, X_MEAS and Y_MEAS, one can

divide the magnitude of the measured value |MEAS| by the magnitude of the

calibration value |CAL| and then solve for the magnitude of the unknown

impedance |Z_UNKN0WN |. The phase of the unknown impedance Z_UNKN0WN is

determined as follows:

The detected values for X and Y are sampled with an analog to

digital (A/D) converter installed in a PC. In this example, a signal monitoring and

processing means, such as a PC, receives the detected signal through an (A/D)

converter at a sample rate sufficient to read the detected sinusoidal signal. For

example, the output from the AGC amplifier could be A/D converted and input to a

PC. The signal monitoring and processing means obtains phase and magnitude

values from the unknown impedance after directly converting the respective

sinusoidal output for extraction of data. In this example, the signal monitoring and

processing means provides the capability of performing a spectral analysis to

monitor, in real time, characteristics such as changes in cell action potential

parameters and cell impedance. By performing real time Fourier analysis, it is

possible to monitor distortion caused by nonlinear electrode effects. Thus, it is a

feature of the invention that the system can perform spectral analysis of the

resulting signals in order to monitor changes in various characteristics in real time. As described generally above, data obtained with the embodiment

can be "normalized" to vaπous conditions in order to monitor the characteristics of

cells which adhere to the respective microelectrodes. For example, the membrane

capacitance and conductance of individual cells which adhere to the

microelectrodes can be monitored, based on various models of the cells. Various

other techniques are described herein.

This embodiment provides high sensitivity and signal resolution for a

large range of applications. One characteristic of this embodiment is that the low

pass filters have a characteristic settling time. Where several electrodes are

monitored sequentially, this characteristic can introduce a minor delay. It is

possible to provide an even faster measurement cycle by heterodyning the

detected signal to a higher frequency and then bandpass filtering to obtain phase

and magnitude information. Such a technique permits real time observation of

extremely rapid variations in electrical characteristics of the cell, such as variations

in transmembrane impedance (caused by opening and closing of ion channels).

A test signal oscillator generates sine and cosine signals,

A sin (ω_test t) and A cos (ω_test t) at a frequency ω_test. The voltage sine signal is

selectively applied across each microelectrode and the reference electrode. The

resulting current is detected using transimpedance amplifier and AGC amplifier.

Mixers multiply the resulting signal B sin (ω_test t+φ) in quadrature by sine and

cosine signals generated by a local oscillator. The signals produced by the local

oscillator have an angular frequency ω_L0, which is the sum of the test frequency

ω_test and an intermediate frequency <_%. As indicated above, the respective outputs

of mixers are represented, respectively, by: (AB/2) cos [ω,_F t+φ]-(AB/2) cos [(2ω_test +ω_IF)t+φ](AB/2) sin [ω_IF t+φ]-(AB/2) sin [(2ω_test

+ω_IF)t+φ]

These outputs are bandpass filtered at the intermediate frequency

(% and the filtered output detected using amplitude modulation detectors to

provide real and imaginary components X=|(AB cos φ)/2| and Y=|(AB sin φ)/2|. The

algebraic sign of X and Y must be determined by a phase-sensitive detector, as is

well known in the art. The unknown impedance between the microelectrode and

the large reference electrode is then calculated in the manner described above.

It is appreciated that in addition to the exemplary embodiments

discussed above, various other alternative embodiments are possible for

impedance measurement. For example, measurement in the time domain can be

used rather than homodyning to extract phase and magnitude information about

each impedance. This can be accomplished, for example, by using a step function

in place of the above-mentioned sinusoidal signal to drive each microelectrode.

Further, the frequency of excitation and the sinusoidal input signal amplitude can

be expanded beyond the exemplary ranges identified above.

Again, various modifications and alternative embodiments will be

apparent to those skilled in the art without departing from the invention. For

example, the microelectrodes and their respective interconnects and bond pads

may comprise any biocompatible conductive substance, such as iridium, activated

iridium, gold, platinum, polysilicon, aluminum, ITO, or TiW, bare or electroplated

with platinum black.

Additionally, the substrate of the integrated device may be

composed of a variety of materials, such as silicon, glass, metal, quartz, plastic, ceramic, polyethylene, or any other suitable type of polymer. It is noted that glass

substrates have been found to provide reduced parasitic capacitance.

Other variations of the structure which are not essential to the

underlying features of the invention include changes in the composition of the

passivation layer. For example, devices made in accordance with the invention

have utilized different passivation layers ranging from 0.5 to 5 μm in thickness.

The passivation layer may comprise any suitable material, including low stress

PECVD silicon nitride, silicon carbide, TEFLON™, polyimide, ceramic, photoresist,

or any type of polymer or thermal plastic, or combination thereof.

Cloning cylinders, housed in Petri dishes, are bonded to the

substrate to define respective wells. The Petri dishes can comprise polystyrene,

glass, polyethylene, TEFLON™, metal, or any other type of polymer. The Petri

dishes may be bound to a chip formed in accordance with the invention using

conventional materials and techniques, such as epoxy, polyurethane, wax, or

thermoplastic, using chemical, thermal, or ultrasonic processes. In this alternate

construction, bondwire connections can be eliminated by use of a substrate

configured to mate directly with a connector, such as an edge card connector or

pads for a standard pin type connector. Of course, this would require the

substrate area to be significantly larger than the active electrode area.

The membrane capacitance, membrane conductance, cell/substrate

separation and action potential parameters of a cell are significant markers

regarding a cell's metabolic state, including general cellular health and ionic

channel activity. The membrane potential, the voltage difference across a cell's

plasma membrane, depends on the distribution of ionic charge. Generally, the distribution of ionic charge determines the electric potential, or voltage. For

example, in a metallic conductor, the mobile particles carrying charge are

electrons; in an aqueous solution, the mobile particles are ions such as Na⁺, K⁺,

CI^", and Ca⁺2. In an aqueous solution, the number of positive and negative

charges are normally balanced exactly, so that the net charge per unit volume is

zero. An unbalanced excess of positive charges creates a region of high electrical

potential, repelling other positive charges and attracting negative charges. An

excess of negative charges repels other negative charges and attracts positive

charge. When an accumulation of positive charges on one side of a membrane is

balanced by an equal and opposite accumulation of negative charges on the other

side of the membrane, a difference of electrical potential is set up between the two

sides of the membrane.

Selective molecular access to the electrodes can be provided by

depositing membranes on them. Several different classes of membranes are

available for use. Nafion acts as a cation exchange membrane (Brazell, et al.,

1987), allowing only uncharged molecules to gain access to the electrodes.

Additionally, various mixtures of cellulose acetate can be prepared which act as

size exclusion membranes, allowing only specific molecular weight species to gain

access to the electrodes. This is critical when monitoring large bioagent

molecules. Often, large bioagent molecules degrade into a variety of break-down

products. It is possible that only the parent bioagent exerts a biological effect and

the break down products do not, however, several of the break down products

may oxidize at potentials very close to the parent molecule when monitored

amperometrically. Using a variety of decreasing size exclusion membranes on the sensors in the array, the concentration of the parent bioagent molecule can be

determined as well as each of its break-down products uniquely.

Charge is carried back and forth across the cell membrane by small

inorganic ions-chiefly Na⁺, K⁺, CI^", and Ca⁺2 --but these can traverse the lipid

bilayer only by passing through special ion channels. When the ion channels

open, the charge distribution shifts and the membrane potential changes. Of

these ion channels, those whose permeability is regulated are the most significant;

these are referred to as gated channels. Two classes of gated channels are of

crucial importance: (1) voltage-gated channels, especially voltage-gated Na⁺

channels, which play the key role in the rapid changes in electrical energy by

which an action potential is propagated along a nerve cell process; and (2) ligand-

gated channels, which convert extracellular chemical signals into electrical signals,

which play a central role in the operation of synapses. These two types of

channels are not particular to neurons: they are also found in many other types of

cells.

One goal of the present invention is to augment semiconductor

sensor technologies with new formulations of membranes containing ionophores,

antibodies and enzymes, to enable the array to monitor a wide range of biological

analytes, environmental toxins, as well as standard blood chemistries (i.e.

electrolytes, antibodies, steroid and protein hormones, anesthetics, a variety of

herbicides, medicinal drugs, drugs of abuse, etc.). One example of monitoring

complex molecules with a membrane is to catalize or inhibit a reaction such as the

measurement of the pesticide Paraoxon. Other complex molecules, such as

neurotoxins and molecules of biological warfare can be detected by immobilizing antibodies and/or enzymes on the surface of an ion-selective membrane, by

performing Enzyme Linked Immuno Sorbent Assays (ELISA), or through the

production of amperometrically detectable reaction products catalyzed by

enzymes causing the formation of electroactive molecules, such as hydrogen

peroxide, from the parent molecule.

Because organophosphates inhibit the reaction mechanism causing

the breakdown of various choline compounds, catalyzed by a variety of

cholinesterase enzymes, these can be used to monitor the presence of Paraoxon.

The membranes must adhere well to the silicon surface to prevent detachment of

the membrane during sampling, rinsing, and washing of sensors in the sampling

chamber. When silicon based, optical detectors are to be utilized, slight pealing of

the membrane can significantly alter the performance of the device. Membranes

that adhere strongly to the silicon surface provide the sensors with a long, useful,

lifetime.

Some examples of membranes include, but are not limited to,

Cellulose Acetate, Poly-Urethane/Poly-Vinyl Chloride, and Silicone Rubber. Each

of these membrane compositions possess differing properties as related to

enzyme and antibody immobilization and adherence to the silicon nitride surface

of microscopic solid-state chemical sensors. Several methods are available for

immobilizing enzymes and antibodies on the surface of the membranes.

Additional techniques amenable to monitoring organophosphorous

containing compounds, including Paraoxon, can also be used. Sensitive assays

using spectrofluorimetry have been reported to have detection limits on the order

of 8 x 10^~7 for Paraoxon (Russell et al., 1999). Enzymes have been incorporated into a hydrophilic polyurethane membrane and deposited on top of hydrophobic

polyurethane membranes (Cho et al., 1999), promoting adhesion to the sensor

surface. Additionally, enzymes can be immobilized on the membrane surface that

cause local changes in pH in the presence of toxins, which can be monitored

utilizing a potentiometric electrode (Mulchandani et al., 1999)

The first method to be employed for the detection of

organophosphorus compounds such as Paraoxon is to monitor the inhibition of the

reaction catalyzed by butyrylcholinesterase, which breaks down butyrylcholine into

choline and butyric acid. Paraoxon has been shown to inhibit this reaction linearly

in proportion to its concentration (Campanella, et al., 1996).

Membrane adhesion has typically been a significant problem

effecting useful lifetime of solid-state chemical sensors. Many of the membranes

utilized for traditional chemical sensors do not adhere well to the silicon-nitride

surface, reducing the yield and lifetime. Membrane adhesion is tested using a Q-

Test II adhesion analyzer. Membrane adhesion is a critical factor and must be

optimized to provide stable electrochemical properties in a flow system. The fluid

flow system, necessary for sample delivery, calibration, washing, and regeneration

of the sensors, tends to cause pealing of the membrane.

To improve membrane adhesion, treatments and modifications of the

sensor's silicon-nitride surface are examined in order to improve

membrane/silicon-nitride cross-linking. These efforts vastly extend the useful

lifetime of commercial devices.

Membranes can be deposited using a set of micropipettes accurate

to 20nL of volume. Other methods known to those of skill in the art can also be used to deposit the membrane. For example, one device, a New Long LS-15TV

screen printing system for patterning membranes and epoxies onto sensor

surfaces can print with +/-5 micron alignment and 25 to 50 micron minimum

feature size.

Other features of the invention will become apparent in the course of

the following descriptions of exemplary embodiments which are given for

illustration of the invention and are not intended to be limiting thereof.

EXAMPLE

A mammalian cell culture chamber 10, with an integrated silicon

sensor array 16, was designed, constructed, and tested. The chamber 10

provides a sterile environment 12 for the culture of cells. The chamber 10 has a

cover 14 that allows free transfer of metabolic gases, minimizes medium

evaporation, and allows sterile transport of the cell culture between an incubator

and test stations (microscope, experimental setup, laminar flow hood, etc.). The

silicon sensor array 16 is a general purpose device which may be modified to

detect and quantify a wide range of analytes depending upon the electronic

method of operation at amperometric sensors and upon the selection of

membrane ionophore for the potentiometric sensors.

The sensor chips are packaged in a ceramic 100-pin pin grid array

(PGA) package (Spectrum Semiconductor Materials, Inc., San Jose, CA) with gold

plated pins and interconnects (Figure 1 and 5). The ceramic package body offers

several advantages when used in tissue culture. First, ceramic material is heat

resistant allowing autoclave temperatures required for sterilization. Second, the thermal properties of ceramic material provide temperature stability when used for

in vitro studies. During the short period when moving the chips from the incubator

to an experimental station (i.e. microscope, laminar flow hood, electronic test

station, etc.) the ceramic material acts as a thermal buffer minimizing temperature

fluctuations at the cells.

Additionally, when forming the sensor array, the completed wire-

bonds are quite fragile and form an electrical connection to the growth media if

exposed. Therefore, an epoxy material, Epoxy Patch 1 C (Dexter Corporation), is

used to seal the bonds. The bonded ceramic packages are heated to

approximately 130°C. Epoxy Patch is carefully applied to the hot packages with

the heat causing the material to liquefy and flow easily around the bonds to seal

them. After curing at 130°C for 30 minutes the result is a hard, durable coating

that provides excellent electrical isolation and completely resists moisture

exposure.

Once the sensor array 16 is secured within the ceramic carrier, a

layer of inert Dow Corning Silicone RTV Sealant 732 (World Precision

Instruments, Inc. Sarasota, FL) or any similar inert silicone or other sealant is

applied leaving a 2 mm x 2 mm window over the actual sensor array 16 (Figure 3).

A small, sterile cloning cylinder (Fisher Scientific, Chicago, IL) is placed over this

window and attached with silicone. Finally, a 35 mm Petri dish (Fisher Scientific,

Chicago, IL), with a hole drilled in the bottom to accommodate the cloning cylinder,

is attached with silicone (Figure 4). This arrangement provides several features:

1) the Petri is clear allowing observation of the culture medium, sensors, and cells;

2) the ability to transport the cells (cultured within the carrier chip) from an incubator to experimental station without compromising sterility; and 3) free

transport of metabolic gases (C0₂, O₂) and, at the same time, minimal evaporation

of the culture medium while housed in an incubator.

Dozens of cell culture chambers 10 with integrated silicon sensor

arrays 16 were constructed and tested in vitro (Figure 9). Cultures of mammalian

cells were maintained for periods greater than 75 days without a single incidence

of contamination. The chambers 10 allowed free exchange of metabolic gases

while minimizing medium evaporation as designed. The chambers 10 are

designed such that it is technically simple to: 1) inoculate with cells; 2) transport

between incubator and experimental station without compromising sterility; and 3)

observe under a microscope.

Throughout this application, various publications, including United

States patents, are referenced by author and year and patents by number. Full

citations for the publications are listed below. The disclosures of these

publications and patents in their entireties are hereby incorporated by reference

into this application in order to more fully describe the state of the art to which this

invention pertains.

The invention has been described in an illustrative manner, and it is

to be understood that the terminology which has been used is intended to be in

the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present

invention are possible in light of the above teachings. It is, therefore, to be

understood that within the scope of the appended claims, the invention may be

practiced otherwise than as specifically described. REFERENCES

Brazell, M.P. Feng, J., Kasser, R.J., Renner, K.J., and Adams, R.N., An improved method for Nafion coating carbon fiber electrodes for in vivo electrochemistry, J. Neurosci. Meth., 22:167-172, 1987;

Campanella, L., Colapicchioni, G., Favero, G., Sammartino, M.P., Tomassetti, M., "Organophosphorus pesticide (Paraoxon) analysis using solid state sensors," Sensors and Actuators B 33(1996 25-33);

Cho, Y.A., Lee, H.S., Cha, G.S., Lee, Y.T., "Farbrication of butyrylcholinesterase sensor using polyurethane-based ion-selective membranes," Biosens Bioelectron 1999 Apr 30;14(4):435-9

Ghosh, P.M. et al., "Monitoring Electropermeabilization in the Plasma Membrane of Adherent Mammalian Cells," Biophys. Journal, 64, 1602-09 (1993);

Giaever, I. et al., "Use of Electric Fields to Monitor the Dynamical Aspect of Cell Behavior in Tissue Culture," IEEE Transactions on Biomedical Engineering, Vol. BME-33, No. 2, Feb. 1986;

Keese, C.R. et al., "A Whole Cell Biosensor Based on Cell-Substrate Interactions," Annual International Conference of the IEEE Engineering in Medicine and Biology Society, Vol. 12, No. 2, (1990);

Keese, C.R., et al., "A Biosensor that Monitors Cell Morphology with Electrical Fields," IEEE Engineering in Medicine and Biology, June/July 1994, 402-08;

Kowolenko, M. et al., "Measurement of Macrophage Adherence and Spreading with Weak Electric Fields," Journal of Immunological Methods, 127, 71-77 (1990);

Lind et al., "Single Cell Mobility and Adhesion Monitoring Using Extracellular Electrodes" Biosensors and Bioelectronics, 6, 359-67 (1991);

Mulchandani, P., Mulchandani, A., Kaneva, I., Chen, W., "Bionsensor for direct determination of organophosphate nerve agents. 1. Potentiometric enzyme electrode," Biosens Bioelectron 1999 January 1 ; 14(1):77-85

Russell, R.J., Pishko, M.V., Simonian, A.L., Wild, J.R., "Poly(ehtylene glycol) hydrogel-encapsulated fluorophore-enzyme conjugates for direct detection of organophosphorus neurotoxins," Analytical Chemistry 1999 November 1 ;71(21):4909-12

United States Patents 4,054,646 4,920,047 5,187,096

Claims

1. A cell culture chamber 10 comprising:

a sterile environment 12 for culture of cells;

a cover 14 means for allowing free transfer of gases; and

a sensor array 16.

2. The cell culture chamber 10 according the claim 1 , wherein

said array is a silicon sensor array 16.

3. The cell culture chamber 10 according the claim 2, wherein

said sensor array 16 is packaged in a ceramic pin grid array.

4. The cell culture chamber 10 according the claim 1 , wherein

said chamber 10 is a size small enough to be viewed under a microscope.

5. The cell culture chamber 10 according to claim 1 , wherein

said sensor array 16 is covered by a moisture resistant layer.