Device and methods for monitoring the status of at least one cell Number:7,435,578 from the United States Patent and Trademark Office (PTO) owispatent A device and methods for monitoring status of at least one cell, wherein the cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein. In one embodiment of the present invention, the device includes a first substrate having a first surface and an opposite second surface, a second substrate supported by the first substrate, the second substrate having a first surface, an opposite second surface, a body portion between the first surface and the second surface, a first side surface and an opposite second side surface, wherein the body portion defines a first passage between the first side surface and the second side surface and an opening on the first surface of the second substrate and in fluid communication with the first passage, and sidewalls positioned above the first surface of the second substrate. In one operation mode, when a first medium is introduced into the first passage, the intracellular space of the cell is in fluid communication with the first passage with the first medium, a sensor measures the response of the cell to the first medium.<H monitoring, methods, Device, and, meth, 7435578.html, Patent, pto, 7435578

Title: Device and methods for monitoring the status of at least one cell

Abstract: A device and methods for monitoring status of at least one cell, wherein the cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein. In one embodiment of the present invention, the device includes a first substrate having a first surface and an opposite second surface, a second substrate supported by the first substrate, the second substrate having a first surface, an opposite second surface, a body portion between the first surface and the second surface, a first side surface and an opposite second side surface, wherein the body portion defines a first passage between the first side surface and the second side surface and an opening on the first surface of the second substrate and in fluid communication with the first passage, and sidewalls positioned above the first surface of the second substrate. In one operation mode, when a first medium is introduced into the first passage, the intracellular space of the cell is in fluid communication with the first passage with the first medium, a sensor measures the response of the cell to the first medium.

Patent Number: 7,435,578 Issued on 10/14/2008 to Wikswo, et al.

Inventors: Wikswo; John P. (Brentwood, TN), Baudenbacher; Franz J. (Franklin, TN), McGuinness; Owen (Brentwood, TN)
Assignee: Vanderbilt University (Nashville, TN)

Appl. No.: 10/755,639

Filed: August 6, 2002

PCT Filed: August 06, 2002

PCT No.: PCT/US02/24911

371(c)(1),(2),(4) Date: January 12, 2004

PCT Pub. No.: WO03/052375

PCT Pub. Date: June 26, 2003

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
60310652	Aug., 2001

Current U.S. Class: 435/288.3 ; 204/403.01; 435/173.4; 435/173.5; 435/173.7; 435/244; 435/285.2; 435/288.4; 435/288.5; 435/30; 435/305.3

Current International Class: C12N 13/00 (20060101); C12M 1/22 (20060101); C12M 1/34 (20060101); C12M 3/00 (20060101)

Field of Search: 435/305.3,288.4,244,285.2,288.2,173.4,173.7,173.5,30 204/403.01

References Cited [Referenced By]

U.S. Patent Documents


5888825	March 1999	Carr et al.
6225109	May 2001	Juncosa et al.
6315940	November 2001	Nisch et al.
6482619	November 2002	Rubinsky et al.
6699697	March 2004	Klemic et al.
2001/0036672	November 2001	Anderson et al.
2002/0182627	December 2002	Wang et al.
2003/0107386	June 2003	Dodgson et al.

Primary Examiner: Beisner; William H.
Assistant Examiner: Bowers; Nathan A
Attorney, Agent or Firm: Morris Manning & Martin LLP Xia, Esq.; Tim Tingkang

Government Interests

The present invention was made with Government support under Grant No. N66001-01-C-8064 awarded by the Defense Advanced Research Projects Administration. The United States Government may have certain rights to this invention pursuant to these grants.

Claims

What is claimed is:

1. A device for monitoring status of at least one cell, wherein the cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein, comprising: a. a first substrate having a first surface and an opposite second surface; b. a second substrate supported by the first substrate, the second substrate having a first surface, an opposite second surface, a body portion between the first surface and the second surface, a first side surface and an opposite second side surface, wherein the body portion defines a first passage therein between the first side surface and the second side surface, and a first opening on the first surface of the second substrate that is in fluid communication with the first passage; c. sidewalls positioned above the first surface of the second substrate; d. a third substrate positioned over the sidewalls and the second substrate and having a first surface and an opposite second surface, wherein the third substrate, the sidewalls and the second substrate define a chamber, and wherein the chamber is in fluid communication with a second passage defined by portions of the sidewalls and the third substrate; e. at least one sensor positioned in the first passage proximate to the first opening, wherein the cell is positioned in the chamber and the intracellular space of the cell is in fluid communication with the first passage through the first opening of the second substrate; f. at least one seal element positioned on the second substrate and proximate to the first opening, for sealing the cell to the second substrate in operation; g. a pair of first controls positioned inside the first passage for controlling the flow of a medium through the first passage; and h. a second control positioned inside the second passage for controlling the flow of a medium through the second passage.

2. The device of claim 1, wherein the membrane of the cell defines a second opening through which the intracellular space of the cell is in fluid communication with the first passage through the first opening.

3. The device of claim 2, farther comprising a punching element positioned underneath the first opening for making the second opening.

4. The device of claim 3, wherein the punching element comprises an electroporation device.

5. The device of claim 2, wherein when a first medium is introduced into the first passage and, the intracellular space of the cell is in fluid communication with the first passage having the first medium, the sensor measures the response of the cell to the first medium.

6. The device of claim 2, wherein when a second medium is introduced into the chamber through the second passage, and at least part of the membrane of the cell is in contact with the second medium in the chamber, the sensor measures the response of the cell to the second medium.

7. The device of claim 2, wherein when a first medium is introduced into the first passage, and a second medium is introduced into the chamber through the second passage, respectively, the intracellular space of the cell is in fluid communication with the first passage having the first medium, and at least part of the membrane of the cell is in contact with the second medium in the chamber, the sensor measures the responses of the cell to the first medium and the second medium.

8. The device of claim 1, wherein the first passage is in fluid communication with a reservoir of a medium.

9. The device of claim 1, wherein the second passage is in fluid communication with a reservoir of a medium.

10. The device of claim 1, wherein the at least one seal element is formed and positioned to substantially encircle the first opening.

11. The device of claim 10, wherein the at least one seal element comprises an ohmic element.

12. The device of claim 1, wherein the body portion of the second substrate further defines an intersection portion where the first passage and the first opening are in fluid communication, and wherein the intersection portion is at least partially formed as a cone shaped portion.

13. A device for monitoring status of a plurality of cells, wherein each cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein, comprising: a. a first substrate having a first surface and an opposite second surface; b. a second substrate supported by the first substrate, the second substrate having a first surface, an opposite second surface, a body portion between the first surface and the second surface, a first side surface and an opposite second side surface, wherein the body portion defines a first passage therein between the first side surface and the second side surface and a plurality of first openings distributed on and over the first surface of the second substrate, wherein each of the plurality of first openings is in fluid communication with the first passage; c. a third substrate positioned over the second substrate, having a first surface and an opposite second surface, and spaced apart from the second substrate thereby defining a space between the second surface of the third substrate and the first surface of the second substrate; d. a plurality of sidewalls positioned between the second substrate and the third substrate thereby partitioning the space between the second substrate and the third substrate into a plurality of chambers above the first surface of the second substrate such that only one of the first openings distributed on and over the first surface of the second substrate is located between the sidewalls of a corresponding chamber, wherein each chamber is in fluid communication with at least one neighboring chamber through a second passage defined over the sidewalls and under the second surface of the third substrate; e. a plurality of sensors positioned in the first passage, each sensor being proximate to a corresponding one of the first openings distributed on and over the first surface of the second substrate, wherein each cell is positioned in a corresponding one of the chambers and the intracellular space of each cell is in fluid communication with the first passage through the first opening located between the sidewalls of a corresponding chamber; f. a plurality of seal elements positioned on the second substrate for sealing a corresponding cell to the second substrate in operation, wherein each seal element is proximate to a corresponding one of the plurality of first openings; g. a plurality of first controls positioned inside the first passage of the second substrate, wherein each chamber has a pair of corresponding first controls for controlling flow of the medium through portions of the first passage that correspond to that chamber; and h. a plurality of second controls, wherein each second control is positioned inside a corresponding second passage for controlling the flow of a medium through that second passage.

14. The device of claim 13, wherein the membrane of each cell defines a second opening, through which the intracellular space of the cell is in fluid communication with the first passage through the first opening located between the sidewalls of a corresponding chamber.

15. The device of claim 14, further comprising a plurality of punching elements, each positioned underneath the first opening located between the sidewalls of a corresponding chamber for making the second opening defined by the membrane of a corresponding cell.

16. The device of claim 15, wherein each punching element comprises an electroporation device.

17. The device of claim 13, wherein when a first medium is introduced into some portion of the first passage and the intracellular space of a cell that is in a chamber corresponding to that portion of the first passage is in fluid communication with the first passage, a corresponding sensor measures the response of the cell to the first medium.

18. The device of claim 13, wherein when a second medium is introduced into a chamber and at least part of the membrane of a corresponding cell in the chamber is in contact with the second medium, a corresponding sensor measures the response of the cell to the second medium.

19. The device of claim 13, wherein when a first medium is introduced into some portion of the first passage and a second medium is introduced into a chamber corresponding to that respective portion of the first passage, respectively, the intracellular space of a corresponding cell in the chamber is in fluid communication with the first passage having the second medium, and at least part of the membrane of the corresponding cell is in contact with the second medium, a corresponding sensor measures the responses of the cell to the first medium and the second medium.

20. The device of claim 13, wherein all of the plurality of sensors are sensors of substantially the same type.

21. The device of claim 13, wherein at least two of the plurality of sensors are different from each other.

22. The device of claim 13, wherein at least one chamber is in fluid communication with a reservoir of a medium through a corresponding second passage.

23. The device of claim 13, wherein the first passage is in fluid communication with a reservoir of a medium.

24. The device of claim 13, wherein each seal element is formed and positioned to substantially encircle its corresponding first opening.

25. The device of claim 24, wherein each seal element comprises an ohmic element.

26. The device of claim 13, wherein all of the plurality of seal elements are seal elements of substantially the same type or there are at least two different types of seal elements in the plurality of sensors.

27. The device of claim 13, wherein the body portion of the second substrate further defines a plurality of intersection portions where the first passage and the plurality of first openings are in fluid communication, respectively, and wherein each intersection portion is at least partially formed as a cone shaped portion.

Description

This application is being filed as a PCT international patent application in the name of Vanderbilt University, a U.S. institution (applicant for all designations except the U.S.), and John P. Wikswo, a U.S. citizen and resident (applicant for the U.S. designation), on 6 Aug. 2002, designating all countries.

Some references, which may include patents, patent applications and various publications, are cited and discussed in the description of this invention. The citation and/or discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any such reference is "prior art" to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety and to the same extent as if each reference was individually incorporated by reference.

FIELD OF THE INVENTION

The present invention generally relates to an apparatus and methods for using biological material to discriminate an agent. More particularly, the present invention relates to an apparatus and methods that utilize a matrix of biological signatures. In one embodiment, the matrix has a plurality of elements and a dimension of N.times.M, where N is the total number of the plurality of cells and M is the total number of the plurality of measurable quantities. Thus, the matrix has in total N.times.M elements, where each element represents a biological signature of one of a plurality of cells in response to an agent, and each biological signature is one of a plurality of measurable quantities. The present invention comprises a method that includes the steps of constructing such a matrix of biological signatures, exposing at least one of the plurality of cells to an agent, measuring the measurable quantities of the at least one of the plurality of cells responsive to the agent, comparing the measured measurable quantities of the at least one of the plurality of cells responsive to the agent with the corresponding biological signatures of the matrix of biological signatures, and identifying the agent from the comparison. The measured measurable quantities can be stored for further processing, analyzing, feed-backing, or the like.

The invention also relates to an apparatus for using biological material to discriminate an agent. In one embodiment, the apparatus includes means for constructing a matrix of biological signatures having a plurality of elements, wherein each element represents a biological signature of one of a plurality of cells in response to an agent, each biological signature being one of a plurality of measurable quantities, and wherein the matrix has a dimension of N.times.M, N being the total number of the plurality of cells and M being the total number of the plurality of measurable quantities; means for exposing at least one of the plurality of cells to an agent. The apparatus further includes means for measuring the measurable quantities of the at least one of the plurality of cells responsive to the agent, means for comparing the measured measurable quantities of the at least one of the plurality of cells responsive to the agent with the corresponding biological signatures of the matrix of biological signatures, and means for identifying the agent from the comparison.

Certain embodiments of the present invention comprise apparatus and methods for monitoring the status of a cell that is metabolically active, wherein each metabolic activity of the cell is characterized by a characterization time. More particularly, the apparatus and methods comprise means and the step for measuring at least one metabolic activity of the cell at a time period shorter than a characterization time corresponding to the measured metabolic activity of the cell, respectively.

Certain other embodiments of the present invention comprise devices and methods for detecting the response of a plurality of cells to at least one analyte of interest. More particularly, the devices and methods comprise means and the steps for contacting the plurality of cells with a plurality of analytes of interest and simultaneously detecting the response of the plurality of cells to the plurality of analytes of interest, respectively.

Certain further embodiments of the present invention comprise devices and methods for device for monitoring status of at least one cell, wherein the cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein. More particularly, the devices and methods comprise means and the steps for providing a medium into the intracellular space of the cell and measuring the response of the cell to the medium, respectively.

Certain other embodiments of the present invention comprise devices and methods for measuring response of at least one cell to a medium, the response of at least one cell to a medium being characterized by a reaction time. More particularly, a device of the present invention comprises a sensor that measures the response of the cell to the medium at a time period shorter than the reaction time.

Certain additional embodiments of the present invention comprise devices and methods for discriminating an agent. More particularly, the devices and methods comprise means and the steps for constructing a decision tree having a plurality of branches, each branch corresponding to at least one defined action, wherein each branch comprises a plurality of successive branches, each successive branch corresponding to at least one defined action, providing a conditioned environment sensitive to the agent, obtaining data from response of the agent to the conditioned environment, extracting features from the obtained data, selecting a branch from the decision tree corresponding to the features, performing on the features at least one defined action corresponding to the branch, producing a classification of the agent, and iteratively repeating any or all steps until the agent is discriminated, respectively.

Certain further embodiments of the present invention comprise devices and methods for discriminating an agent. More particularly, the devices and methods comprise means and the steps for providing a plurality of L parameters, L being an integer, each parameter being related to the status of the agent, fitting the plurality of L parameters into a set of ith order differential equations, i=1, . . . , N, N being an integer, obtaining a plurality of L features corresponding to L parameters, respectively, from the set of ith order differential equations, separating the L features into a plurality of classes with a corresponding confidence level, providing a plurality of L+1 parameters, each parameter being related to the status of the agent, fitting the plurality of L+1 parameters into a set of ith+1 order differential equations, obtaining a plurality of L+1 features corresponding to L+1 parameters, respectively, from the set of ith+1 order differential equations, separating the L+1 features into a plurality of classes with a corresponding confidence level, and iteratively repeating any or all steps until a plurality of classes for the agent is separated with a desired corresponding confidence level, respectively.

Certain other embodiments of the present invention comprise devices and methods for discriminating an agent. More particularly, the devices and methods comprise means and the steps for providing a broad spectrum assay having a plurality of L cell lines, L being an integer, each cell line being able to respond to the agent, measuring responses of cell line i, i=1, . . . , L, to the agent, separating the responses into class m, m=1, . . . , O, O being an integer and the total number of available classes, with a corresponding robustness factor, selecting cell line j, j=1, . . . , L but .noteq.i, from the knowledge of class m, measuring responses of cell line j, j=1, . . . , L but .noteq.i, to the agent, defining a set of feature extraction algorithms from the measured response of cell line j, j=1, . . . , L but .noteq.i, selecting cell line k, k=1, . . . , L but .noteq.i and .noteq.j, measuring responses of cell line k, k=1, . . . , L but .noteq.i and .noteq.j, to the agent, separating the responses into class n, n=1, . . . , O, O being an integer and the total number of available classes, with a corresponding robustness factor, and iteratively repeating any or all steps until a class for the agent with a desired robustness factor is obtained, respectively.

BACKGROUND OF THE INVENTION

The biological cell may act as a parallel processing, non-linear, multistate, analog computer. This analog computer can occupy a volume of less than 10.sup.-16 m.sup.3 and is primarily powered only by sugars, fats, and oxygen. The complexity of these computers is evidenced by the attempts to model ongoing biochemical processes based on Mycoplasma genitalium, a microbe with the smallest known gene set of any self-replicating organism (http:\\www.e-cell.org). However, even this simplest model requires hundreds of variables and reaction rules, and a complete model even for a mammalian cell would be much more complex, requiring in excess of 10.sup.5 variables and equations.

Because the cell behaves as an analog computer, it can be programmed. Historically, a limited set of interventions has allowed physiologists and engineers to study living cells and characterize the feedback control systems that govern cell function. With the advent of genetic engineering, it is now possible to reprogram the genetic machinery of a cell, for example to turn a particular gene on or off, or to produce large quantities of a particular biochemical. However, there has been little efforts and progress for inserting man-made devices into the control system of a single living cell so as to convert the cell into a programmable computational engine.

Therefore, among other things, there is a need to merge cellular biophysics, microcircuits and microfluidics, and information technology to create, among other things, programmable microsystems that can be used for sensing, feedback, control and analysis of a single cell and/or an array of interconnected and instrumented living cells.

Additionally, current bio-sensors use biological molecules for specific agent detection via specific binding reactions. However, wide-spectrum detection is expensive, requiring a priori threat knowledge and a large quantity of specific cells. Assays are susceptible to overload from multiple threats and false detection and from non-pathogenic "spoof" organisms. Furthermore, addressing new threats involves a lengthy, costly design process. In addition, conventional assays lack cellular machinery to increase sensitivity.

Therefore, among other things, there is also a need to develop new systems and methods that are capable of providing a complete bio-functional signature of a CBW agent, environmental contaminant, unknown drug, or other threats for better, fast, sensitive accurate and efficient detection.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a device for monitoring status of at least one cell, wherein the cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein. In one embodiment, the device includes a first substrate having a first surface and an opposite second surface, a second substrate supported by the first substrate, the second substrate having a first surface, an opposite second surface, a body portion between the first surface and the second surface, a first side surface and an opposite second side surface, wherein the body portion defines a first passage between the first side surface and the second side surface and an opening on the first surface of the second substrate and in fluid communication with the first passage, and sidewalls positioned above the first surface of the second substrate.

The device also includes a third substrate having a first surface and an opposite second surface. The third substrate, the sidewalls and the second substrate define a chamber that is in fluid communication with a second passage defined by portions of the sidewalls and the third substrate. The device further includes at least one sensor positioned in the first passage proximate to the opening, wherein the cell is positioned in the chamber and the intracellular space of the cell is in fluid communication with the first passage through the opening of the second substrate.

The membrane of the cell defines an opening through which the intracellular space of the cell is in fluid communication with the first passage through the opening of the second substrate. The device further includes a punching element positioned underneath the opening of the second substrate for making the opening defined by the membrane of the cell. The punching element can be a mechanical device such as a pressure-based suction device or an electroporation device such as an electric potential sucking device.

In one operation mode, when a first medium is introduced into the first passage, the intracellular space of the cell is in fluid communication with the first passage with the first medium, the sensor measures the response of the cell to the first medium.

In another operation mode, when a second medium is introduced into the chamber through the second passage, at least part of the membrane of the cell is in contact with the second medium in the chamber, the sensor measures the response of the cell to the second medium.

In yet another operation mode, when a first medium is introduced into the first passage and a second medium is introduced into the chamber through the second passage, respectively, the intracellular space of the cell is in fluid communication with the first passage with the first medium and at least part of the membrane of the cell is in contact with the second medium in the chamber, the sensor measures the responses of the cell to the first medium and the second medium.

The first passage is in fluid communication with a reservoir of a medium. The device further includes a pair of first controls positioned inside the first passage for controlling the flow of a medium through the first passage.

The device second passage is in fluid communication with a reservoir of a medium. The device also includes a second control positioned inside the second passage for controlling the flow of a medium through the second passage.

In another aspect, the present invention relates to a device for monitoring status of a plurality of cells, wherein each cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein. In one embodiment, the device includes a first substrate having a first surface and an opposite second surface, a second substrate supported by the first substrate, the second substrate having a first surface, an opposite second surface, a body portion between the first surface and the second surface, a first side surface and an opposite second side surface, wherein the body portion defines a first passage between the first side surface and the second side surface and a plurality of openings distributed on and over the first surface, each opening being in fluid communication with the first passage, and a third substrate having a first surface and an opposite second surface and spaced apart from the second substrate thereby defining a space between the second surface of the third substrate and the first surface of the second substrate.

The device also includes a plurality of sidewalls positioned between the second substrate and the third substrate thereby partitioning the space between the second substrate and the third substrate into a plurality of chambers above the first surface of the second substrate such that only one of openings distributed on and over the first surface is located between the sidewalls of a corresponding chamber, wherein each chamber is in fluid communication with at least one neighboring chamber through a second passage defined by portions of the corresponding sidewalls and the third substrate. The device further includes a plurality of sensors positioned in the first passage, each sensor being proximate to a corresponding one of openings distributed on and over the first surface of the second substrate. The plurality of sensors can be substantially the same. Or, alternatively, at least two of the plurality of sensors can be different from each other.

Each cell is positioned in a corresponding one of the chambers and the intracellular space of each cell is in fluid communication with the first passage through the opening located between the sidewalls of a corresponding chamber. The membrane of each cell defines an opening through which the intracellular space of the cell is in fluid communication with the first passage through the opening located between the sidewalls of a corresponding chamber. The device further includes a plurality of punching elements, each positioned underneath an opening located between the sidewalls of a corresponding chamber for making the opening defined by the membrane of a corresponding cell. Each punching element can be a mechanical device such as a pressure-based suction device or an electroporation device such as an electric potential sucking device. Punching elements can be same or different.

In one operation mode, when a first medium is introduced into some portion of the first passage, the intracellular space of a cell that is in a chamber corresponding to that portion of the first passage is in fluid communication with the first passage with the first medium, a corresponding sensor measures the response of the cell to the first medium.

In another operation mode, when a second medium is introduced into a chamber, at least part of the membrane of a corresponding cell in the chamber is in contact with the second medium, a corresponding sensor measures the response of the cell to the second medium.

In yet another operation mode, when a first medium is introduced into some portion of the first passage and a second medium is introduced into a chamber corresponding to that portion of the first passage, respectively, the intracellular space of a corresponding cell in the chamber is in fluid communication with the first passage with the first medium and at least part of the membrane of the corresponding cell is in contact with the second medium, a corresponding sensor measures the responses of the cell to the first medium and the second medium.

The first passage is in fluid communication with a reservoir of a medium. The device further includes a plurality of first controls positioned inside the first passage for controlling the flow of a medium through the first passage, wherein for each chamber, a corresponding pair of the first controls controls the flow of the medium through portions of the first passage under a corresponding chamber.

At least one chamber is in fluid communication with a reservoir of a medium through a second passage. The device also includes a plurality of second controls, each positioned inside a corresponding second passage for controlling the flow of a medium through that second passage.

In a further aspect, the present invention relates to a method for monitoring the status of at least one cell, wherein the cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein. In one embodiment, the method includes the steps of confining the cell in a chamber, making an opening in the membrane of the cell, providing a first medium into the intracellular space of the cell through the opening in the membrane, and measuring the response of the cell to the first medium. The method further includes the steps of providing a second medium into the chamber such that at least part of the membrane of the cell is in contact with the second medium and measuring the response of the cell to the second medium.

In yet another aspect, the present invention relates to a device for monitoring the status of at least one cell, wherein the cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein. In one embodiment, the device includes means for confining the cell in a chamber, means for making an opening in the membrane of the cell, means for providing a first medium into the intracellular space of the cell through the opening in the membrane, and means for measuring the response of the cell to the first medium. The device further includes means for providing a second medium into the chamber such that at least part of the membrane of the cell is in contact with the second medium and means for measuring the response of the cell to the second medium.

In another aspect, the present invention relates to a method for monitoring the status of at least one cell, wherein the cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein. In one embodiment, the method includes the steps of confining the cell in a chamber, making an opening in the membrane of the cell, providing a first medium into the intracellular space of the cell through the opening in the membrane, providing a second medium into the chamber such that at least part of the membrane of the cell is in contact with the second medium and measuring the response of the cell to the second medium. The method further includes the step of measuring the response of the cell to the first medium.

In yet another aspect, the present invention relates to a device for monitoring the status of at least one cell, wherein the cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein. In one embodiment, the device includes means for confining the cell in a chamber, means for making an opening in the membrane of the cell, means for providing a first medium into the intracellular space of the cell through the opening in the membrane, means for providing a second medium into the chamber such that at least part of the membrane of the cell is in contact with the second medium and means for measuring the response of the cell to the second medium. The device further includes means for measuring the response of the cell to the first medium.

In a further aspect, the present invention relates to a method for controlling the physiological status of at least one cell, wherein the cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein and controls its physiological status through an internal cellular control mechanism. In one embodiment, the method includes the step of providing at least one medium to the cell such that at least part of the membrane of the cell is in contact with the medium to override the internal cellular control mechanism. The medium may have an agent.

In one operation mode, the method further includes the steps of confining the cell in a chamber and making an opening in the membrane of the cell. The providing step further comprises the steps of supplying a first medium into the intracellular space of the cell through the opening in the membrane, and supplying a second medium into the chamber such that at least part of the membrane of the cell is in contact with the second medium. The method further includes the steps of measuring the response of the cell to the second medium, and adjusting the composition of the second medium from the response to affect the overriding of the internal cellular control mechanism. Moreover, the method further includes the steps of measuring the response of the cell to the first medium, and adjusting the composition of the first medium from the response to affect the overriding of the internal cellular control mechanism.

In another operation mode, the method further includes the steps of monitoring the concentration of at least one selected component of the medium and adjusting the composition of the medium from the monitored concentration of at least one selected component of the medium to affect the overriding of the internal cellular control mechanism. Additionally, the method further includes the steps of s measuring the response of the cell to the medium, and adjusting the composition of the medium from the response to affect the overriding of the internal cellular control mechanism.

In yet another aspect, the present invention relates to a device for controlling the physiological status of at least one cell, wherein the cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein and controls its physiological status through an internal cellular control mechanism. In one embodiment, the device includes means for providing at least one medium to the cell such that at least part of the membrane of the cell is in contact with the medium to override the internal cellular control mechanism.

The device further includes means for confining the cell in a chamber and means for making an opening in the membrane of the cell. In one embodiment, the providing means includes means for supplying a first medium into the intracellular space of the cell through the opening in the membrane and means for supplying a second medium into the chamber such that at least part of the membrane of the cell is in contact with the second medium. The device further includes means for measuring the response of the cell to the second medium and means for adjusting the composition of the second medium from the response to affect the overriding of the internal cellular control mechanism. Moreover, the device further includes means for measuring the response of the cell to the first medium and means for adjusting the composition of the first medium from the response to affect the overriding of the internal cellular control mechanism.

Additionally, the device includes means for monitoring the concentration of at least one selected component of the medium and means for adjusting the composition of the medium from the monitored concentration of at least one selected component of the medium to affect the overriding of the internal cellular control mechanism. The device further includes means for measuring the response of the cell to the medium and means for adjusting the composition of the medium from the response to affect the overriding of the internal cellular control mechanism.

In a further aspect, the present invention relates to a method for controlling the physiological status of at least one cell, wherein the cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein and controls its physiological status through an internal cellular control mechanism. In one embodiment, the method includes the steps of providing at least one medium to the cell such that at least part of the membrane of the cell is in contact with the medium, monitoring at least one selected component of the medium, and adjusting the composition of the medium from the monitored concentration of at least one selected component of the medium to deliver or remove analytes to the intracellular space through the membrane to affect the internal cellular control mechanism.

In another aspect, the present invention relates to a device for controlling the physiological status of at least one cell, wherein the cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein and controls its physiological status through an internal cellular control mechanism. In one embodiment, the device includes means for providing at least one medium to the cell such that at least part of the membrane of the cell is in contact with the medium, means for monitoring at least one selected component of the medium, means for adjusting the composition of the medium from the monitored concentration of at least one selected component of the medium to deliver or remove analytes to the intracellular space through the membrane to affect the internal cellular control mechanism.

These and other aspects will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows a multicellular bio-silicon hybrid microsystem according to one embodiment of the present invention.

FIGS. 2A-2B show a PicoCalorimeter or a device according to one embodiment of the present invention: A. side view and B. top view.

FIGS. 3A-3C show a Microbottle or a device according to one embodiment of the present invention: A. side view; B. top view; and C. sectional view along line A-A in FIG. 3A.

FIGS. 4A-4C show a Microbottle or a device according to another embodiment of the present invention: A. side view; B. top view (with lid removed); and C. sectional view along line A-A in FIG. 4A.

FIGS. 5A-5C show a Microbottle or a device according to yet another embodiment of the present invention: A. side view; B. top view; and C. sectional view along line A-A in FIG. 5A.

FIGS. 6A-6D show a Picocalorimeter or a device according to one embodiment of the present invention: A. side cross-sectional view along line D-D in FIG. 6C; B. side cross-sectional view along line C-C in FIG. 6C; C. cross-sectional view along line A-A in FIGS. 6A and 6B; and D. cross-sectional view along line B-B in FIGS. 6A and 6B.

FIGS. 7A-7C show a physiometer or a device according to one embodiment of the present invention: A. side sectional view; B. cross-sectional view along line A-A in FIG. 7A; and C. cross-sectional view along line B-B in FIG. 7B.

FIG. 8 illustrates an integrated bio-silicon-hybrid system design environment according to one embodiment of the invention.

FIG. 9 shows a bio-functional signature matrix according to one embodiment of the present invention.

FIG. 9A schematically shows a bio-functional signature matrix of FIG. 9 in another form according to one embodiment of the present invention.

FIG. 10 shows data of parathion (open symbols) and paraoxon (filled symbols) on metabolic activity of human hepatocyte and neuroblastoma cells according to one embodiment of the present invention.

FIGS. 11A-11C schematically show a sensor head for multispectral readout according to one embodiment of the present invention: A. side sectional view; B. bottom view; and C. perspective view.

FIGS. 12A-12C schematically show a Nanophysiometer or a device according to one embodiment of the present invention: A. side cross-sectional view along line A-A in FIG. 12B; and B. top view; and C. exploded view of part B in FIG. 12A.

FIGS. 13A-13C schematically show a Nanophysiometer or a device according to another embodiment of the present invention: A. side view; and B. cross-sectional view along line A-A in FIG. 13A; and C. enlargement view of part B in FIG. 13B.

FIG. 14 schematically shows an optical setup for fluorescence measurements associated with a Nanophysiometer according to one embodiment of the present invention.

FIG. 15 schematically shows response of optical beacons to a binding event as a means to identify the expression of particular mRNA in response to toxins and agents according to one embodiment of the present invention.

FIG. 16 illustrates an example of cellular pathways that can be monitored according to one embodiment of the invention.

FIGS. 17A-17B illustrate an example of toxin discrimination by simultaneous monitoring of multiple metabolic signals following the exposure of cells to some toxins according to one embodiment of the invention: A. to DNP; and B. to Cyanide.

FIGS. 18A-18B show discrimination of toxins/agents by monitoring characteristic temporal response of cellular phenotypes to toxins, according to one embodiment of the present invention: A. for Macrophage; and B. for Hepatocyte.

FIGS. 19A-19B schematically show discrimination by characteristic responses in a conditioned environment according to one embodiment of the present invention: A. no phenobarbital preexposure; and B. with phenobarbital preexposure.

FIG. 20 shows discrimination by characteristic reaction kinetics of metabolic pathways according to one embodiment of the present invention.

FIG. 21 shows the effect of soman on an action potential of a neuron according to one embodiment of the present invention.

FIG. 22 is a flowchart illustrating a Process to define a differential discrimination process according to one embodiment of the invention.

FIG. 23 illustrates two signal classification algorithms s according to one embodiment of the invention.

FIG. 24 schematically shows a diagnostics path or process according to one embodiment of the present invention.

FIGS. 25A-25B show a Picocalorimeter or a device according to another embodiment of the present invention: A. side cross-sectional view along line A-A in FIG. 25B; and B. tilted view from the bottom.

FIGS. 26A-26B show an iridium oxide pH electrode forming on a platinum interdigitated microelectrode array according to one embodiment of the present invention: A. a photomicrograph of the electrode array with platinum, iridium oxide, and platinum microstrips on a glass substrate; B. a pH calibration of the sensor.

FIGS. 27A-27B show a Nanophysiometer or a device according to one embodiment of the present invention: A. side cross-sectional view; and B. cross-sectional view along line A-A in FIG. 27A.

FIGS. 28A-28C show a Nanophysiometer or a device according to another embodiment of the present invention: A. top view; and B. exploded of part A in FIG. 28A; and C. cross-sectional view along line B-B in FIG. 28B.

FIGS. 29A-29C shows a Nanophysiometer or a device according to yet another embodiment of the present invention: A. top view; and B. exploded of part A in FIG. 29A; and C. cross-sectional view along line B-B in FIG. 29B.

FIG. 30 shows a Nanophysiometer or a device according to a further embodiment of the present invention in a top view.

FIGS. 31A-31E illustrate the utilization of NanoPhysiometer electrochemical sensors and their temporal response to changes in pH and oxygen according to one embodiment of the present invention: A. the average pH as a function of time in a 100 pL well containing a single cell with no flow; B. same as FIG. 31A, except plotted as a function of logarithmic time to show that the response is constant until the protons have time to diffuse from the cell to the electrode; C. the time taking for the pH to drop by a certain amount; D. the results of the test of the Nanophysiometer with a platinum interdigitated array that senses oxygen; and E. an individually addressable interdigitated microelectrode array.

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments of the invention are now described in detail. Referring to the drawings, like numbers indicate like parts throughout the views. As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein and throughout the claims that follow, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise. Additionally, some terms used in this specification are more specifically defined below.

Definitions

The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. For example, conventional techniques of molecular biology, microbiology and recombinant DNA techniques may be employed in accordance with the present invention. Such techniques and the meanings of terms associated therewith are explained fully in the literature. See, for example, Sambrook, Fitsch & Maniatis. Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (referred to herein as "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins, eds. 1984); Animal Cell Culture (R. I. Freshney, ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. E. Perbal, A Practical Guide to Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994). See also, PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, Inc., New York (1990); Saiki et al., Science 1988, 239: 487; and PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, Ed., Stockton Press.

Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the devices and methods of the invention and how to make and use them. For convenience, certain terms are highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term is the same, in the same context, whether or not it is highlighted. It will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.

As used herein, "about" or "approximately" shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term "about" or "approximately" can be inferred if not expressly stated.

The term "agent" is broadly defined as anything that may have an impact on any living system such as a cell. For examples, the agent can be a chemical agent. The chemical agent may comprise a toxin. The agent can also be a biological agent. Moreover, the agent may comprise at least one unknown component, which may be identified by practicing the present invention. Additionally, the agent may comprise at least one known component, whose interaction with cells or other components of an environment may be detected by practicing the present invention. The agent can also be a physical agent. Other examples of agent include biological warfare agents, chemical warfare agents, bacterial agents, viral agents, other pathogenic microorganisms, emerging or engineered threat agents, acutely toxic industrial chemicals ("TICS"), toxic industrial materials ("TIMS") and the like. Examples of chemical agents that may be related to practicing the present invention include Mustard (that may be simulated with chloroethyl ethyl sulphide (endothelia cells in PC)), GB-Sarin (that may be simulated with Disopropylfluorophosphate (DFP)), VX (that may be simulated with Malathion) or the like. Examples of viral agents (and their simulants) that may be related to practicing the present invention include MS2, Hepatitus or simulant or attenuated virus, Retroviruses alphaviruses or the like. Examples of bacterial agents (and their simulants) that may be related to practicing the present invention include Bacillus globigii or Bacillus subtilis as spore formers similar to anthrax, Erwinia herbicola as a simulant for vegetative bacteria (not sporagenic), E. coli or the like. Additional examples of agents can also be found in the following exemplary list of agents: Botulinum Toxin (seven immunological types: A, B, C1, C2, D, E, F, G) Staphylococcus enterotoxin B Saxitoxin Ricin (Ricinus communis) Epsilon toxin of Clostridium perfringens Mycotoxins Aflatoxins that inhibit DNA and RNA synthesis Anatoxin A Microcystins Cholera Toxin Tetrodotoxin Substance P Bacillus anthracis (Anthrax) Yersinia Pestis, (gram-negative coccobacillus causing the zoonotic infection Plague) Clostridium botulinum Francisella tularensis (a gram-negative, facultative intracellular bacterium that causes the zoonosis Tularemia) Brucella spp (spp=several different species?) Burkholderia mallei (Glanders) Burkholderia pseudomallei Chlamydia psittaci Shigella dysenteriae Salmonella spp Vibrio cholerae Cryptosporidium parvum Clostridium perfringens Hepatitis C Variola major (smallpox) Filoviruses/Arenaviruses Alphaviruses Cephalomyelitis Viruses Nipah Virus (a new paramyxovirus) Hantavirus Tick-borne hemorrhagic fevers Dengue (Breakbone or Dandy Fever) fever virus Enteric Viruses Hepatcytes and Hepatitis A Lymphocytes Erythrocytes Endothelial cells HL1 (Cardiac) Secretory cell (depolarize and it secretes things) Beta=insulin PC12 neural cells HELA (Helen Lane) HEK293 Human Epithial Kidney cells Coxiella burnetti Ricksettia prowazekii VX, V-gas G-series (GF-cyclohexyl sarin, GD-Soman, GB-Sarin, GA-Tabun) Mustard Agents HN-1-Nitrogen Mustard HN-2-Nitrogen Mustard (N-Oxide Hydrochloride) Sulfar Mustard Adamsite Arsines Lewisite Hydrogen Cyanide Cyanogen Chloride BZ (Benzphetamine) LSD (Lysergic Acid Diethylamide) (enable comment for this) Chlorine Phosgene CN (2-Chloroacetophenone) Fuel & Combustion Products (Jet Fuels) JP-4 JP-8 TMPP Herbicides/Pesticides Methyl Parathion (an organophosphorus insecticide) Volatile Organic Carbons (VOC) Benzene Toluene (methylbenzene) Xylene Heavy Metals Lead Chromium Mercury Halogens Fluorine Bromine Cyanides Isocyanates cyanides (as CN) Hydrogen Chloride Sulfur Dioxide Oxides of Nitrogen (NOx) Vinyl Chloride Barium Nitrate Hydrazine DBNP-di-tris-butyl-nitrophenol.

The term "toxin" is broadly defined as any agent that may have a harmful effect or harmful effects on any living system such as a cell. Examples of toxins that may be related to practicing the present invention include cyanide, endotoxin, okadaic acid, Phorbol Myristate Acetate ("PMA"), microcystin, Dinitrophenol ("DNP"), Botulinum toxin (a common threat agent; inhibit transmitter release, whole cell MB), Staphylococcus enterotoxin B, ricin (inhibits protein synthesis and ribosmone, OT), mycotoxins, aflatoxins, cholera toxin (activates Cl pump, vesicle MB, NBR), Saxatoxin or tetrodotoxin (Na channel blocker, vesicle MB), Microcystins (hepatocyte metabolism in PC) and organophosphates. Other examples of toxins may be also discussed somewhere else in the specification.

Additional examples of toxins can also be found in the market. For example, the following is an exemplary list of toxins with their corresponding product number that are readily available from a commercial source at gotnet.com:

TABLE-US-00001 PRODUCT PRODUCT DESCRIPTION NUMBER Adenylate Cyclase Toxin from Bordetella pertussis 188 Alpha Toxin from Staphylococcus aureus 120 Anthrax Lethal Factor (LF), Recombinant from Bacillus anthracis 171 Anthrax Protective Antigen (PA), Recombinant from Bacillus anthracis 172 Anti-Choleragenoid, Goat Antibody for Cholera Toxin B Subunit 703 Anti-Exotoxin A, Goat Antibody for Exotoxin A from Pseudomonas aeruginosa 760 Anti-Toxin A, Goat Antibody for Toxin A from Clostridium difficile 752 Anti-VACh Transporter Saporin Conjugate 770 Biotin, Cholera Toxin B Subunit Conjugated 112 Bordetella pertussis, Adenylate Cyclase Toxin 188 Bordetella pertussis, Filamentous Hemagglutinin 170 Bordetella pertussis, Pertussis Toxin, Liquid in Glycerol Buffer 179A Bordetella pertussis, Pertussis Toxin, Lyophilized in Buffer 180 Bordetella pertussis, Pertussis Toxin, Lyophilized, Salt Free 181 Bordetella pertussis, Pertussis Toxin A Protomer 182 Bordetella pertussis, Pertussis Toxin B Oligomer 183 Botulinum Neurotoxin Type A from Clostridium botulinum 130A Botulinum Neurotoxin Type A Heavy Chain 132 Botulinum Neurotoxin Type A Light Chain 131 Botulinum Neurotoxin Type A Toxoid 133 Botulinum Neurotoxin Type B from Clostridium botulinum 136A Botulinum Neurotoxin Type B Heavy Chain 138 Botulinum Neurotoxin Type B Light Chain 137 Botulinum Neurotoxin Type B Toxoid 139 Cholera Toxin, Azide Free 100 Cholera Toxin from Vibrio cholerae 101 Cholera Toxin A Subunit 102 Cholera Toxin B Subunit 103 Cholera Toxin B Subunit, Low Salt 104 Cholera Toxin B Subunit Conjugated to Fluorescein Isothiocyanate 106 Cholera Toxin B Subunit Conjugated to Horseradish Peroxidase 105 Cholera Toxin B Subunit Conjugated to Tetramethylrhodamine B 107 Isothiocyanate Cholera Toxin B Subunit Conjugated to Phycoerythrin 109 Cholera Toxin B Subunit Conjugated to Biotin 112 Cholera Toxin B Subunit, Recombinant 114 Clostridium botulinum, Botulinum Neurotoxin Type A 130A Clostridium botulinum, Botulinum Neurotoxin Type A Heavy Chain 132 Clostridium botulinum, Botulinum Neurotoxin Type A Light Chain 131 Clostridium botulinum, Botulinum Neurotoxin Type A Toxoid 133 Clostridium botulinum, Botulinum Neurotoxin Type B 136A Clostridium botulinum, Botulinum Neurotoxin Type B Heavy Chain 138 Clostridium botulinum, Botulinum Neurotoxin Type B Light Chain 137 Clostridium botulinum, Botulinum Neurotoxin Type B Toxoid 139 Clostridium botulinum, Exoenzyme C3 143 Clostridium difficile, Anti-Toxin A, Goat Antibody for Toxin A from Clostridium difficile 752 Clostridium difficile, Toxin A 152 Clostridium difficile, Toxin A Toxoid 153 Clostridium difficile, Toxin B 155 Clostridium tetani, Tetanolysin 199 Clostridium tetani, Tetanus Toxin 190 Clostridium tetani, Tetanus Toxin C-Fragment 193 Clostridium tetani, Tetanus Toxoid 191 Corynebacterium diphtheriae, Diphtheria Toxin CRM Mutant 149 Corynebacterium diphtheriae, Diphtheria Toxin, Unnicked 150 Corynebacterium diphtheriae, Diphtheria Toxoid 151 Diphtheria Toxin CRM Mutant 149 Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae 150 Diphtheria Toxoid 151 Enterotoxin Type B from Staphylococcus aureus 122 Escherichia coli J5 (Rc), Lipopolysaccharide 301 Escherichia coli K12, D31m4, Primarily Diphosphoryl Lipid A 402 Escherichia coli K12, D31m4 (Re), Lipopolysaccharide 302 Escherichia coli K12 strain LCD25, [.sup.3H]Lipopolysaccharide 510 Escherichia coli K12 strain LCD25, Lipopolysaccharide 314 Escherichia coli O111:B4, Lipopolysaccharide 201 Escherichia coli O55:B5, Lipopolysaccharide 203 Escherichia coli, Stable Toxin 118 Exoenzyme C3 from Clostridium botulinum 143 Exotoxin A fro