# Patent

## Title
System for rapid chemical activation in high-throughput electrophysiological measurements

## Abstract

An ultraviolet light source is added to a high-throughput electrophysiogical measurement system to allow for rapid chemical stimulation via caged compound release to a plurality of measurement samples. The integrated electrophysiogical measurement system includes a computer-controlled data collection system, an integrated electronics head for making parallel electrical measurements, and an integrated fluidics head used in part to transfer test compounds into the measurement process. This light source, and associated light coupling to a plurality of test samples, is used in conjunction with the system to make effectuate high-throughput electrical measurements with respect to fast-acting, chemically-activated electrophysiological events. The UV-source modification allows for rapid stimulation and measurement of multiple fast ligand-gated ion channel events in parallel.

## Background
REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of U.S. patent application Ser. No. 09/631,909 filed Aug. 4, 2000, now U.S. Pat. No. 6,488,829, entire contents of which are incorporated herein by reference.


FIELD OF INVENTION
The present invention relates generally to the field of electrophysiology, wherein electrical measurements are made on biological cells and cell membranes to understand interactions between specific membrane components such as ion channels and transporters. More particularly, the invention relates to apparatus and methods for rapidly introducing an activating chemical agent into multiple measurement wells in parallel, thereby enabling the study of fast ligand-gated electrophysiological events in a high-throughput manner.
BACKGROUND OF INVENTION
The electrical behavior of cells and cell membranes is of profound importance in basic research as well as in modem drug development. As described in the above-reference parent applications, a specific area of interest in this field is in the study of ion channels and transporters. Ion channels are protein-based pores found in the cell membrane that are responsible for maintaining the electro-chemical gradients between the extra cellular environment and the cell cytoplasm.
Quite often these membrane channels are selectively permeable to a particular type of ion, e.g. potassium or sodium. The channel is generally comprised of two parts; the pore itself, and a switch mechanism that regulates (gates) the conductance of the pore. Examples of regulation mechanisms include changes in transmembrane voltage or the activation or deactivation of a membrane receptor via a chemical ligand. Ion channels are passive elements in that once opened, ions flow in the direction of existing chemical gradients. Ion transporters are similar in that they are involved in the transport of ions across the cell membrane, however they differ from ion channels in that energy is required for their function and they tend to actively pump against established electrochemical gradients.
An interesting and technically challenging aspect of ion channels involves their rapid and quite diverse signaling kinetics. Many ion channels can be activated and then de-activated in a few milliseconds. This requires that the instrumentation used in their analysis have the ability to following these changes with a fairly high temporal bandwidth, on the order of 10 kHz.
For an instrument to resolve these kinds of changes it is not only necessary that the recording apparatus have the required temporal bandwidth, but in addition the method of stimulating the ion channel event must also be fast. The electrical recording aspect of this problem is involved but readily achievable since high-bandwidth operational amplifiers are readily available.
The issue of achieving a rapid stimulus deserves additional explanation. As previously mentioned, some ion channels are activated by voltage. In these cases the same electronics used to record ion channel currents can also be used to control the voltage stimulus. This type of measurement is common in the industry and is referred to as a voltage clamp. In this case the time bandwidth of the stimulus, an electrical signal, is inherently fast enough so as not to degrade the kinetics of the voltage-gated ion channel signals.
Another class of ion channel events relies on chemical or “ligand” gating. These kinetic channel events are activated by specific chemical messengers such as the release of intracellular calcium, adenosine 3′,5′—monophosphate (cyclic AMP or cAMP) or acetylcholine (ACh). It is beyond the scope of this application to discuss all of the potential signaling chemicals that are of biological or therapeutic interest and the above serve only as examples. It should be mentioned, however, that in some cases the chemical activation of an ion channel is extra-cellular in its initiation, and in other cases it is intra-cellular. This implies that not only is it important that the compound can be released on the time scale of tens of milliseconds, but in some cases it is desirable to have it introduced within the membrane of a living cell.
One technique for accomplishing rapid stimulation of ligand-gated channels utilizes photo-activatable or “caged” compounds. This term refers to chemicals which are chemically altered such that the active nature of the compound is suppressed (“caged”) until photo-activated, usually by a short pulse of ultra-violet (UV) light of wavelength in the range of 240 and 400 nm.
The photolysis of such compounds is very fast and thereby can rapidly (in some cases in microseconds) release the active species of the compound. The underlying chemistry for making various common biological chemicals photoa-activatable is well-developed and the “caged” version of many compounds are commercially available for purchase through companies such as Molecular Probes of Eugene, Oreg.. In addition, when intracellular application is required, the caged version can often be made cell permeable such that it can be loaded into the cytoplasm of the cell for rapid intra-cellular activation at a later time
The technique of using pulsed UV illumination of a biological sample to rapidly release chemicals is fairly common in the fields of rapid cellular imaging and single-well patch-clamp electrophysiology. Parpura describes a system that utilizes a micro-manipulated optical fiber to deliver UV energy for flash photolysis in support of microscopic imaging studies. U.S. Pat. No. 5,936,728, describes a flash photolysis system for use in a scanning microscope allowing for automatic alignment of the ultraviolet directed beam and the detected image point in time. To date, there has been no utilization of this technique in the field of high-throughput (i.e., non-patch clamp) electrophysiology as described in the parent to this application.
SUMMARY OF THE INVENTION
This invention improves upon, and extends, the teachings of U.S. Pat. No. 6,488,829, which resides in high-throughput electrophysiological measurements. This integrated electrophysiogical measurement system includes a computer-controlled data collection system, an integrated electronics head for making parallel electrical measurements, and an integrated fluidics head used in part to transfer test compounds into the measurement process.
To this basic configuration, the instant invention introduces additional components facilitating computer control of a pulsed ultra-violet (UV) light source. This light source, and associated light coupling to a plurality of test samples, is used in conjunction with the other instrumentation to make effectuate high-throughput electrical measurements with respect to fast-acting, chemically-activated electrophysiological events.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a drawing which shows a high-throughput screening system according to the invention including a UV illumination module and integrated fluidics head for parallel operations; and
FIG. 2 depicts a preferred embodiment of a UV illumination technique whereby the light energy is directed via optical fibers to the biological samples.



DETAILED DESCRIPTION OF THE INVENTION
In contrast to prior-art patch clamp techniques, wherein a glass pipette is used to form a high-resistance electrical seal with a single biological membrane, commonly assigned U.S. Pat. No. 6,488,829 describes a technique wherein a plurality of minute apertures in a two-dimensional substrate provide the requisite sealing function. The advantage of this approach is that it eliminates the need for micromanipulation of an electrode assembly by a skilled user, while providing a format suitable for achieving multiple electrical seals in parallel, dramatically increasing the measurement throughput of the device.
To review, a specific embodiment of the system architecture enabling electrophysiological measurements to be conducted simultaneously on a plurality of cells (e.g. an N×M grid) is shown in FIG. 1. This architecture is comprised of several subsystems including a measurement platform (2) containing positions for a multi-well electrophysiogical measurement substrate (4), wash stations (6), reagent stations (8) and trays for containing potential drug candidates (10).
Included with this measurement platform is a 3-D mechanical gantry system (12) capable of independently moving a multi- or single-channel fluidics head (14) and electronics read head (16). The fluidics head and the electronics head could, without loss of function, both be comprised of single probes, N×1 (1-dimensional) as shown here or N×M (2-dimensional) geometries. The gantry system allows for the transfer of potential drug candidates to the various N×M “wells” of the multi-well measurement substrate using the fluidics head (14) as well as for spatially selectable electrical recording via a plurality of electrodes (18). Also shown is a data storage and processing unit (20) and user interface CRT display (22).
The present invention includes a UV light source module (24) which is capable of coupling light through a light coupler (26) into the measurement substrate (4) concurrently with the ability to electronically record currents from the same wells. This adaptation allows for the rapid and direct activation (through UV flash photolysis of a caged compound) and simultaneous electrical recording of time critical, ligand-activated ion channel or ion transporter events.
FIG. 2 illustrates one specific embodiment of this invention, whereby a UV light source (24) is controlled by the central processing unit (20). CPU (20) is preferably capable of controlling the optical pulsewidth and intensity of the source such that the timing, duration and light energy of the ultraviolet exposure can be automatically controlled. The light is preferably fed via optical fibers (26) to the sample through the electronic read head assembly (16). As a further alternative, the light may be coupled to the biological sample by other means, for example, by free space optics or evanescent wave coupling through the base of the substrate. Conventional optical elements such as mirrors, beam splitters, diffusers, collimators, telescopic optics, and the like may also be used as appropriate in place of, or in addition to the components previously described.

## Claims

1. An electrophysiological measurement apparatus for analyzing a biological material, comprising:
a multi-well plate having a plurality of fluid chambers, wherein each fluid chamber is configured to hold a biological material to be analyzed;
a thin plastic substrate having an array of apertures in alignment with the chambers of the multi-well plate;
wherein the substrate is bonded to the multi-well plate such that the chambers are open at the top and sealed at the bottom, except for the apertures;
wherein the apertures are smaller in diameter than the biological material, thereby enabling a high-resistance seal to be formed between a biological material present in each chamber and a corresponding aperture; and
wherein the substrate has a glass coating at least in the region where the high-resistance seal is formed between the biological material and the substrate; a fluid plenum to receive the multi-well plate such that at least the substrate is immersed;

a first electrode disposed in the fluid plenum;
at least one second electrode adapted to fit into the top openings of the fluid chambers of the multi-well plate;
a light source for illuminating one or more of the chambers so as to facilitate the rapid release of a caged compound present therein; and
electrophysiological measurement circuitry in electrical communication with the electrodes.

2. The electrophysiological measurement apparatus of claim 1, wherein the light source is an ultraviolet light source.

3. The electrophysiological measurement apparatus of claim 1, wherein a single aperture is associated with each chamber of the multi-well plate.

4. The electrophysiological measurement apparatus of claim 1, wherein the substrate includes mylar or polyimide.

5. The electrophysiological measurement apparatus of claim 1, wherein the diameters of the apertures are in the range of 1 to 10 micrometers.

6. The electrophysiological measurement apparatus of claim 1, wherein the apertures are tapered.

7. The electrophysiological measurement apparatus of claim 1, wherein the multi-well plate is sealed to the fluid plenum, enabling a differential pressure to be applied relative to the fluid in each chamber, thereby causing the material in each chamber to migrate to a respective aperture.

8. The electrophysiological measurement apparatus of claim 1, wherein the fluid plenum includes a chemical reagent causing a biological material in each chamber to permeabilize in the vicinity of the aperture.

9. The electrophysiological measurement apparatus of claim 1, further comprising a mechanism for moving the at least one second electrode into the chambers of the multi-well plate, so as to automate the measurement of the material contained therein.

10. The electrophysiological measurement apparatus of claim 1, further comprising:
a plurality of second electrodes in alignment with a plurality of the chambers of the multi-well plate; and
a mechanism for moving the plurality of second electrodes into the chambers of the multi-well plate to perform simultaneous measurements on the material contained therein.

11. The electrophysiological measurement apparatus of claim 1, further comprising a system for transferring fluids from one or more sources to the chambers of the multi-well plate.

12. The electrophysiological measurement apparatus of claim 1, further comprising a mechanism for directing the light from the source to the multi-well plate in an automated and spatially selectable manner.

13. The electrophysiological measurement apparatus of claim 1, further comprising a mechanism for directing the light from the source to spatially selected wells in conjunction with a plurality of electrodes, thereby providing for the simultaneous electrical recording of the biological materials during and subsequent to illumination of the materials.

14. The electrophysiological measurement apparatus of claim 1, further comprising an electronic control of optical pulse width and intensity, such that the timing, duration, and energy of the light directed from the light source onto the chambers can be automatically controlled.

15. The electrophysiological measurement apparatus of claim 1, further comprising a guide for coupling the light from the light source directly into the biological materials.

16. The electrophysiological measurement apparatus of claim 15, wherein the guide includes one or more optical fibers.

17. The electrophysiological measurement apparatus of claim 16, wherein the guide includes at least one optical component selected from the group consisting of mirrors, beam splitters, diffusers, collimators, and telescopic optics.

18. The electrophysiological measurement apparatus of claim 1, further comprising at least one of a cell, organelle, and vesicle positioned in at least one of the fluid chambers.

19. The electrophysiological measurement apparatus of claim 1, wherein the system is adapted to measure at least one of an electrical potential difference and a current across at least a portion of a biological material sealed across at least one of the apertures.

20. An electrophysiological measurement apparatus for analyzing a biological material, comprising:
a multi-well plate having a plurality of fluid chambers, wherein each fluid chamber is configured to hold a biological material to be analyzed;
a thin substrate having an array of apertures in alignment with the chambers of the multi-well plate;
wherein the substrate is bonded to the multi-well plate such that the chambers are open at the top and sealed at the bottom, except for the apertures; and
wherein the apertures are smaller in diameter than the biological material, thereby enabling a high-resistance seal to be formed between a biological material present in each chamber and a corresponding aperture;

a fluid plenum to receive the multi-well plate such that at least the substrate is immersed;
a first electrode disposed in the fluid plenum;
at least one second electrode adapted to fit into the top openings of the fluid chambers of the multi-well plate;
a light source for illuminating one or more of the chambers so as to facilitate the rapid release of a caged compound present therein;
electrophysiological measurement circuitry in electrical communication with the electrodes; and
a mechanism for moving the at least one second electrode into the chambers of the multi-well plate, so as to automate the measurement of the material contained therein.

21. The electrophysiological measurement apparatus of claim 20, further comprising:
a plurality of second electrodes in alignment with a plurality of the chambers of the multi-well plate; and
a mechanism for moving the plurality of second electrodes into the chambers of the multi-well plate to perform simultaneous measurements on the material contained therein.

22. The electrophysiological measurement apparatus of claim 20, wherein the light source is an ultraviolet light source.

23. The electrophysiological measurement apparatus of claim 20, wherein a single aperture is associated with each chamber of the multi-well plate.

24. The electrophysiological measurement apparatus of claim 20, wherein the substrate includes mylar or polyimide.

25. The electrophysiological measurement apparatus of claim 20, wherein the diameters of the apertures are in the range of 1 to 10 micrometers.

26. The electrophysiological measurement apparatus of claim 20, wherein the apertures are tapered.

27. The electrophysiological measurement apparatus of claim 20, wherein the multi-well plate is sealed to the fluid plenum, enabling a differential pressure to be applied relative to the fluid in each chamber, thereby causing the material in each chamber to migrate to a respective aperture.

28. The electrophysiological measurement apparatus of claim 20, wherein the fluid plenum includes a chemical reagent causing a biological material in each chamber to permeabilize in the vicinity of the aperture.

29. The electrophysiological measurement apparatus of claim 20, further comprising a system for transferring fluids from one or more sources to the chambers of the multi-well plate.

30. The electrophysiological measurement apparatus of claim 20, further comprising a mechanism for directing the light from the source to the multi-well plate in an automated and spatially selectable manner.

31. The electrophysiological measurement apparatus of claim 20, further comprising a mechanism for directing the light from the source to spatially selected wells in conjunction with a plurality of electrodes, thereby providing for the simultaneous electrical recording of the biological materials during and subsequent to illumination of the materials.

32. The electrophysiological measurement apparatus of claim 20, further comprising an electronic control of optical pulse width and intensity, such that the timing, duration, and energy of the light directed from the light source onto the chambers can be automatically controlled.

33. The electrophysiological measurement apparatus of claim 20, further comprising a guide for coupling the light from the light source directly into the biological materials.

34. The electrophysiological measurement apparatus of claim 33, wherein the guide includes one or more optical fibers.

35. The electrophysiological measurement apparatus of claim 34, wherein the guide includes at least one optical component selected from the group consisting of mirrors, beam splitters, diffusers, collimators, and telescopic optics.

36. The electrophysiological measurement apparatus of claim 20, further comprising at least one of a cell, organelle, and vesicle positioned in at least one of the fluid chambers.

37. The electrophysiological measurement apparatus of claim 20, wherein the system is adapted to measure at least one of an electrical potential difference and a current across at least a portion of a biological material sealed across at least one of the apertures.

38. An electrophysiological measurement apparatus, comprising:
a first fluid chamber, configured to contain a cell or membrane to be measured;
a second fluid chamber;
a thin plastic substrate separating the two chambers, the substrate having an aperture formed therethrough which is smaller in diameter than the cell or membrane, thereby enabling a high-resistance seal to be formed between the cell or membrane and the substrate, the substrate further having a glass coating at least in the region where the high-resistance seal is formed between the biological material and the substrate;
a light source for illuminating one or more of the chambers so as to facilitate the rapid release of a caged compound present therein;
an electrode disposed in each of the fluid chambers; and
electrophysiological measurement circuitry in electrical communication with the electrodes.

39. An electrophysiological measurement apparatus, comprising:
a first fluid chamber, configured to contain a cell or membrane to be measured;
a second fluid chamber;
a thin substrate separating the two chambers, the substrate having an aperture formed therethrough which is smaller in diameter than the cell or membrane, thereby enabling a high-resistance seal to be formed between the cell or membrane and the substrate;
a light source for illuminating one or more of the chambers so as to facilitate the rapid release of a caged compound present therein;
an electrode disposed in each of the fluid chambers;
electrophysiological measurement circuitry in electrical communication with the electrodes; and
a mechanism for moving at least one of the electrodes into a corresponding at least one of the fluid chambers, so as to automate the measurement of the material contained therein.

40. The electrophysiological measurement apparatus of claim 39, further comprising:
a plurality of first chambers, each configured to contain a cell or membrane to be measured, forming a multi-well plate, wherein the substrate has a plurality of apertures, and wherein the apertures are in alignment with the plurality of first chambers;
wherein the substrate has a plurality of apertures, in alignment with the plurality of first chambers; and
wherein the mechanism for moving at least one of the electrodes moves the electrodes into contact with the first chambers.