BACKGROUND OF THE INVENTION The precise manipulation of microscopically small particles, such as cells and liposomes, is necessary for many cellular analysis methods. Many of these methods require the precise placement of cells at predetermined positions. For instance, Patch Clamp measurements require that a cell be positioned on a small fluid-filled hole as disclosed in B. Sakmann and E. Neher, Eds., Single-Channel Recording (lest ed. 1983).

In another example, the observation of a cytosol of a single cell using confocal microscopy requires that the cell be positioned in the focal spot of an objective lens of a microscope. Many other methods require the precise positioning of cells and microscopic particles, such as fluorescence correlation spectroscopy of cells and cell/lipid membranes, and the introduction and/or capture of genetic material from selected cells.

Automatic techniques exist that can precisely position a microscopic particle using the charge that exists on that particle as disclosed in PCT Publication WO 99/31503 to Vogel and Schmidt, and Schmidt et al., 39 Angew. Chem. Int. Ed. 3137 (2000). However, demand still exists for a fast and reliable manual positioning technique that is generally applicable to most microscopic materials, including individual cells.

SUMMARY A micropositioner apparatus is disclosed for the positioning of individual cells, or other microscopic particles, at precise locations on a carrier so as to facilitate experimentation on the cell or particle. The micropositioner includes a carrier for supporting cells and electrodes for establishing an electrical field that leads to a either a flow of fluid or the creation of a dielectrophoretic force, which urges the cells onto the carrier at a predefined position or positions.

The carrier includes a reference side, a cell side and a channel that extends between and connects the reference side to the cell side. The cell side is in physical contact with a fluid compartment (= cell compartment) containing fluid, which contains cells or other small particles to position. The reference side is in contact with a fluid compartment containing reference buffer (=reference compartment). The channel inside the carrier connects these two compartments. One electrode is positioned on the cell side of the carrier and the other is positioned on the reference side of the carrier. The electrodes do not have to be physically attached to the carrier but must touch the fluid inside the cell and reference compartment. Two modes of positioning exist: (1) positioning by electrodendosmotic flow and by (2) dielectrophoresis.

For (1), the electrodes establish a voltage difference between the cell side and the reference side. The resulting electrical field along the inside and near the channel leads to electrodendosmotic flow (EOF) of the medium from the cell side through the channel to the reference side. EOF requires the channel walls or parts thereof or the surface area near the channel entrance to be electrically charged. The flow of the medium urges the cell to move towards the channel until the cell is positioned at the cell side entrance to the channel.

For (2), the electrodes apply an alternating voltage, which causes a spatially inhomogenous and timely alternating electrical field around the channel. The field strength increases approaching the channel. By the phenomenon known as dielectrophoresis, the electrical field exerts a force directly on the particle or cell that increases in proximity to the channel. This force urges the particle into position at the entrance of the channel.

In another aspect, the micropositioner can be used to conduct patch clamp recordings on the positioned cell. The cell or a membrane patch thereof covers the entrance to the channel and separates the medium in the cell compartment from the medium in the reference compartment. The electrodes can be used to apply a voltage across the cell or cell membrane and to register any changes in cell/membrane conductance due to the introduction of stimuli into the medium, such as ion channel agonists or antagonists, hormones or cytokines. The electrodes can also perforate the cell by the application of a relatively high voltage for a certain time. Perforating the cell allows whole cell recording and allows genetic material to be captured from, or introduced into, the cell.

BRIEF DESCRIPTION OF THE DRAWINGS Having thus described the invention in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale, and wherein: Figure 1 is a schematic depicting a first embodiment of a micropositioner using electroendosmotic flow to position a cell; and Figure 2 is a schematic depicting a second embodiment of a micropositioner using dielectrophoretic forces to position a cell.

DETAILED DESCRIPTION OF THE INVENTION The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like numbers refer to like elements throughout.

Figure 1 schematically depicts a first embodiment of a micropositioner according to the present invention. The micropositioner includes a carrier 11, a cell compartment 13, a reference compartment 14 and a channel 12 extending through the carrier between

the cell compartment and the reference compartment. The entire or part of the carrier 11 is preferably electrically insulating, thus avoiding an electrical current flow through the carrier material from the cell compartment 13 to the reference compartment 14. The micropositioner also includes electrodes 15 comprising an electrode for positioning and measurement, called recording electrode 16 positioned in the cell compartment 13 and a reference electrode 17 in the reference compartment 14 of the carrier 11. The channel 12 of the micropositioner is surrounded by a fluid buffer medium 18 in which cells 19 are in suspension in the cell compartment 13. Electroendosmotic flow (EOF) is motivated by the electrodes 15 and causes the medium 18 to flow from the cell compartment 13 through the channel 12 to the reference compartment 14. The flow of the medium 18 urges the cells 19 in the direction of the channel 12 until one of the cells is positioned at the channel in a known location for subsequent testing and measurement. Several factors dictate the strength and direction of the EOF, the most important of which are the voltage applied by the electrodes 15, the ionic strength of the medium 18, the pH of the fluid medium 18, and the electrical charge density of the channel walls and dimensions of the channel 12.

The characteristics of the carrier 11 and its channel 12 establish a condition in which capillary electrophoresis occurs and which allows an EOF to be generated.

Preferably the channel 12 is sufficiently narrow and comprises electrically charged channel walls (zeta potential X 0) 23. The channel 12 includes a cell compartment entrance 21, a reference compartment entrance 22 and is limited inside the carrier 11 by walls 23. The zeta potential and the polarity of the voltage applied to the electrodes 15 determine the direction of the EOF: An electrical net charge of the walls of the channel 12 establishes a distinctive distribution of ionic species in any ionic solution, such as the medium 18. Using negatively charged channel walls, the result is a layer of more tightly bound cations immediately adjacent to the channel walls 23 and a more loosely associated layer 20 that is predominately anionic in nature. EOF occurs when an electric field causes movement of the more loosely bound layer 20. For negatively charged channel walls, movement of the more loosely bound anionic layer and consequently EOF from the cell to the reference compartment requires a negative potential at the recording

electrode. Vice versa, for positively charged channel walls, a positive potential is required.

The length of the channel 12 is preferably within an order of magnitude of the cells 19 or smaller, typically within a range of 1 to 100 um. A shorter channel 12 increases the voltage drop per length unit, i. e. the electrical field, but reduces the length of the loosely bound layer 20 and consequently the amount of medium 18 that is directly moved by the electrical field. No particular channel length is a cutoff for EOF. Even a micropositioner with a channel 12 with a length of less than 1 um has a significant EOF, which is caused by the voltage drop in the channel AND around the channel entrances 21 and 22. The EOF outside the channel results from the strong voltage drop near the micropositioner surface located in the vicinity of the channel entrance for short channels.

For EOF generated in the vicinity of the channel entrance, the same considerations as for EOF generated inside the channel apply.

The channel 12 preferably has a diameter at the cell compartment entrance21 that is less than the diameter of the cells 19 themselves, typically around 1 to 10 0, um. The small diameter of the cell compartment entrance causes the cells to become lodged at the cell compartment entrance 21 and prohibits the cells 19 from flowing through the channel 12. The known diameter and location of the cell compartment entrance 21 allows the cells 19 to be positioned at a very well defined place in a Cartesian coordinate system or other coordinate system as desired.

The channel 12 and portion of the carrier 11 surrounding the channel can include an adhesive (not shown) to improve the positioning of the cells. Various charged compounds can attract cells 19 and cause them to adhere to the channel 12 and/or channel entrance 21 by interacting with the cell surface. Examples of these include surface treatments with Poly-L-Lysine, biotin-avidin (for molecular interaction) or glutaraldehyde (for covalent bonding).

The electrodes 15 are preferably redox electrodes constructed of Ag/AgCl or Pt and connected in series to a voltage supply and current recorder for recording the electrical characteristics of the positioned cell 19. The electrodes 15 preferably operate at a potential difference of less than 1 V in order to avoid electroporation on the

positioned cell 19. The electrodes 16 and 17 can be arbitrarily placed in the compartments 13 and 14, respectively, because the voltage drop across the channel 12 is usually significantly higher than any other voltage drops or electrode impedances in the local area.

To increase the EOF by means of increasing the potential difference driving EOF, the recording electrode can be split in one EOF driving electrode (=EOF electrode) and one electrode used for the following measurements (=measurement electrode), such as voltage clamp of the cell. Both electrodes are normally not connected. The EOF driving part is placed inside the channel 12 and the measurement electrode is placed in the cell compartment. EOF is generated by a voltage difference between EOF and reference electrode. Since the cells are not placed between these electrodes, they will not experience the resulting electrical field. Consequently, the potential difference can be very high (V » 1 V, i. e. much higher than cell-physiological voltages) for EOF positioning. After cell positioning, measurements are conducted using the measurement electrode in the same way the original recording electrode is used for measurements.

After positioning, the EOF electrode may be connected to the reference potential (usually ground), i. e. to the reference electrode, to not introduce noise to the recording system.

The EOF direction is dictated in large part by the charge of the channel walls 23 and the channel entrance 21 and 22, as well as the polarity of the voltage applied by the electrode 15. For instance, EOF is directed into the cell compartment entrance 21 in a carrier 11 and channel 12 constructed entirely of unmodified Si02 and a negative voltage applied by the recording (or EOF) electrode because of the silanol groups on the channel walls 23 reducing the movement of adjacent cations and consequently leading to a higher net-flux of anions, which under the given voltage polarity points towards the reference side. Reversing the charge of the channel walls 23 reverses the EOF direction, causing the medium 18 to flow from the reference compartment 14 to the cell compartment 13.

The buffer medium 18 filling the cell and reference compartments 13 and 14 comprise a solution in which cells may be viably suspended, including but not limited to, a Ringer Solution or PBS. In some cases it can be of advantage to use different buffers in both compartments, e. g. if using precious compounds. In such a case an individual cell

buffer and reference buffer are used. The compartments 13 and 14 are usually formed by the surface tension of the small fluid volumes on a hydrophilic/hydrophobic patterned carrier 11 surface. The carrier 11 surface may also contain small grooves for the fluid compartments 13 and 14. Small grooves are important if small compartment volumes are used or the compartment temperatures are high (e. g. 37 C) to reduce the air exposed compartment medium surface and consequently to reduce evaporation of the medium.

The medium 18 in the cell compartment 13 preferably contains cells 19, or it can also contain other microparticles of interest such as portions of the cells, products created by the cells, liposomes, etc. Note, however, that the size of the cell compartment entrance21 will be dictated by the size of the particles or cells that the user wishes to position for analysis.

One advantage of EOF is that it doesn't require the cells 19 or microparticles to be charged because it is the flow of the medium 18 that moves the cells and microparticles. The cells and microparticles enter the area of significant EOF, which may only extend for several micrometer into the cell compartment, randomly by such phenomena as convection, sedimentation or diffusion and are drawn toward the cell compartment entrance as the medium 18 flows in the direction of the cell compartment entrance21. EOF is considered significant when it is the main determinant of the direction of cell movement. Cell concentrations in the medium are preferably on the order of 104 to 106 cells/ml to optimize the efficiency of cell positioning. A higher density of cells generally results in a greater chance of a cell being in a position to be attracted to the cell compartment entrance21 by the EOF. Lower concentrations avoid more a contamination of the carrier surface with debris contained in the cell suspension, but take longer for positioning. The cell density is usually a compromise between these factors.

Figure 2 depicts a schematic of a second embodiment of the present invention that uses Dielectrophoresis (DEP) in place of EOF to position cells and microscopic particles. The micropositioner of the second embodiment is similar to the first embodiment. However, DEP positioning does not require (1) A surface charges on or about the channel 12.

(2) A long channel 12, since positioning of cells is mediated by direct interaction'Alternating Field-Cell'and not indirectly by a medium flow.

The channel length can be down to several nanometer, just ensuring that the device is sufficiently insulating if electrical experiments are performed.

(3) A strong limitation of positioning voltages, even if cells are experiencing the resulting strong electrical field, since they are alternating (ACV).

(4) Redox type electrodes for positioning, since for alternating voltages the electrode capacitance allows the flow of capacitive currents.

The frequency of the applied voltage is preferably between 10 kHz and 1 GHz and the magnitude is preferably below 10 V. In the 10 kHz to 1 GHz frequency range the electrode impedance is negligible even for non-redox electrodes 15 (such as those using Au) so that the main voltage drop occurs in and around the channel 12. Because the voltage drop outside the channel creates the positioning field, the channel length is preferably very short, that is less than 20 , m. The voltage drop around the channel 12 creates an inhomogeneous electrical field around the channel, which increases in magnitude in proximity to the channel. The field exerts a DEP force on the cells 19 (and other microparticles, which have dielectrical properties different from the medium they are suspended in) around the channel entrance, thereby drawing them towards the channel, i. e. the point of the highest field strength. The conditions for which cells are attracted (and not repelled, for instance) depend largely on the dielectrical properties of the cells and medium as well as the chosen frequency of the electrical positioning field.

These conditions are usually experimentally found.

Note that DEP is different than EOF, which exerts a force on the fluid medium 18 and not the microscopic particles or cells themselves. The magnitude of the DEP force exerted depends on the size and material composition of the cells and particles, and the frequency of the applied voltage used to generate the field. Thus particles or cells of a certain size or composition can be selectively positioned. Also, the selection of an appropriate frequency and magnitude of voltage allows the exclusion of certain debris.

As noted above for the first embodiment, treatment of the cell compartment entrance21

can be used to ensure that the cell 19 remains positioned after the DEP force is removed.

Surface treatment may also support the electrically tight adhesion of cells to the channel entrance, which is required for electrical measurements. For patch clamp recordings the required seal resistances are usually above 1 GOhm.

Another advantage of the second embodiment is that electrolysis and other effects that damage the cells 19 are much less likely when using an alternating voltage. Thus, a much higher field strength can be used.

A third embodiment of the current invention advantageously combines a direct voltage with a superimposed alternating voltage to position the cells 19 by DEP in a counter EOF stream of medium 18, thereby preventing contamination of the carrier 11 surface prior to positioning. The EOF is reversed so as to flow from the reference compartment 14 to the cell compartment 13 while the DEP force moves selectively the cell 19 against the EOF stream to the cell compartment entrance21 of the channel 12.

The EOF propels debris such as lipids, proteins and organelles away from the cell compartment entrance 21, thereby preventing contamination.

Once a cell 19 is positioned at the entrance to the channel 12, a range of analyses can be performed on the individual cell. For instance, the entrance to the channel 12 could be positioned within the focal spot of an objective lens of a confocal microscope for measurement and visual inspection. For positively charged carrier 11 surfaces, electrically tight adhesion will cause most cells 19 to stick to the surface by charge interaction. Electrically tight adhesion to the channel 12 entrance forces all current flowing into or out of the channel through the cell 19 thereby allowing electrical measurement of the cell 19 and its cell membrane. The application of a voltage pulses in the order of 1 V over several milliseconds will open the cell membrane to allow genetic material be captured from, or introduced to, the cell. The genetic material may include, but is not limited to, mRNA, plasmids and viruses. In addition, perforating the patch of cell membrane attached to the channel entrance 21 makes the entire cell electrically accessible and allows whole cell recording, as opposed to just the current flow through the attached membrane patch. These measurements usually entail the attachment of a

voltage clamp circuit to the electrodes as described in B. Sakmann and E. Neher, Eds., Single-Channel Recording (lSt ed. 1983).

The positioned cell 19 can also be subjected to selected stimuli by adding compounds such as ion channel agonists and antagonists, hormones and cytokines to either side, or both sides of the cell. Giant liposomes containing protein channels that allow the passage of RNA and DNA could be positioned at the cell compartment entrance21. Measurement of the changing current by a current recorder during passage of the RNA and DNA allows deduction of the nucleotide sequence of the RNA and DNA. Positioning the cell or giant vesicle 19 so that its membrane covers the cell compartment entrance 21 allows the reconstitution of natural or artificial ion channels contained within the membrane. These ion channels can be used as diagnostic tools to detect metabolites in the medium 18 that modify the conductance or gating kinetics of the ion channels. Note that many other applications would benefit from precise micropositioning of cells and microparticles and the previous list is meant to be exemplary, not limiting.

A forth embodiment of the positioning device combines multiple positioning devices according to embodiment 1, 2 and 3 to one single positioning device. Individual positioning sites can be individually controlled by individual electrodes and fluid compartments. However, an efficient realization of a multipositioner unifies the reference compartments and reference electrodes to one single reference compartment and one reference electrode, thereby reducing the number of electrodes and compartments required by a factor of nearly 2. Individual positioning and measurements are performed by direct access to the individual cell compartments. The advantage of this forth embodiment is the possibility to integrate many micropositioner on one carrier 11. As an example, using silicon/silicon dioxide/silicon nitride waver, individual compartments can be as close as 1 mm in x-y direction on the waver (chip) surface and be produced simultaneously.

Many modifications and other embodiments of the invention will come to mind to one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings.

Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.