CN1529814A - Device and method for determining and/or monitoring electrophysiological properties of ion channels - Google Patents

Abstract

An apparatus and method for determining and/or monitoring electrophysiological properties of ion channels in ion channel-containing structures uses AC-driven positioning electrodes to drive an object to be analyzed to a measurement position. The measurement location may comprise an aperture located between two compartments containing the solution, each compartment comprising a respective measurement electrode. The aperture includes a cohesive area at its periphery and is sized such that an object to be analyzed cannot pass through the aperture. Alternatively, the measurement location may comprise a measurement electrode surrounded by an adhesive area suitably dimensioned to accommodate the object, to which the object may be adhered, and a second measurement electrode remote from the measurement electrode.

Description

A device and method for determining and/or monitoring electrophysiological properties of ion channels

technical field

The present invention relates to a device and method for determining and/or monitoring the electrophysiological properties of ion channel-containing structures, typically lipid membrane-containing structures such as ion channels of cells, by establishing electrophysiological measurement structures in which In structure, the cell membrane forms a high-resistance seal around the measuring electrodes, enabling the determination and monitoring of the current flow through the cell membrane. The device of the invention is typically part of a measurement system for studying electrical events in cell membranes, for example a device for implementing the patch clamp technique for studying ion transport channels in biological membranes. In particular, the present invention designs a device for a patch-clamp measurement system that has high throughput and uses only a small amount of test compound, only a small amount of liquid carrier, and is capable of testing simultaneously and independently on many Parallel experiments are performed on cells to perform many experiments in a short period of time.

Background technique

The general concept of electrically isolating a membrane and studying ion channels in that membrane under voltage-clamp conditions is presented by Neher, Sakmann, and Steinback in "Extracellular patch clamping, an method of electric current", outlined in PfluegerArch.375; 219-278, 1978. They found that by squeezing a pipette containing acetylcholine (ACh) against the surface of the muscle cell membrane, they were able to see discrete jumps in the electrical current attributable to the opening and closing of ACh-activated ion channels. However, they were limited in their work by the fact that the resistance of the pipette's glass-to-membrane seal (10-50 MΩ) was very small relative to that of the channel (10 GΩ). The electrical noise generated by this seal is inversely related to resistance and is large enough to mask the current flowing through ion channels, which are less conductive than ACh channels. This also prevents the voltage in the pipette from clamping to a value different from the voltage of the electrolysis cell, as that would result in a high current through the seal.

It was then found that by firing a glass pipette and by applying suction to the inside of the pipette, a very high resistance (1-100 GΩ) seal could be obtained using the surface of the cell. This giga-seal reduces the magnitude of the noise to a level at which most channels of biological interest can be studied and greatly expands the voltage range over which these studies can be performed. This improved seal is called a "giga-seal" and the pipette is called a "membrane pipette". A more detailed description of the giga-seal can be found in O.P.Hamill, A.Marty, E.Neher, B.Sakmann & F.J.Sigworth: "A modified patch-clamp technique for high-resolution current recording from cell- and cell-free membrane patches", Found in Pflügers Arch. 391, 85-100, 1981. For their work in developing the patch clamp technique, Neher and Sakmann received the 1991 Nobel Prize in Physiology or Medicine.

Ion channels are transmembrane proteins that facilitate the migration of inorganic ions across cell membranes. Ion channels are involved in various processes such as generation and synchronization of action potentials, synaptic transmission, hormone secretion, muscle contraction, etc. Many drugs exert their special effects through the regulation of ion channels. Examples are antiepileptic compounds such as diphenylhydantoin and lamotrigine that block voltage-associated Na ⁺ channels in the brain, antihypertensive drugs such as nifedipine that block voltage-associated Ca2 ⁺ channels in smooth muscle cells pyridine and diltiazone, and stimulators of insulin release that block ATP-regulated K ⁺ channels in the pancreas such as glibenclamide and tolbutamide. In addition to chemically induced modulation of ion channel activity, patch clamp techniques have enabled scientists to perform manipulations using voltage-dependent channels. These techniques include adjusting the polarity of the electrodes in the patch pipette and changing the composition of the salt to moderate the concentration of free ions in the electrolyte.

The patch clamp technique represents a major development in biology and medicine, as it allows the measurement of ion flux through individual ion channel proteins and also allows the study of the response of individual ion channels to pharmaceuticals. Briefly, in standard patch clamp techniques, thin (app. 0.5-2 μm diameter) glass pipettes are used. The tip of the patch pipette is squeezed against the surface of the cell membrane. The pipette tip seals tightly to the cell and insulates a little ion channel protein in the patch of membrane.

The activity of these channels can be measured individually by removing the remainder of the cell ("single-channel" recording), or alternatively, the membrane can be ruptured (e.g. by applying subatmospheric pressure in the pipette) to give Highly conductive access to the cell interior, thus allowing measurement of channel activity across the cell membrane ("whole cell" recording). An intermediate position between the two is the "cell-attached" mode, where the cells are attached to the pipette using a giga-seal, but the membrane membrane inside the pipette remains intact.

During single-channel and whole-cell recordings, the activity of individual channel subtypes can be characterized by imposing a "voltage clamp" across the membrane. In the voltage clamp technique, the membrane current is recorded at a constant membrane potential. Or—more precisely—the amplifier delivers exactly the current necessary to maintain the membrane potential at a level determined by the experimenter. Therefore, the current generated by the opening and closing of ion channels does not allow recharging of the membrane.

The temporal resolution and voltage control in such experiments is impressive, often in the millisecond or even microsecond range. However, a major obstacle to the use of the patch clamp technique as a general method in pharmacological screening has been the limited number of compounds (typically at most 1 or 2) that can be tested per day. And, the very slow rate of solution change that can be achieved around cells and membrane patches may pose a major obstacle.

The major limitations determining the throughput of the patch clamp technique are the positioning and clamping of the cells and pipette, and the characteristics of the solution delivery system that directs the dissolved compound to the cells and the patch.

In a typical patch-clamp setup, cells are placed in experimental vessels that are continuously perfused with saline solution. The establishment of cell-pipettor connections in these containers is time-consuming and cumbersome. Compounds are applied by changing the inlet of a valve connected to a small supply bottle. The requirements for support liquid and samples to be tested are high.

High-throughput systems for performing patch-clamp measurements have been proposed, typically comprising a substrate with multiple positions suitable for immobilizing cells in a measurement structure where the electrical properties of the cell membrane can be determined.

US 5,187,096, Rensselaer, discloses a device for monitoring the cell substrate impedance of cells. Cells are cultured directly on the electrodes, which are then covered with many cells, therefore, measurements on individual cells cannot be performed.

WO 98/54294, Leland Stanford discloses a device comprising a substrate having a well comprising an array of electrodes. The substrate with wells and electrodes (metal electrodes) is made of silicon using CVD (chemical vapor deposition) and etching techniques, and includes a silicon nitride "passivation" layer surrounding the electrodes. Cells are cultured directly on the electrode array. The substrate is suitable for measuring electrophysiological properties, and various proposed measurement schemes are disclosed.

WO 99/66329, Cenes, discloses a device comprising a substrate with perforations arranged in wells and electrodes provided on each side of the substrate. The substrate is made by perforating a silicon substrate with a laser and may be coated with an anti-stick material on the surface. The substrate is adapted to establish a giga-seal with the cells by positioning the cells over the perforations using suction that creates a flow of liquid through the perforations, providing an anti-adhesion layer around the perforations, or by directing the cells electrically. Cells can be permeabilized by EM fields or chemical methods to provide whole-cell measurement configurations. All perforations in the well, and thus all measurable cells, share one working electrode and one reference electrode, see Figure 1, so measurements on individual cells cannot be performed.

WO 99/31503, Vogel et al., discloses a measurement device with apertures arranged in wells on a substrate (carrier) and separating two compartments. The measuring device comprises two electrodes located on either side of the aperture and suitable for positioning cells at the opening of the aperture. The substrate can have hydrophobic and hydrophilic regions to guide the positioning of cells at the aperture openings. Cell localization by means of electrophoretic movement of cells towards the aperture is also disclosed.

Manipulation of cells by dielectrophoresis (DEP) is disclosed in, for example, US 6,149,789, Benecke et al; US 5,454,472, Benecke et al; US 5,795,457, Pethig et al; US 5,858,192 Becker et al and US 6,059,950, Dames et al. The theory involved is e.g. US 5,814,200, Pethig et al. and G. Fuhr et al. 'Motion of cells in a time-varying field - principle and potential', 'Electrical operation of cells', U. Zimmerman, G.A. Neill, eds ., CRC Press, Boca Raton, described in USA1996.

Particles in the medium are polarized by an electric field applied to the medium, and charges are induced at particle boundaries. At the same time, if the medium is polarizable, charges will accumulate at the boundary of the medium adjacent to the particle to an extent opposite to those induced on the particle. If the particle has a greater polarizability than the medium, then the net dipole, the sum of the dipole induced in the particle and the dipole effectively present in the space occupied by the particle in the medium, will be parallel to the field; If the medium has a greater polarizability than the particles, the net dipoles will be antiparallel. In a diverging field there will be a net force on the particle: parallel dipoles will give a net force towards the larger field region - positive DEP; antiparallel dipoles will give a net force away from them - negative DEP.

In an AC field, the polarization of the particle, and thus the induced net dipole, will be frequency dependent. The polarization of the medium hardly changes. Therefore, the time-averaged DEP force will also depend on frequency (and, among other parameters, the conductivity and permittivity of the medium, the size of the particles, the effective conductivity and permittivity). Cells and vesicles are thin films of complex dielectric properties, meaning that at low and high frequencies they appear to have a lower polarizability than a physiologically conductive typical aqueous medium, and at intermediate frequencies they appear to have a lower polarizability than Physiological conductivity is typical of aqueous media with high polarizability. Thus, for a given medium in the correct conductivity range, the same particles can be used to obtain negative DEP at low and high frequencies, and positive DEP at intermediate frequencies. Additionally, by using an electrode array, where the electrode pairs are energized by an AC potential at a frequency in the negative DEP range such that the cells float on the electrode array, and using a well-chosen phase sequence, it is possible to apply A time-averaged force, thus causing the particles to move horizontally relative to the array. This can be used to achieve rotation ('electrorotation') or translation ('traveling wave dielectrophoresis') (TWD), or to position particles relative to motion induced by another force, such as flow of the surrounding medium.

US 6,149,789, Benecke et al., disclose a patterned electrode array that will move cells in a given direction predetermined by the geometry of the array. This can be used to move cells to a given location, or to separate distinct cells from a mixed population. In a relevant aspect of the present application, a 'transformable filter' is disclosed wherein, to achieve separation of cells from other types, cells are moved to the center of an array of concentric circular electrodes where they can pass through apertures (at hydrostatic pressure down) and flow.

US 5,454,472, Benecke et al. disclose another embodiment for continuous separation of microparticles using the TWD principle. A TWD field in one direction is combined with a second force having a component in an orthogonal direction to dislodge particles with selected properties out of the flow pattern followed by the other. In particular, a scheme is disclosed in which particles are moved by an array of TWD electrodes on the substrate in a first compartment parallel to the substrate having apertures therein. The second compartment is connected to the first compartment by an aperture, and the electrophoretic force is provided by a field perpendicular to the direction of motion of the TWD field between an electrode in the second compartment and another electrode in the first compartment. Acts to draw particles through the aperture into the second compartment.

US 5,795,457, Pethig et al. disclose a means by which a reaction between particles suspended in a liquid can be induced by simultaneously applying more than one non-uniform field to the suspension, which reaction is broadly defined as covering chemistry, biochemical or physical interactions. Also disclosed in the concept of extraction by this method is a device similar to the concept disclosed in the above US 5,454,472, Benecke et al., wherein a device is provided through which liquid and particles can pass, the difference from Benecke et al. being the AC DEP field Used to move particles through an aperture, not a DC field.

US 5,858,192, Becker et al., discloses a DEP cell manipulation device comprising a helical electrode array for guiding cells towards or away from the center of the helix by TWD. A port in the center is exposed through which cells and fluids can pass. A sensing element, such as a biosensor, adjacent to the central port is disclosed.

US 6,059,950, Dames et al discloses a helical electrode array substantially similar to Becker et al, the difference between the two publications lies in the details of the method of energizing the electrodes.

Another device and method for cell sorting is disclosed in US 5,814,200, Pethig et al. Although it is not particularly relevant to the present invention, this disclosure is equally useful, and an overview of DEP theory established in the prior art is given.

Summary of the invention

The present invention provides a device and method for performing electrical measurements on structures containing ion channels, such as cell membranes and artificial membranes, which is capable of high throughput and substantially automated operation, is more reliable, and can be used substantially more than existing devices. Smaller amounts of test compound.

The invention provides a device comprising one or more measurement sites suitable for immobilizing an object, such as a cell, comprising a membrane comprising ion channels such that the membrane forms a giga-seal around the measurement electrode at the measurement site relative to the adhesion area. This enables the determination or monitoring of electrophysiological properties of cell membranes using the measuring electrode and a reference electrode placed in the liquid surrounding the subject. Alternatively, the measurement location may comprise an aperture, in which case the membrane forms a giga-seal around the aperture, which communicates with a liquid-filled compartment in which the measurement electrodes are located. The liquid will then pass through the pore to touch the cell membrane and can be measured in a similar way. The adhesive area may be of any shape - in a preferred embodiment it will be approximately circular.

It should be understood that the term "object" used in this specification refers to any object including a structure comprising an ion channel, such as a lipid membrane comprising an ion channel or an artificial membrane comprising an ion channel. A cell is an example of such a structure, and is used for clarity in this specification especially with reference to the prior art, examples of electrophysiological properties are current flow through ion channels or capacitance of membranes containing ion channels. Whole-cell and cell-attached configurations are believed to also apply to other closed objects including membranes, such as vesicles.

The present invention provides a means of locating an object at a measurement location. In the prior art described above, where the measurement location comprises an aperture, positioning is achieved by using, for example, entrainment of objects in a liquid flow through the aperture, as in US 4,055,799 (Coster et al.), or by contacting a liquid-filled Electrophoresis (EP) excited by a DC field between the measuring electrode of the compartment and the reference electrode contacting the liquid surrounding the object, as in WO 99/31503 (Schmidt et al.), by US 5,506,141 (Weinreb et al.) and by WO01 /25769 (Sophion) discloses that this is achieved by moving the object towards the measurement location. This has many disadvantages. The object must have a net charge: this is true for most cells, e.g. most eukaryotic cells have a negative surface charge at the surrounding medium at physiological pH, but for vesicles special processing steps are required to give a charged surface. A DC potential difference between the electrodes will cause a DC current to flow, resulting in an induced current process at the electrodes, which can be unfavorable, for example due to reversible electrode materials such as Ag/AgCl switching between oxidized and reduced forms (Ag to AgCl and vice versa), and possible electrolysis of the solution at an irreversible electrode such as Pt causes problems. The limited capability of small reversible electrodes is especially disadvantageous if the electrodes have to be very small, eg if the measuring electrodes are to be surrounded by a giga-seal, as disclosed in our earlier application WO 01/25769. And, if the aperture exists, the locating force, i.e. the attractive force of the object towards the oppositely charged electrode, is not self canceling at the aperture - even when located at the aperture, the object will still be attracted, and if it is sufficiently deformable, it Movement through the aperture can continue (as occurs for capsules as disclosed in WO 99/31503). This would introduce a limit on the field strength and thus the rate of motion of the object, and possibly take the necessary controls to detect the presence of the object at the aperture and switch off the field to prevent damage to the object.

The invention discloses that the object is advantageously positioned at the aperture or at the measuring electrode by means of dielectrophoresis (hereinafter abbreviated as DEP). In the presence of an aperture, this is achieved by applying an AC potential between two electrodes in solution contacting opposite sides of the aperture; for a film with significant AC resistance, the AC field will be largest and most divergent at the aperture, In the same way as with respect to the DC field used in WO 99/31503 and US 5,506,141, objects in the vicinity of the aperture will therefore have an ) in the force. For embodiments including a measuring electrode and no aperture is present, an AC potential is applied between the measuring electrode and a reference electrode in contact with the solution in which the subject is suspended.

As described for the prior art (see e.g. G. Fuhr et al., 'Cell movement in a time-varying field - principle and potential', 'Electrical manipulation of cells', U. Zimmerman, G.A. Neill, eds., CRC Press, Boca Raton, USA 1996), the direction and relative value of the force will depend on the AC frequency, the conductivity of the surrounding solution and the nature of the object. To achieve localization of the object at the aperture or measuring electrode, the AC frequency and conductivity of the solution will be determined according to the type of object in use to induce a positive DEP effect. The DEP force is only applied in the region of the divergent field, so once objects have reached a region of a more uniform (albeit higher) field, eg in contact with the electrode or aperture towards which they are moving, they experience less force. This is in contrast to the case of positioning at an aperture by EP or suction, in which case when the object is at the aperture it will experience a greater force than if it is close to but spaced from the aperture. By engineering the AC fields an object will experience in different parts of the device and at different times during its motion, DEP can be used to achieve more precise object localization than EP, for example through a combination of positive and negative DEP effects.

The frequency of the AC field required to achieve positive and negative DEP forces on a given subject can be estimated using prior art publications (see, eg, Fuhr et al., Figure 4, above). Typically, negative DEP is only found at any given frequency if the conductivity of the surrounding medium is greater than that of the object's interior; positive DEP is typically found at higher frequencies if the conductivity of the surrounding medium is lower than that of the object's interior, Negative DEP is found at lower frequencies. Large DEP forces are mainly found when the conductivity of the medium is significantly smaller than that of the interior of the object. Therefore, the object localization method according to the invention using positive DEP is preferably carried out using a suspension medium with a conductivity lower than that of a standard physiological medium, the osmolarity of the medium being controlled by using sugar inclusions such as manna Alcohol, as known in the art, to maintain viability, when the subject is cells, followed by replacement of the medium with a different solution that more closely approximates normal physiological composition. The devices of the invention preferably include means to deliver the cells in a suspension medium and effectively replace the solution once the giga-seal has been achieved. The optimum conductivity and frequency of operation of each embodiment of the invention will depend on the subject in use. Typical ranges for positive and negative DEP described in the prior art are as follows. For typical mammalian cells, type MDA-MB-231 human breast cancer cells in a medium with a conductivity of 56 mS/m, negative DEP and TWD effects are found in the range 10-100 kHz, positive DEP at higher frequencies. For a medium of 406 mS/m, negative DEP and TWD are found in the range 30 kHz-10 MHz, and positive DEP is found above that range (US 5,858,192, Becker et al.). For 3T3 fibroblasts in media with a conductivity of 10 mS/m, the separation frequency between negative and DEP is cited in US 6149789, Benecke et al. as approximately 100 kHz. The conductivity of a 0.15M NaCl solution is close to that of a typical cell growth medium, approximately 1400mS/m.

The present invention proposes that, using an AC field applied between a measuring electrode and a reference electrode, whether or not the measuring location comprises an aperture, or between any other pair combination of electrodes provided for purposes related to the measuring location, by dielectrophoresis ( DEP) to locate objects. DEP can be used alone or in combination with other driving means for moving objects such as EP, entrainment in flow, gravity or centrifugal sedimentation, or magnetic localization assisted by magnetic markers of the object. Object positioning by DEP forces includes forces acting directly on the object itself, or on additional marker entities associated with or attached to the object, or a combination of the object and marker entities.

The process of object positioning involves moving the object from the bulk of the suspended liquid into the vicinity of the measurement location, and then moving the object to a location such as to create a giga-seal at that location. The present invention proposes that one or both of these two steps are realized by DEP. For example, the movement of an object from a majority liquid to position is conceived to be achieved by deposition or by positive DEP (as described above for other applications in the prior art); the movement to establish a giga-seal is achieved by positive DEP, or if Using the aperture, this is achieved by entrainment in the suction flow through the aperture. Alternatively, the movement from the majority of the liquid towards the position can be achieved by negative DEP (traveling wave DEP, abbreviated as TWD) to induce lateral movement of the object relative to the surface providing the position. Using DEP to move the subject close to the aperture, followed by suction down onto the aperture to form a seal, has the advantage of avoiding the need for a large flow of liquid through the aperture and the significant pressure differential and time this would require.

Another significant advantage provided by the present invention is the ability to use DEP to move the subject towards the measurement location in preference to any debris present in the solution that may tend to stick near the aperture or measurement electrodes thereby reducing the likelihood of achieving a giga-seal . The net time-averaged force on an object of characteristic size r moving through a liquid ⁽ e.g. a sphere of radius r) caused by DEP is proportional to r (see e.g. US 5,814,200, Pethig et al., incorporated herein by reference), So the force increases enormously with the size of the object. Most impurities in the solution will be smaller than the object under test and so will experience less force. Consequently, the debris will tend to move slower than the object under test towards the measurement location, in contrast to the movement achieved by flow entrainment which will also tend to move the debris.

In the case of an aperture, a slight counterflow can be provided through the aperture (as is done in conventional electrophysiology experiments) to maintain an unobstructed aperture and surrounding adhesion area while the subject moves towards the aperture using DEP. A lower DEP force on the debris will cause it to be impassable due to fluid flow. In the case of a measurement electrode on a substrate, the DEP-induced fast movement of the object towards the electrode will reduce the likelihood of debris deposition, a slow fluid flow can be maintained over the electrode surface which will tend to prevent debris deposition, or The electrodes can be operated in an orientation chosen to prevent debris deposition. Counterflow through the aperture may be created by a hydrostatic pressure difference across the aperture. Alternatively, it can be provided by electro-osmotic flow (EOF) between the two sides of the aperture driven by a DC potential difference across the aperture superimposed on the AC DEP signal. For example, if the rear side of the aperture is held at a more positive potential than the front side, for an aperture material with a positive coefficient of electroosmotic mobility, then liquid will flow through the aperture from the rear to the front. If the object is charged and enters the DC field, there will also be an EP effect on the object, which will also work for negatively charged objects such as cells to approach the aperture from the front side (i.e. in the opposite direction to the EOF direction ) to move cells, which may be advantageous. The DC potential and the magnitude of the AC DEP signal are chosen to give the best combination of these effects. If the EP effect acting on the object is not desired, the EOF can be produced using electrodes close to the aperture on the front side, so that the field hardly, if any, penetrates into the liquid.

The present invention provides a means of determining that a giga-seal has in fact been achieved by monitoring the impedance between the measurement electrode and the reference electrode at the measurement location. Impedance measurements are optionally used to monitor the proximity of the subject to the measurement location depending on the force applied to the subject and (in the case of an aperture) the choice of positioning method - eg switching between DEP and suction if desired , thus using feedback to control the localization process. The application of reverse flow through the apertures as described above is optionally also controlled in this way.

The present invention also provides means to deliver a test sample (typically a pharmacological drug candidate) to each measurement location so that experiments can be performed on each subject fixed at the location. The sample delivery means may be arranged to deliver individual samples to individual locations, or to group locations together such that the same sample is delivered to more than one location. Such a set of positions in the device of the present invention is called the test limit.

The invention also provides means for performing a large number of individual experiments in a short period of time. This is accomplished by providing a system that includes multiple test boundaries and control components that can coordinate the operation of the test boundaries and related functions. Each test boundary may include measurement locations, means for locating an object at each location, means for establishing a giga-seal, means for selecting a location where a giga-seal has been established, measurement electrodes and one or more Reference electrode. Thereby, independent experiments can be performed in each experimental boundary. Because of the small size of the test boundary, the invention allows measurements to be performed using only a small amount of support liquid and test sample.

Therefore, according to one aspect, the present invention provides a device for performing electrical measurements on a subject comprising ion channels in a medium, comprising:

A substrate having a first surface defining the boundaries of a first compartment suitable for retaining a first solution, one or more measurement locations located on the first surface, each location comprising: in the first surface, a an aperture communicating with the second compartment retaining the second solution; an adhesive region surrounding the aperture to which an object can adhere to form a high resistance seal between the first compartment and the second compartment;

a first measuring electrode located in said first compartment, which in use contacts a first solution;

a second measurement electrode located in the second compartment, which in use contacts the second solution;

measurement means, electrically connected to the first and second measurement electrodes, adapted to perform electrical measurements on an object adhered to the adhesion area at the measurement location; and

Object localization means comprising a first localization electrode located in the first compartment and adapted to provide an AC signal to the electrode to form an AC field in the first solution, the AC field acting to move the object by dielectrophoresis towards the measurement Location.

In one solution, the first measuring electrode and the first positioning electrode can be formed as a single electrode structure. In another solution, the second measuring electrode and the first positioning electrode can be formed as a single electrode structure.

According to another aspect, the invention provides a device for performing electrical measurements on an object comprising ion channels in a medium, comprising:

A substrate having a first surface which, in use, is in contact with a first solution and on which one or more measurement sites are located, each site comprising: a first measurement electrode; an adhesive region surrounding the first measurement electrode , an object can be adhered to the adhesion region to form a high resistance seal between the first measurement electrode and the first solution; and a conductive track for connecting the first measurement electrode to the measurement instrument while keeping it insulated from the first solution;

a second measuring electrode which, in use, is in contact with the first solution;

Measuring means, which are electrically connected to the first measuring electrode and the second measuring electrode, and are adapted to perform electrical measurements on an object adhered to the adhesion area at the measurement location;

Object localization means comprising a first localization electrode on the substrate and adapted to provide an AC signal to the localization electrode to form an AC field in the first solution, the AC field acting to move the object by dielectrophoresis towards the measurement location .

In one solution, the first measuring electrode and the first positioning electrode can be formed as a single electrode structure. In another solution, the second measuring electrode and the first positioning electrode can be formed as a single electrode structure.

According to another aspect, the present invention provides a test structure for use in performing electrical measurements on an object comprising ion channels in a medium, comprising:

A substrate having a first surface on which one or more measurement locations are located, each measurement location comprising:

an aperture in the first surface and in communication with the second surface of the substrate;

a sticky area around the aperture to which an object can stick to form a high resistance seal thereto;

At least one positioning electrode substantially surrounding or adjacent to the adhesive layer region for applying an AC signal thereto to form an AC field near the measurement location, the AC field acting to dielectrophoretically move the object towards the measurement location.

In one aspect, the test structure further includes a first measurement electrode formed on or near the first surface, and a second measurement electrode formed on or near the second surface.

According to another aspect, the present invention provides a test structure for use in performing electrical measurements on an object comprising ion channels in a medium, comprising:

A substrate having a first surface on which one or more measurement locations are located, each measurement location comprising:

a first measuring electrode;

an adhesion area surrounding the first measurement electrode to which an object may adhere to form a high resistance seal thereto;

at least one positioning electrode substantially surrounding or adjacent to the adhesive layer region for applying an AC signal thereto to form an AC field in the vicinity of the measurement location, the AC field acting to move the object by dielectrophoresis towards the measurement location such that it Adheres to the stick area to form a high resistance seal.

In one version, the test structure further comprises a second measurement electrode on the first surface, separated from the first measurement electrode by at least the adhesive region.

According to another aspect, the invention provides a method for performing electrical measurements on an object comprising ion channels in a medium, comprising the steps of:

providing a first solution comprising a suspended object to be measured to a first surface of a substrate having an aperture therein in communication with a second surface of the substrate;

providing a second solution to the second surface to establish fluid contact between the first and second surfaces;

testing that fluid contact has been achieved therebetween by measuring electrical continuity between the first and second electrodes respectively located on or adjacent to the first and second surfaces;

The object to be measured in the first solution is driven by dielectrophoresis to a measurement location with an adhesive area surrounding the aperture.

In another embodiment, the method provides the further step of testing the resistance of the seal at the measurement location on the substrate by measuring the electrical impedance between said first and second electrodes. In another embodiment, the method provides the further step of establishing a measurement structure of a subject by applying one or more electrical pulses between the first and second electrodes, and performing measurements on said subject.

According to another aspect, the invention provides a method for performing electrical measurements on an object comprising ion channels in a medium, comprising the steps of:

providing a first solution comprising a suspended object to be measured to a first surface of a substrate having a first measurement electrode thereon, and an adhesive region surrounding said first electrode;

providing a second measurement electrode in electrical connection with the first solution; and

The object to be measured in the first solution is driven to the measurement position by dielectrophoresis, so that the object adheres to the adhesion area to form a high-resistance seal therewith.

In another embodiment, the method provides the further step of testing the resistance of the seal at the measurement location on the substrate by measuring the electrical impedance between said first and second electrodes. In another embodiment, the method provides the further step of establishing a measurement structure of a subject by applying one or more electrical pulses between the first and second electrodes, and performing measurements on said subject.

Objects containing ion channels in solution can be directed towards the measurement location on the substrate by DEP alone, by entrainment in the flow in the first solution, or by both. The flow in the first solution may be induced by a hydrostatic pressure differential applied from outside the substrate or by suction generated by suction means located on or outside the substrate. Examples of suitable pumping means include electroosmotic flow, or other electrokinetic effects such as electrocapillarity, control of pressure from a pressurized fluid, by chemical reaction or boiling, formation of vapor or gas in the area in contact with the solution, by pressure Electrically driven suction, or other means that will be apparent to those skilled in the art.

The sticky area provided at the measurement site may be the surface of the substrate itself, and is preferably patterned to give a localized sticky area near the measurement electrode or aperture, so that the object sticks to the site without forming completely around the measurement electrode or aperture The possibility of gigabit sealing is minimized. Alternatively, the substrate material may be such that a giga-seal is not formed, whereas an adhesion layer known to form a giga-seal to the cell or capsule such as silica or glass such as borosilicate may be deposited near the aperture and Form a pattern.

In this context, the term "giga-seal" generally means a seal of at least 1G ohms, but for certain types of measurements where currents are large, lower values may be sufficient.

Whole-cell configurations can be obtained by applying a pulse, or a series of potential difference pulses, between the measurement and reference electrodes at a position where the subject is held there with a giga-seal. If an aperture is present, pulses can be applied between electrodes in contact with the solution on either side of the aperture. The realization of the whole-cell approach is shown by monitoring the impedance of the object at the location - the capacitance between the electrodes will increase as the capacitance of the object's membrane appears in the circuit. A series of pulses can be of gradually increasing duration and/or potential difference over time until a whole-cell approach is achieved. This process can be monitored by the system to learn which pulse protocol is most effective for a given subject, which protocol is then applied to all subjects used in a given experiment.

Alternatively, a whole-cell configuration can be obtained by subjecting the portion of the ion channel-containing structure closest to the measuring electrode to an interaction with the pores forming the substance.

Alternatively, if the aperture exists, the whole-cell configuration can be achieved by making the structure containing the ion channel surrounded by a giga-seal by means of a pulse of suction applied through the aperture using, for example, one of the above-mentioned suction means. Partially broken to obtain.

In both methods above, the achievement of a whole-cell approach can be monitored electrically, and the process can be controlled as described for the electrical pulse method.

Other membrane configurations known in the art of electrophysiology, such as the use of 'outside-out' or 'outside-in' patches excised from the membrane, are also contemplated as part of the present invention.

Depending on the specific embodiment of the device, in particular the substrate comprising the measurement site, the addition of the object and the support liquid is performed in one of the following ways. In one embodiment, the test boundaries are accessible from above, and droplets of support liquid and objects can be provided to each test limit by means of a dispensing or pipetting system. Systems such as ink jet printer heads or foam jet printer heads may be used. Another possibility is the 'nQUAD' aspiration dispenser or any other dispensing/pipetting device suitable for taking small liquid doses. Alternatively, the support liquid and object are coated entirely on the substrate (e.g., by pouring the object-containing support liquid over the substrate or immersing the substrate in the object-containing support liquid), thereby providing the support liquid and object to the substrate. Limits for each trial. If small amounts are used, the handling of the liquid on the substrate should preferably be performed in a high humidity atmosphere to avoid evaporation problems.

In a preferred embodiment, each assay boundary is a chamber accessed by one or more microchannels. This would give more control over the interaction with the external environment, and a more controlled and smaller application of liquid to the measurement location. Faster test solution transfers will be obtained than priming with the pipette alone. In a particularly preferred embodiment, a suspension of the test subject and a sample of the test compound are loaded sequentially from the liquid distribution system into the microfluidic access channel, and then flow sequentially through the measurement location to a waste holding tank, which Preferably provided on a substrate or a component mounted on or mounted to a substrate.

In another aspect of the method, the cells are cultured directly on the substrate while immersed in the growth medium. In the best case, the cells will form a homogeneous monolayer (depending on the type of cells being grown) over the entire surface, except for areas where the surface is deliberately made unsuitable for cell growth. However, in the present invention it is envisaged that a giga-seal is formed around each aperture that contacts the common test boundary, so the formation of a 'tight bond' between cells in this layer is not necessary, unlike in WO 99/ As disclosed in 66329 (Cenes).

In yet another aspect of the method, artificial membranes with integrated ion channels can be used in place of cells. Such artificial membranes can be made from saturated solutions of lipids by placing small pieces of lipids on the pores. This technique is described in detail in, for example, Christopher Miller, "Ion Channel Reconstruction", Plenum 1986, p.557. Bimolecular lipid membranes can form on the pore if the pore size is appropriate and a polar liquid such as water is present on both sides of the pore. The next step is to incorporate the protein ion channels into the bilayer. This can be achieved by providing lipid vesicles with integrated ion channels on one side of the bilayer membrane. The sac may be drawn by, for example, an osmotic gradient to fuse with the bilayer whereby ion channels are incorporated into the bilayer. Alternatively, capsules comprising ion channels may be positioned individually at the measurement sites.

Brief description of the drawings

Embodiments of the invention will now be described, by way of example only, with reference to the following drawings, in which:

Figure 1a is a cross-sectional view of a first embodiment of the device according to the invention showing the measurement locations in detail;

Figure 1b is a plan view of the device shown in Figure 1a;

Figure 2 is a diagram of a measurement system comprising the apparatus shown in Figures 1a and 1b;

Figure 3 is a cross-sectional view of a second embodiment of the device showing the measurement locations in detail;

Figure 4 is a cross-sectional view of a third embodiment of the device showing the measurement locations in detail;

Figure 5a is a cross-sectional view of a fourth embodiment of the device showing the measurement locations in detail;

Figure 5b is a plan view of the device shown in Figure 5a;

Figure 5c is a plan view of a fifth embodiment of the device which also looks in section as in Figure 5a;

Figure 5d is a plan view of a sixth embodiment of the device which also looks in section as in Figure 5a;

Figure 6a is a cross-sectional view of a seventh embodiment of the device showing the measurement locations in detail;

Figure 6b is a plan view of the device shown in Figure 6a;

Figure 7a is a cross-sectional view of an eighth embodiment of the device showing the measurement locations in detail;

Figure 7b is a plan view of the device shown in Figure 7a;

Figure 8 is a plan view of a ninth embodiment of the device showing the measurement locations in detail;

Figure 9 is a cross-sectional view of a tenth embodiment of the device showing the measurement locations in detail;

Figure 10 is another cross-sectional view of the device shown in Figure 9 showing features around the measurement location;

Fig. 11 is a cross-sectional view of an eleventh embodiment of the device showing the measurement locations in detail.

DESCRIPTION OF THE PREFERRED EMBODIMENT

FIG. 1 a shows a sectional view and FIG. 1 b shows a plan view of one embodiment of a measuring location according to the invention. A measurement site 1 for fixing an object 2 to be tested is formed on a substrate 12 . The measurement location comprises a first surface on which one or more compartments or "trial boundaries" 36, each in the form of a well, are defined, and a second compartment comprising one or more second compartments 6. surface. In the embodiment shown in Figure 1, only a single test boundary and a single second compartment are shown, but it should be understood that there may be multiple test boundaries and multiple second compartments each provided on a single substrate. compartment, and more than one substrate may be provided in each device. The test limit 36 communicates with the second compartment through an aperture 30 formed in the separation membrane 4 . The second compartment communicates with one or more liquid supply channels 7 defined either in the substrate 12 or by alignment of features in one or both of the substrate and backing or mounting element 10 middle. The electrode 8 is provided in the test confinement 36 and the second electrode 16 is provided in the second compartment 6, which in use are in contact with the test confinement and the liquid in the second compartment.

In use, the test boundary is filled with liquid comprising object 2 and the second compartment is filled with electrolyte to achieve liquid conductivity between electrode 16 and aperture 4 . The object 2 is positioned in the aperture 30 and forms a giga-seal around it by means of the positioning means of the present invention. In the embodiment in Figure 1, a giga-seal is formed to the material 31 forming the membrane 4, the adhesion area 18 is defined by the coating 46 to which the object will not adhere, leaving only the area of the material 31 adjacent the aperture exposed. Alternatively, as shown in later embodiments, material 31 may be such that a giga-seal is not formed and an adhesion layer may be deposited and patterned around the aperture. If the substrate 12 is conductive, the electrodes 16 and the contact electrolyte in the second compartment 6 may optionally be insulated from the substrate by an insulating layer 43, which itself may be the same material 31 as that forming the film; This will allow more than one electrode 16 to be provided on a common substrate while remaining insulated from each other.

In some embodiments, advantageously, the second compartment 6 and the electrode 16 are electrically common; in this case, the electrode 16 is indicated as a reference electrode. In other embodiments, the test limit 36 is advantageously electrically common, and the electrode 8 is represented as a reference electrode. Although one of each of electrodes 8 and 16 is shown in Figure 1, less than one reference electrode per measurement location, or only one common reference electrode may be provided.

The device shown in Figure 1 can be manufactured in different ways from a variety of different materials. The essential feature is that the material near the aperture in the adhesion region is suitable for forming a giga-seal to the object comprising the lipid membrane. Such materials include silicon, plastics, pure silica and other glasses such as quartz or borosilicate, or doped with materials selected from Be, Mg, Ca, B, Al, Ga, Ge, N, P, As and their An oxide of one or more dopants of silicon dioxide. The adhesive material may itself form a film comprising pores, or may be deposited on or inserted into the film-forming material.

The minimum size of the device is similar to the size of the object to be tested, so eg for a typical mammalian cell on the order of 10 μm in diameter, the diameter of the giga-seal will preferably be 5 μm or less. Therefore, in the presence of apertures, the diameter of the apertures is preferably 5 μm or less, more preferably 2 μm or less; the outer diameter of the adhesive region is preferably 10 μm or less, in the presence of measuring electrodes, by The diameter of the measurement electrode surrounded by the adhesive area is preferably 5 μm or less, more preferably 3 μm or less. These dimensions will be larger if larger objects such as oocytes are to be tested, and smaller for smaller objects.

The dimensions are such that standard microfabrication methods suitable for the purpose are advantageous. The preferred method of fabrication of the embodiment in FIG. 1 is based on standard microfabrication processing techniques known in the art using silicon substrate 12 . A typical sequence of processing is as follows:

1. Silicon nitride is deposited (by low pressure chemical vapor deposition - LPCVD) on both sides of the silicon wafer (12), preferably both sides are polished. This layer (31) forms a thin film comprising apertures (30) on the front side and forms an etch mask to define the backside etch through the silicon.

2. Deposit Au/Cr on the backside of the wafer, then photolithography and etch to form an Au/Cr mask for etching the silicon nitride to pattern the subsequent silicon hole etch.

3. Plasma etch the nitride (CF4/O2) to form the silicon etch mask.

4. KOH etch to form the subsequent well 6 (which will be pyramidal as is known in the art as shown in Figure 1) and membrane.

5. Back-aligned IR lithography - aligns the front side mask with the rear etch mask. The apertures 30 in the front side are patterned.

6. Plasma etch Nitride (CF4/O2) - open the aperture.

7. Deposit an optional insulating layer 43 on the exposed silicon on the rear side of the compartment 6 if desired - silicon nitride deposited by LPCVD.

8. Using SU-8 Photolithography - This applies a layer (46) of SU-8 photopatternable epoxy on the film to define the adhesion area 18.

9. Deposit metal to form electrodes 16, if desired.

10. Partial sawing of the wafer for cutting after final stage plasma cleaning.

11. Oxygen plasma cleaning - removes residue from the surface of the adhesive layer.

12. Using, for example, capillary wicking of adhesives to join silicon and plastic parts, or fusion bonding between chips and parts, to mount the chips thus formed into insulating housing parts, such as molded plastic parts, to provide liquid Delivery and Handling.

The test limit 36 may be defined by one or more isolating members mounted on or to the substrate 12 and may be in the form of an opening as shown in FIG. . In the simple opening configuration in Figure 1, the test limit is defined by the insulating part 38, only depicted in simplified form to show its relationship to measurement location 1 and its function to limit the area of liquid applied to the test side of the device. The walls 37 of the test boundary are shown vertical in the Figures, but in practice advantageously will be sloped, or have variable sides or undercuts, so that they are wider near the plane of the membrane than at the opening, as is suitable for the liquid and The manipulation by which a test subject suspended in a liquid is added to the test boundary. The rear side of the substrate 12 may also have additional components mounted on or to the substrate 12, such as having one or more liquid channels 7 formed therein or cooperating with the substrate 12 to form such channels. Part 10.

Alternative embodiments (not shown) are also contemplated as part of the present invention, and instead of the well 36 and associated electrodes, it includes one or more fluidic channels passing through the aperture, thus allowing suspended objects and other liquids to pass through rather than moving. Liquid manipulation delivered to test subjects. Such flow devices are known in the art, for example as disclosed in WO 0020554 (AstraZeneca). Electrodes 8 are then included in contact with fluidic channels known in the art, either close to the aperture or remote from the aperture, depending on the desired operating characteristics of the device and the conductivity of the solution in use.

A measuring system according to the invention is shown in Figure 2, comprising the measuring position device shown in Figure 1, the measuring system 200 comprising:

The device of Fig. 1, as shown in 202, the measurement position on the substrate 12 is installed in the part 10, and the part 10 is used to determine the fluid passage 7 to the second compartment 6 (Fig. 1), the test limit on the front side uses flow-through arrangement, reached with liquid supplied through channel 204, object 2 fixed in place at the measurement location, and electrodes 8 and 16 in contact with the liquid on both sides of the membrane;

Electrolyte reservoir 210 connected to channel 7, fluid connection 214 capable of supporting pressure and comprising means of suction 212 and means of creating a differential pressure between channel 7 on the rear side of the measurement location and channel 204 on the front side . This means is shown in Figure 2 to include a valve 216, but could equally include additional suction or means to use a hydrostatic head, leading to a waste container 218 leading to air after the valve;

Another liquid supply means 220 for the front side, which is used to supply at least (i) a suspension of the object to be tested, (ii) an electrolyte for the conductivity test and (iii) a compound to be tested. Advantageously, the liquid supply means comprises a liquid dispensing system which can provide the three types of liquids sequentially without entrainment of air bubbles, which for example further comprises a tray 221 of the liquid supply, and a sampler head 223 which can be moved to select the appropriate sequence of liquids . The liquid supply means are connected to the channel 204 via a liquid supply connection 224 comprising suction means 222 and from there to a vented waste container 228;

Switching means 240, which may connect electrodes 8 and 16 to electrical measuring means 242, or DEP signal generating means 246, or in some cases both together; and

Control and recording means 248, which control the operation of all parts of the system and record the results produced by the measurement means.

The suction means 212, 222 may comprise any practically applicable fluid displacement means detailed in the description of the invention, and the suction means may be located in any practically suitable location in the fluid connection lines 214, 224.

The operating sequence of the system is as follows:

1. Before any liquid flows into the front side channel 204, the device 202 is first primed by flowing electrolyte from the reservoir 210 through channel 7.

2. When the rear side liquid line 214 is flooded and full, the valve 216 is closed and the suction means 212 operates to build up an overpressure in line 214 above atmospheric. The pressure required is not exact, but is sufficient to flush air out of the second compartment 6 in the device shown in FIG. 1 a and move the electrolyte up the aperture 30 in the membrane 4 .

3. After this has been achieved, the pre-prepared electrolyte is selected by the liquid distribution system 220 and flowed through the channel 204 .

4. The measuring means 242 then test that the continuity has been obtained between the channels 204 and 7 through the aperture: ie the aperture is ready to receive a test subject.

5. The valve 216 is then opened to equalize the pressure between the front and rear of the aperture - optionally positive pressure is maintained by means of suction to maintain a slow flow through the aperture from the rear side to the front side which is used to avoid the aperture being blocked by debris ;

6. The liquid dispensing system 220 selects the suspension liquid containing the test subject and flows it into the channel 204 .

7. Switching means 240 selects DEP signal generator 246, which applies an AC signal to the electrodes at a frequency that induces a positive DEP force on the object, used to pull the object toward the aperture until it sticks;

8. Preferably, the measuring means 242 tests for the presence of an object at the aperture by measuring the impedance of the aperture. Optionally, the localization process of the object is controlled using feedback from the output of the measurement means to the localization means.

9. Giga-seals can form naturally. Alternatively, formation may be assisted by applying a slight negative pressure at the aperture to draw the object down onto the sealing surface, or by applying an electrical potential across the aperture, or both;

10. Then, the measurement means 242 tests the establishment of the gigabit seal. Alternatively, where a giga-seal does not form naturally, the process can be controlled using feedback from the output of the measurement means.

11. Optionally, whole-cell configuration is achieved by methods as described above, for example by applying a brief negative pressure in channel 7 .

12. Once the giga-seal and (optionally) whole-cell configuration is confirmed, the system initiates the baseline and conventional experimental sequence of compound application via the liquid distribution system and records the results using the control means 248 .

In practice, additional measurement locations will be included in the system, and the above steps will be performed for each location in the system. Some redundancy is expected - not every position will form a successful giga-seal to the subject where necessary, or a successful whole-cell measurement configuration, and these will be recorded by the system and excluded from subsequent trials .

Figure 3 shows an embodiment using positive DEP to position a subject in a similar manner as in Figure 1, but with the additional feature of at least one additional electrode that simplifies subject positioning. Further electrodes 40 used in DEP localization of objects are formed on the surface of the thin film material 31 . The electrodes 40 are located around the aperture and are preferably circularly symmetrical about the aperture, for example in the form of a ring or arc, so as to give a strong diverging field in the region of the aperture that is completely or substantially symmetrical about the aperture. The electrodes 40 are connected by conductor tracks 41 in contact with suitable contact means (not shown) to form off-chip electrical contacts. The DEP electrode 40 is preferably coated in at least the area around the aperture with a sealing material 44 capable of forming a giga-seal to the object 2 . This is then used to form a giga-seal around the aperture after the object has been attracted to the electrode by the positive DEP. Areas of material 31 other than the sticking area are then preferably coated with an insulating coating 46 to which the object is not easily sealed, to reduce the chance of the object sticking to the sealing area at times other than when it is centered. possibility. The diameter of the opening in layer 46 is chosen to maximize the likelihood that the object to be adhered will entirely cover the aperture and that the object will not readily adhere in such a way as to contact the object sealed to the aperture or impede access thereto. Thus, the diameter is preferably smaller than the diameter of the test object, more preferably smaller than or equal to half the diameter of the object.

Another reason for the advantage of coating the electrodes 40 and tracks 41 is to reduce the possibility of electrochemical reactions between them and the solution. In the embodiment shown in FIG. 3, encapsulation material 44 is extended over an extended area of the chip surface, and coating material 46 is positioned over encapsulation material 44, the exposed areas of encapsulation material 44 being covered by the adjacent apertures in coating material 46. The width of the opening is determined. If the coating 46 has suitable dielectric properties, the additional advantage is obtained that the field experienced by the object in the solution, generated by the electrode 40 and the contact track 41, is significantly reduced in those regions remote from the aperture. , thereby confining the strong positive DEP effect to the region adjacent to the aperture.

Other electrodes, eg for measurement, may also be provided on the material 31 and insulated from the solution by the layer 46 . This can be done, for example, to avoid the need for the electrodes 8 to be formed or mounted on the upper part 38 .

In use, an object deposited in the well 36 in a liquid (not shown) is drawn by the positive DEP towards the AC energized electrode 40 . The second or counter electrode for the AC DEP signal may be electrode 8 or an alternative electrode located in contact with the solution in which the subject is suspended. Those objects closest to the electrode 40 will be drawn towards the electrode. Since the electrodes are at least substantially circularly symmetric about the aperture, and therefore the field is also substantially circularly symmetric about the aperture, objects will tend to be drawn down towards the electrodes in such a way that they deposit on the electrodes and cover the aperture. As the positive DEP force draws the object toward the electrode, it encounters the surface of the sealing material 44 overlying the electrode and seals to the electrode. Exact centering of the object is not necessary if the aperture is covered and a giga-seal is achieved completely around it. Therefore, the adhesive layer 44 must completely surround the aperture, even if the electrode 40 does not completely surround the aperture. Once the seal is formed, the signal forming positive DEP can be turned off, if desired, and the seal will hold the object in place.

The device of Figure 3 is substantially similar to that of Figure 1 and may be fabricated by a similar process with an additional step after Step 6 in the earlier fabrication sequence:

6a. Metal deposition to form electrodes 40 and contact tracks 41

6b. Photolithography to pattern the counter electrode 40 and contact tracks

6c. Deposit insulating/adhesion layer 44 by sputtering - eg silicon dioxide or glass

FIG. 4 shows another embodiment in which the central electrode is located underneath a thin film of material 31 . This design is suitable for thin films that have good properties for transporting electric fields, eg they are thin and/or have a high relative permittivity. In this way, the field lines from the electrode 40 pass through the thin film material and then diverge towards the distant reference electrode so that the same positive DEP effect pulls the subject down towards the electrode. Therefore, the material 31 can be directly used as a sealing surface of an object without applying a sealing material on the electrodes. Anti-sticking material 46 is used to define the sticking area as a ring around the aperture, as before. The electrodes 40 are connected to the outside world by contact tracks 41 which are preferably insulated from the substrate 12 (in this case the substrate is conductive) by an insulator layer 43 .

In another alternative embodiment, the function of the electrode 40 on the underside of the membrane may be performed by the electrode 16 in the well below the membrane. With respect to an electrode contacting the solution on the upper side of the membrane, such as electrode 8, an AC potential applied to this electrode can induce a positive DEP field through the pores, as described above with respect to FIG. field if it has good dielectric properties. These properties can be controlled by patterning regions of the film to give greater field transport through regions adjacent to the holes than elsewhere, so confine the DEP force to that region. A coating of insulating polymer around the adhesion area can be used to achieve this.

In other preferred embodiments, additional electrodes are provided to induce motion of the object caused by the DEP towards the aperture from a region remote from the aperture. A positive DEP produced at or near the aperture will pull the object towards the aperture, but at an end distance from the aperture, the divergence of the positive DEP field and thus the force acting on the particle will be small. The movement of the object towards the aperture can be achieved using the traveling wave dielectrophoresis (TWD) principle described in the prior art using negative DEP in a linear array of electrodes. The positioning of the object can then be determined by positive DEP at the aperture as described for the embodiment shown in Figure 1, by suction forces induced by flow through the aperture, for example, by hydrostatic or electroosmotic flow, or by positive DEP combined with flow. Advantageously, an electrode 40 is provided adjacent to the aperture, connected to a separate power source and energized to induce positive DEP towards it, as in the above embodiment. For a given object type and solution, negative and positive DEP effects are formed by applying low and high frequency fields, respectively. The extent of the field is relatively short so that the positive DEP field can be applied in the central region of the negative DEP TWD field without adversely affecting the negative DEP TWD field. Alternatively, a detection control may be included to detect the presence of an object in the vicinity of the aperture and initiate positive DEP or suction positioning. Such means may include optical observation of objects near the aperture, and/or measurement of electrical properties of one or more electrodes near the aperture.

Figure 5 shows a preferred embodiment including a negative DEP TWD array to move objects towards the aperture. Figure 5a is a cross-sectional view at XX of the structure shown in plan view in Figure 5b. In the embodiment in Figure 5a, where the components are numbered as in Figure 3, additional electrodes 50a-h are shown on either side of the aperture. The structure shown in Figure 5a forms part of the bottom of the well or fluid channel in the same manner as in Figure 3, but the scale of the drawing is now such that only the base of the well or channel is shown. Electrode 50 forms part of a TWD structure known in the art and comprises an array of electrodes which are sequentially energized to suspend objects negatively dielectrophoretically above the array and to cause them to move in solution parallel to the array. The electrodes are arranged such that they are perpendicular to at least one axis through the aperture, so directing the subject towards the aperture. Plan view 5b shows aperture 30 surrounded by electrodes 40 connected to pads by tracks 41; electrodes 50a-h are then arranged in an arc substantially around the aperture, connected to further pads by conductor tracks 52a-h. Then, application of an appropriately selected four-phase AC field as disclosed in, for example, US 6149789 (Benecke et al.) or US 5795457 (Pethig et al.) will drive the object radially towards the center of the arc, i.e. the aperture. Electrodes 40 are provided adjacent to the aperture, as before, and can be used for positive DEP attraction of the subject to the aperture. Alternatively, if this aspect of the invention is not required, for example if positive DEP attraction by the aperture itself is to be used, then the inner electrode 40 may be omitted, or used as the innermost element of the TWD array.

The arrangement of electrodes and tracks in Figure 5b is advantageous because it requires only a single layer of conductor tracks and no vias or crossings. The number of electrodes arranged around the aperture is arbitrary and selected according to the area in which objects need to be collected and moved towards the aperture. If the density of objects in the carrier solution is high, then a small radius of collection is sufficient, requiring relatively few electrodes. In this case, there are 8, and they will be combined in groups of 4 to achieve the optimal TWD field - ie, a and e, b and f, c and g, d and h will be performed collectively. If a large number of electrodes are required, it would be advantageous to use two metallization layers and vias to connect between them. Figure 5c shows the same electrode pattern as Figure 5b, now connected to fewer common conductor tracks: eg electrodes 50a and 50e are connected to track 53a, 50b and 50f are connected to track 53b and so on. The tracks are insulated from each other by vias 55 leading to crossings 57 in the second conductive track layer. If an increasing number of electrodes are used in the array, then the common connection pattern will expand such that each electrode is connected to electrodes four more electrodes from the aperture, i.e. 50a, e, i, m, etc. (not shown) will all be connected to track 53a. Then five tracks and pads will suffice.

Figure 5d shows a preferred embodiment where the TWD array is arranged as a series of concentric circles, contacted by conductor tracks on a second conductive layer insulated from the first conductive layer. This embodiment has the advantage that there are no areas where TWD is inactive, as it does in the areas where TWD is inactive in Figures 5b and 5c. The concentric electrodes are connected by vias 55 to conductive tracks 53 on the second, lower level in the structure. The electrodes are connected to their respective conductor tracks in the same way as for the embodiment in Fig. 5c.

In another preferred embodiment shown in plan view in FIG. 6b and in cross-section at X-X on the plane in FIG. Disclosed spiral arrangement, four electrodes 60, 62, 64 and 66 are provided in a staggered arrangement and are fed through connector tracks 61, 63, 65, and 67 respectively from the AC source such that between adjacent electrodes There is an increasing 90 degree phase difference between them. This causes the object to move along a radial path towards the center of the spiral. The object is then positioned at the aperture by the means described above.

Figure 7b shows a plan view of another embodiment, and Figure 7a shows a cross-sectional view at X-X on the plan view, wherein a helical TWD array is provided as in the embodiment shown in Figure 6, in addition, a central electrode 40 is provided, with Depends on the positive DEP localization of the object at the aperture (the aperture is not shown in Figure 7b). Electrodes 60, 62, 64, 66 are connected to tracks 61-67, as before, except that there is now a fifth helical electrode in the set, which consists of alternating, pilot apertures between two of the TWD helical electrodes The conductor track 41 of the ring electrode 40 . This is a particularly advantageous electrode array for connecting the center electrode 40 without the need for a second level conductive layer. However, track 41 may lead to electrode 40 on a second stage below spiral electrodes 60-66, if desired.

Other configurations of TWD arrays for moving objects towards the aperture are contemplated as part of the invention. The embodiments described above have an array arranged substantially around the aperture that moves objects from a range of large angles towards the aperture. However, embodiments including arrays of moving objects from one or more cardinal directions are also contemplated. For example, FIG. 8 shows in plan view an apparatus in which a subject is moved by TWD through a channel 90 using an array of electrodes 80, 82, 84, 86, etc. etc. are driven by four AC lines 81, 83, 85, 87 connected as known in the prior art such as US 6149789 (Benecke et al.) or US 5795457 (Pethig et al.). The apertures are preferably located in the region where the channel is narrow, as shown in FIG. 8 . The connection to the drive lines requires two levels of contact layers, with vias 89 leading between the two levels. Alternative configurations of linkages will be apparent to those skilled in the art.

Another embodiment of the invention is shown in Figures 9 and 10, where positive DEP is used to position the object at the planar electrode rather than the aperture. FIG. 10 shows a measurement location 100 on a substrate 102 . Figure 9 shows the measurement location in a near-sighted manner; Figure 10 shows the measurement location as part of a test apparatus 104, comprising: a substrate 102 with the measurement location; a component 106 formed as part of the substrate or mounted on the substrate in The test limits are determined in the form of a well 120 in which the measurement location 100 is located; a reference electrode 108, which is installed in the well to contact the liquid in the well, is connected by a conductive track 110 to a contact area 112 kept away from the solution, and is covered by a protective layer 114 to insulate. The measurement site 100 comprises a working electrode area 122 connected by a conductive track 116 to a contact area 118 which is also remote from the solution. Referring to FIG. 9, details of the measurement positions are as follows. Working electrode region 122 is defined by openings in insulating protective layer 124 . The conductor track 116 exposed in the opening is coated with a material selected to give a stable electrochemical potential in the cell support solution containing chloride ions, preferably formed from a silver layer 130 which in turn is coated with a silver chloride layer 132 The Ag/AgCl electrode. Surrounding the opening in layer 124 is a region 134 onto which a thin film of object 140 may be formed giga-sealed. If the material forming layer 124 is itself suitable for the formation of a giga-seal, no further modification of the material is required in this area. Additional material 134 is advantageously provided to enhance the seal to the object. Examples of such sealing materials include silicon dioxide or glass, preferably deposited by sputtering. The design of the measurement location is such that, in contrast to the situation in the prior art, no close proximity is required between the top surface of the electrode (shown here by the surface of layer 132) and the object membrane when the object is sealed to the sealing area. of the seal. In fact, a layer of solution is advantageously included in the space 142 between the surface of the layer 132 and the subject film. This layer of solution is used to apply a well-defined potential derived from the electrode potential to the subject membrane, and is also used to maintain a mild solution environment in the immediate vicinity of the membrane, rather than at the point where cells are bonded to the electrode typically. There is a changing environment in the technical approach.

In use, an AC potential is applied to electrode 122 relative to a remote reference electrode, such as electrode 108 in FIG. Frequency of. The AC field will diverge strongly over the electrodes in the same way from the previous embodiment; objects will be drawn towards the electrodes by positive DEP, and the strong divergence of the field near the electrodes will mean that they will readily reach the space directly above the electrodes . When drawn down towards the electrodes, they encounter and form a giga-seal to the seal area, which encloses the electrode. The quality of the seal can then be checked by conventional methods known in electrophysiology. Modifications can be made to the embodiment in Figure 9 to improve the possibility and quality of the seal, eg the surface of the seal material 134 is advantageously raised above the surface layer 132 of the electrode. This serves to keep the cell membrane away from the surface of the electrode, which is advantageous because silver ions are less prone to being metabolized by toxic cells. For this purpose, another material containing the electrolyte, such as a hydrogel, may be included, covering the surface of the Ag/AgCl electrode, to reduce the diffusion of Ag+ ions in solution towards the cells.

In contrast to localization by dielectrophoresis (EP) as disclosed in our earlier application WO 01/25769, object localization by DEP is advantageous because the size of the measurement electrode surrounded by the adhesion area must be small - for typical mammalian The cells, typically 5 μm or less in diameter - and the electrodes must be reversible to establish a stable measurement potential with respect to the solution. Therefore, the ampacity of the electrodes is limited by the transition of the material between its oxidized and reduced forms (Ag to AgCl and vice versa). This limits the amount of current that can pass in a given DC direction, thus limiting the extent to which electrophoresis can be used to locate cells. However, ACDEP avoids this problem. The capacity of small electrodes is sufficient for procedures of electrophysiological measurements.

FIG. 11 shows another embodiment in which a measurement site 100 is provided as before on a substrate 102 , similar elements being numbered similarly as in FIGS. 9 and 10 . The measurement locations in FIG. 11 are envisaged to be included in a test rig similar to that shown in FIG. 10 . Furthermore, in FIG. 11 , an additional electrode 150 is provided which completely or substantially surrounds the working electrode 122 . The electrode 150 is intended to provide a positive DEP attraction that draws the subject down to the measurement location, and is connected to external circuitry by conductive tracks 152 leading to a contact area 156 insulated from the solution by an insulating layer 154 . This embodiment has the advantage that the working electrode is not used for DEP object localization; thus avoiding the current flowing through the electrode during the process, which, when AC, can still increase the rate of dissolution of the AgCl electrode surface, thereby increasing the rate of dissolution from the solution. Possibility of Ag+ ions to poison cells [2]. A layer of sealing material 134 is applied over the DEP electrode 150 to give a sealing surface where cells can form a giga-seal after they have been drawn towards the electrode.

In another embodiment (not shown, but readily apparent from the above embodiments), additional positioning electrodes are provided around the central electrode 150 in the same manner as in the earlier embodiments of FIGS. 4-8 above including apertures. . The movement of objects by means of a negative DEP TWD towards a central region where they can be located and where a giga-seal can be formed is advantageous in a similar manner using a positive DEP.

In the embodiments shown above in Figures 9-11, the overall system and sequence of operations shown in Figure 2 are slightly modified. Because there is no aperture to a second liquid-filled compartment, it is not possible to use suction to position cells, assist giga-seal formation, or achieve whole-cell configuration. The first point must be achieved by DEP alone, EP disclosed in our earlier application WO 01/25769, or a combination of both; the formation of the giga-seal can be assisted by the potential difference between the measuring and reference electrodes. Whole-cell configuration is achieved by pulses of potential differences between the measuring and reference electrodes, and/or application of pore-forming compounds in the vicinity of the measuring electrodes. Parts of the above apparatus and sequence of operations related to the application of liquid flow at the rear side of the aperture, or the control of the differential pressure across the aperture, are simply omitted. Other aspects, such as the detection of objects in the measurement position or the feedback control of the positioning process are applicable.

Other implementations are within the scope of the appended claims.

Claims (66)

1. A device for performing electrical measurements on an object (2) comprising ion channels in a medium, comprising: A substrate (12) having a first surface defining the boundaries of a first compartment (36) suitable for retaining a first solution, on which one or more measurement locations (1) are located, each location comprising : an aperture (30) in the first surface communicating with a second compartment suitable for retaining a second solution; an adhesive region (18) surrounding the aperture to which the object can be adhered to in the first compartment ( 36) form a high-resistance seal with the second compartment (6); a first measuring electrode (8) located in said first compartment, which in use contacts the first solution; a second measuring electrode (16) located in the second compartment, which in use contacts the second solution; measurement means (200, 242), electrically connected to the first and second measurement electrodes (8, 16), adapted to perform electrical measurements on the object (2) adhered to the adhesion area at the measurement location; and Object localization means (246) comprising first localization electrodes (8, 40, 50, 60...) and adapted to provide an AC signal to the electrodes to form an AC field in the first solution, the AC field acting to The object is moved towards the measurement position by dielectrophoresis. 2. The device according to claim 1, wherein the object localization means (246) further comprises a second localization electrode (40) located in the first or second compartment. 3. The device according to claim 1 or claim 2, wherein the first measuring electrode (8) and the first positioning electrode are provided as a single electrode structure. 4. Apparatus according to claim 1 or claim 2, wherein the second measuring electrode and the first positioning electrode are provided as a single electrode structure. 5. Apparatus according to any one of claims 1 to 3, wherein one of said measurement electrodes is electrically common to more than one of said measurement locations. 6. The device according to any preceding claim, wherein the sticking area (18) comprises a first area (44) directly surrounding the aperture (30), the object (2) can stick to this area, the sticking area is not stuck to by the object The second area (46) on it is to surround. 7. The device according to claim 6, wherein the aperture (30) has a diameter of 5 microns or less. 8. The device according to claim 7, wherein the aperture (30) has a diameter of 2 microns or less. 9. The device of claim 6, claim 7 or claim 8, wherein the first region has a diameter of 10 microns or less. 10. An apparatus for performing electrical measurements on an object (140) comprising ion channels in a medium, comprising: a substrate (102) having a first surface which in use is in contact with a first solution and on which one or more measurement locations (100) are located, each location comprising: a first measurement electrode (130 ); an adhesive region (134) surrounding the first measurement electrode to which the object can be adhered to form a high resistance seal between the first measurement electrode and the first solution; and a conductive track (116) for attaching the first measurement electrode a measuring electrode is connected to the measuring instrument while keeping it isolated from the first solution; a second measuring electrode (108), which in use is in contact with the first solution; a measuring device (200, 242) electrically connected to the first measuring electrode and the second measuring electrode and adapted to perform electrical measurements on the object (140) adhered to the adhered area at the measuring location; an object localization device (246) comprising a first localization electrode (150) on the substrate and adapted to provide an AC signal to the localization electrode to form an AC field in the first solution, the AC field acting to pass Dielectrophoresis moves the object towards the measurement location. 11. The device of claim 10, wherein the first positioning electrode (150) substantially surrounds or is adjacent to the adhesion area. 12. Apparatus according to claim 10 or claim 11, wherein the first positioning electrode and the second measuring electrode are formed as a single electrode structure (108). 13. Apparatus according to any one of claims 10 to 12, wherein the adhesion region (134) comprises a first region immediately surrounding the first measurement electrode, to which the object can be adhered, the first region consisting of a region to which the object does not adhere. The second area to surround. 14. The apparatus of claim 13, wherein the first measurement electrode has a diameter of 5 microns or less. 15. The apparatus of claim 14, wherein the first measurement electrode has a diameter of 3 microns or less. 16. The device of claim 13, claim 14 or claim 15, wherein the first region has a diameter of 10 microns or less. 17. Apparatus according to claim 10, wherein the adhesion area (134) and the first measurement electrode (130) are spatially separated such that in use the volume (142) is between the object (140) adhered to the adhesion area and the first measurement electrode formed between. 18. Apparatus according to any preceding claim, wherein the object positioning means (246) further comprises entrainment means (222) for generating a flow of the first solution in the first compartment (36). 19. Apparatus according to claim 18, wherein said entrainment means comprises a suction pump. 20. Apparatus according to any preceding claim, wherein the measuring means (200, 242) further comprises a pulse generator (246) adapted to apply an electrical pulse or sequence of pulses between said measuring electrodes until an electrode between predetermined levels of impedance. 21. A device according to any preceding claim, further comprising dispensing means (221) for delivering the subject and the first solution in the vicinity of the measurement location. 22. The apparatus according to claim 21, wherein the distribution means comprises a plurality of microchannels formed in the first surface of said substrate, each microchannel communicating with a measurement location. 23. Apparatus according to any preceding claim, wherein the measurement location further comprises a confinement wall (38, 106) separating the measurement location from an adjacent measurement location. 24. Apparatus according to claim 23, wherein the confinement wall comprises a deposited or adhesive layer applied to the substrate and patterned thereon to define said measurement location. 25. Apparatus according to any preceding claim, further comprising one or more positioning electrodes arranged around each measurement location in a configuration so as to be able to apply a diverging electric field about the measurement location. 26. The apparatus of claim 25, further comprising a plurality of electrodes arranged around each measurement location, adapted to generate a negative dielectrophoretic force to drive the object towards the vicinity of the respective measurement location. 27. Apparatus according to any one of claims 1 to 9, wherein said object positioning means further comprises entrainment means for creating flow of the first solution through said aperture. 28. An experimental structure for use in making electrical measurements on an object comprising ion channels in a medium, comprising: A substrate (12) having a first surface on which one or more measurement locations (1) are located, each measurement location comprising: an aperture (30) located in the first surface and communicating with the second surface of the substrate; an adhesive region (18) around the aperture to which the object can be adhered to form a high resistance seal; at least one positioning electrode (40, 50, 60...), which substantially surrounds or is adjacent to the adhesive layer region, configured for applying an AC signal to form an AC field in the vicinity of the measurement location, the AC field acting to move by dielectrophoresis The object is oriented towards the measurement position. 29. The test structure according to claim 28, further comprising a first measuring electrode (8) formed on or near the first surface; and a second measuring electrode (8) formed on or near the second surface 16). 30. A test structure according to claim 28 or claim 29, wherein the positioning electrode (40) comprises a conductive layer around the aperture on the substrate, and the adhesion area comprises a dielectric layer (44) on the positioning electrode. 31. A test structure according to claim 28 or claim 29, further comprising an anti-adhesive region (46) surrounding the adhered region. 32. A test structure according to any one of claims 28 to 31, wherein the pores have a diameter of 5 microns or less. 33. A test structure according to claim 32, wherein the diameter of the pores is 2 microns or less. 34. A test structure according to any one of claims 28 to 33, wherein the adhered areas have a diameter of 10 microns or less. 35. A test structure according to claim 28 or claim 29, comprising a plurality of measurement locations, each measurement location being separated by an insulating wall (38). 36. A test structure according to claim 35, wherein the insulating wall comprises a field layer deposited on said substrate, each of a plurality of wells in said field layer defining one of said measurement locations. 37. A test structure according to any one of claims 28 to 36, wherein the apertures (30) are formed in the film material (4) on the first surface, bridging a considerable portion of the larger through-holes formed in the substrate material, but Not all, and wherein the positioning electrodes (40) are formed on the underside of the film material (31). 38. A test structure according to claim 28 or claim 29, further comprising a plurality of positioning electrodes arranged in an array adjacent to each measurement location, adapted to enable application of traveling wave dielectrophoresis to the subject to drive the subject towards the aperture. 39. A test structure according to claim 38, wherein the electrode array comprises a series of concentric tracks (50) extending around the measurement location. 40. A test structure according to claim 39, wherein the concentric track is a partial ring around the measurement location. 41. A test structure according to claim 39, wherein the concentric track is a full circle around the measurement location. 42. A test structure according to claim 38, wherein the electrode array comprises a series of concentric helical tracks (60). 43. A test structure according to claim 42, wherein the electrode array further comprises conductive tracks to said at least one positioning electrode. 44. A test structure according to claim 38, wherein the array of electrodes comprises a linear array. 45. An experimental structure for use in making electrical measurements on an object comprising ion channels in a medium, comprising: A substrate (12) having a first surface on which one or more measurement locations (1) are located, each measurement location comprising: a first measuring electrode (130); an adhesive region (14) around the first measurement electrode to which an object can be adhered to form a high resistance seal; and At least one positioning electrode (40, 50, 60...), which substantially surrounds or is adjacent to the adhesive layer region, is configured for applying an AC signal to form an AC field in the vicinity of the measurement location, the AC field acting to detect by dielectrophoresis Move the object towards the measurement location so that it adheres to the adhered area to form a high resistance seal. 46. The test structure according to claim 45, further comprising a second measurement electrode (108) on the first surface, separated from said first measurement electrode at least by said adhesive region. 47. A test structure according to claim 45 or claim 46, wherein the diameter of the first measurement electrode is 5 microns or less. 48. The apparatus of claim 47, wherein the diameter of the first measurement electrode is 3 microns or less. 49. A device according to any one of claims 45 to 48, wherein the adhered areas have a diameter of 10 microns or less. 50. Apparatus according to claim 45 or claim 46, wherein the adhesion area (134) and the first measurement electrode (130) are spaced apart such that in use the volume (142) is between an object (140) adhered to the adhesion area and formed between the first measuring electrodes. 51. The test structure of claim 45, further comprising a plurality of positioning electrodes arranged in an array adjacent to each measurement location, adapted to be capable of applying traveling wave dielectrophoresis to the object to drive the object toward the first measurement electrode. 52. A test structure according to claim 51, wherein the electrode array comprises a series of concentric tracks (50) extending around the measurement location. 53. A test structure according to claim 52, wherein the concentric track is a partial ring around the measurement location. 54. A test structure according to claim 52, wherein the concentric track is a full circle around the measurement location. 55. A test structure according to claim 51, wherein the electrode array comprises a series of concentric helical tracks (60). 56. A test structure according to claim 55, wherein the electrode array further comprises conductive tracks to said first measurement electrodes. 57. The test structure of claim 51, wherein the array of electrodes comprises a linear array. 58. A method for making electrical measurements on an object comprising ion channels in a medium, comprising the steps of: providing a first solution comprising a suspended object to be measured to a first surface of a substrate having an aperture therein in communication with a second surface of the substrate; providing a second solution to the second surface to establish fluid contact between the first and second surfaces; testing that fluid contact has been achieved between the first and second electrodes by measuring electrical continuity between the first and second electrodes respectively located on or adjacent to the first and second surfaces; as well as The object to be measured in the first solution is driven by dielectrophoresis to a measurement location with an adhesive area surrounding the aperture. 59. The method of claim 58, further comprising the step of testing the resistance of the seal at the measurement location on the substrate by measuring the electrical impedance between said first and second electrodes. 60. The method of claim 58 or claim 59, further comprising the step of establishing a measurement configuration of the subject by applying one or more electrical pulses between the first and second electrodes, and performing measurements on said subject . 61. The method of claim 58, further comprising the step of enhancing adhesion of the object to the adhesion area by applying reduced pressure to the second solution. 62. The method of claim 58, further comprising the step of establishing the measurement configuration of the subject by applying the pore forming compound to a region of the subject enclosed by the high resistance seal and in contact with the second solution. 63. The method of claim 58, further comprising the step of providing the test compound in solution into the vicinity of the subject while operating the measuring device to observe the subject's electrical response to the presence of the compound and to electrical stimulation provided by the measuring device. 64. A method for making electrical measurements on an object comprising ion channels in a medium, comprising the steps of: providing a first solution comprising a suspended object to be measured to a first surface of a substrate having a first measurement electrode thereon, and an adhesive region surrounding said first electrode; providing a second measurement electrode in electrical contact with said first solution; and The object to be measured in the first solution is driven to the measurement position by dielectrophoresis, so that the object adheres to the adhesion area to form a high-resistance seal therewith. 65. The method of claim 64, further comprising the step of testing the resistance of the seal at the measurement location on the substrate by measuring the electrical impedance between said first and second electrodes. 66. The method of claim 64 or claim 65, further comprising the step of establishing a measurement configuration of the subject by applying one or more electrical pulses between the first and second electrodes, and performing measurements on said subject .

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