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WO2003057819A1 - Pointes de pipette de cuvette d'electroporation, reseaux de cuvettes a plusieurs puits, appareil gabarit d'electrode concu pour une automatisation et utilisations de ceux-ci - Google Patents

Pointes de pipette de cuvette d'electroporation, reseaux de cuvettes a plusieurs puits, appareil gabarit d'electrode concu pour une automatisation et utilisations de ceux-ci Download PDF

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Publication number
WO2003057819A1
WO2003057819A1 PCT/US2003/000343 US0300343W WO03057819A1 WO 2003057819 A1 WO2003057819 A1 WO 2003057819A1 US 0300343 W US0300343 W US 0300343W WO 03057819 A1 WO03057819 A1 WO 03057819A1
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WO
WIPO (PCT)
Prior art keywords
electrode
electrical potential
receptacle
species
pipette tip
Prior art date
Application number
PCT/US2003/000343
Other languages
English (en)
Inventor
James B. Finley
Robert Lawrence Frost
Michael Harrington
Thomas P. Lewis
Original Assignee
Uab Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Uab Research Foundation filed Critical Uab Research Foundation
Priority to AU2003206415A priority Critical patent/AU2003206415A1/en
Priority to CA002472686A priority patent/CA2472686A1/fr
Publication of WO2003057819A1 publication Critical patent/WO2003057819A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/50—Means for positioning or orientating the apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
    • C12M33/06—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles for multiple inoculation or multiple collection of samples
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion

Definitions

  • This invention relates generally to improved devices and methods for the electroporation of species, such as, but not limited to, cells, organelles or vesicles. These improved methods and devices are adapted for automation and allow high through-put electroporation of species.
  • transformation In molecular biology research and the practice of biotechnology, it is often desired to transfer DNA or other molecules from a solution into a living cell or other membrane-bounded species.
  • introduction of plasmid DNA into a bacterial cell generally termed "transformation,” is used to introduce non-native genetic material into a host cell.
  • transformation, or related processes are necessary for gene cloning, use of siRNA and DNA library construction.
  • Other processes, for which transfer of material into or out of membrane bounded vesicles, cells or organelles include but are not limited, to production of cloned proteins, tests on the effects of inserting materials into cells and collection of material from inside cells.
  • One principle in common of each of these is that for many substances, there is no effective transfer across the untreated membrane of a cell, vesicle or organelle.
  • Electroporation the transient formation of small holes in dielectric membranes in response to electric fields can be used to render otherwise impermeable membranes permeable to substances such as, but not limited to, drugs, nucleic acids and proteins (Neumann et al., EMBO J. 1:841-845 (1982); BioTechniques 6(7): 6750 (1988)).
  • the use of the electric field techniques used to electroporate cells can be used to fuse vesicles or cells. This technique, as well as others illustrating the state of the art, can be found in U.S. Patent No. 4,849,089 and references found therein.
  • electroporation involves applying a brief, high-voltage pulse to a sample of containing membrane bounded species, such as cells. If, for example, a population of cells are subjected to such a brief, high-voltage pulse in the presence of DNA, the process will result in DNA uptake by a subpopulation of surviving organisms.
  • the peak electric field strength used for electroporation of bacterial cells is typically in the range of 0.3 to 30 kV/cm. Modifications for the purposes of optimizing the technique of electroporation for specific and varied cells types are well known to those of skill in the art; for example, low transformation efficiencies by electroporation can often be increased by systematic evaluation of several key variables.
  • These variables include the cell density, the solution used to suspend the cells, the voltage and time of the electrical pulse, and the temperature at which the process is performed.
  • systems that perform electroporation are widely known to those of skill in the art and include those manufactured by or for Amaxa, BioRad, BTX, Eppendorf and others.
  • Control of the electrical pulse used in electroporation can include the use of techniques known to those of skill in the art. Examples of these techniques illustrating the skill include those described by U.S. Patent Nos. 6,150,148 and references incorporated therein.
  • Electroporation systems are commercially available. Most are designed to use a square plastic cuvette with metal electrodes embedded in the sides. Each cuvette has a fixed spacing between the electrodes. Typically, cuvettes are available with electrode spacing from 0.1 to 4.0 cm, resulting in a required peak pulse of 0.1 to 3.0 kV. This pulse is usually produced by discharge of a high voltage capacitor.
  • cells are prepared at a certain cell density in a specific buffer. A measured amount of solution containing cells treated for the process (typically 50-100 microliters) is removed and mixed with plasmid DNA.
  • solutions, cell and DNA are typically delivered by manual pipetting and are placed either directly into an electroporation cuvette, in which case they are mixed in the cuvette, or are mixed together in a separate container and then transferred to the electroporation cuvette.
  • the cuvette is then covered and inserted by hand into a chamber adapted for the electroporation cuvette.
  • the chamber contains electrical contacts that, when current is applied, conduct the current pulse from the capacitor to the cuvette. After the pulse has been applied, the cuvette is removed by hand from the chamber and the solution, containing cells and DNA is then removed from the cuvette by pipetting for transfer to a separate container.
  • U.S. Patent No. 5,422,272 by Papp et al. (hereafter, "the '272 patent") describes an attempt to provide an improved means for electroporation.
  • the described device which combines the functions of a pipette tip and an electroporation cuvette, while an improvement over the then-state-of-the-art, still requires the manual manipulation of the device and is not amenable to automation.
  • the pipette tip of Papp et al. is adapted for use with a hand held version of the electrical apparatus required to perform the pipetting and the electroporation steps. This does not suggest any aspect of the present invention and is, furthermore, not adaptable to further automation.
  • this invention in a first aspect, relates to a pipette tip that includes a receptacle having a proximal opening having a size suitable for receiving a distal end of a shaft and a distal tip opening having a size suitable for picking up and/or dispensing a fluid from the pipette tip, an electrode assembly including two or more electrodes, the electrodes being spaced one from the other to form a cavity wherein the fluid may be transiently retained, and an electrode contact assembly including two or more electrode contacts, wherein the electrode contacts are conductively connected to the electrodes.
  • the invention in a second aspect, relates to a device for performing electroporation.
  • the device can include a receptacle support, wherein the receptacle support is adapted to support a removably attachable receptacle comprising two or more electrode contacts, wherein the two or more electrode contacts include a first electrode contact and a second electrode contact.
  • the device can include; at least one actuator, whereby the device can provide a relative negative pressure or a relative positive pressure to the volume of the removably attached receptacle; two or more electrical potential contacts, wherein the two or more electrical potential contacts include a first electrical potential contact for conductively contacting the first electrode contact and a second electrical potential contact for conductively contacting the second electrode contact; and a controller unit for executing a procedure on the removably attachable receptacle, wherein the procedure includes the application of the relative negative or the relative positive pressure to the volume of the receptacle.
  • the invention in a third aspect, relates to a device-implemented method for electroporating a species.
  • This method can include the steps of: attaching a removably attachable receptacle to any device made according to the second aspect of the invention; providing a fluid sample, wherein the fluid sample includes the species to be electroporated; applying a negative pressure to the removably attachable receptacle, thereby drawing in a portion of the fluid sample; and applying an electrical potential to the removably attachable receptacle, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species.
  • the invention in a fourth aspect, relates to a device-implemented method for introducing a molecule into a species.
  • This method can include the steps of: attaching a removably attachable receptacle to any device made according to the second aspect of the invention; providing a fluid sample, wherein the fluid sample includes the species to be electroporated; applying a negative pressure to the removably attachable receptacle, thereby drawing in a portion of the fluid sample; applying an electrical potential to the removably attachable receptacle, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species; and providing molecules in the fluid sample, wherein the molecules are introduced into the electroporated species.
  • the invention in a fifth aspect, relates to a computer program product for controlling a device for performing electroporation.
  • the computer program product can include a computer-readable medium on which is recorded a procedure for controlling a device for performing electroporation.
  • the procedure can include application of a relative negative or a relative positive pressure to the volume of a removably attached receptacle.
  • the invention in a sixth aspect, relates to an assembly for holding a plurality of removably attachable volumetric containers.
  • the assembly can include a rack having an upper surface member defining a plurality of holes in a regular pattern.
  • This plurality of holes can be formed to receive and hold volumetric containers.
  • the assembly can be adapted to maintain any containers held in the rack at a selected temperature.
  • the invention in a seventh aspect, relates to a device for performing electroporation.
  • the device can include: a receptacle support, wherein the receptacle support is adapted to support a removable receptacle comprising two or more electrode contacts; two or more electrical potential contacts, wherein the two or more electrical potential contacts include a first electrical potential contact for conductively contacting the first electrode contact and a second electrical potential contact for conductively contacting the second electrode contact; and a controller unit for executing a procedure on the removable receptacle.
  • the procedure can include; transfer of the removable receptacle from a storage area to the receptacle support; provision of a fluid sample in the removable receptacle, wherein the fluid sample includes the species to be electroporated and molecules to be introduced into the species; application of an electrical potential to the removable receptacle, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species, thereby introducing the molecules into the species therein; transfer of the removable receptacle transferred to the receptacle support earlier from the receptacle support; and optionally, repetition of each of these steps to repeat the process defined by the procedure.
  • the invention relates to a device-implemented method for introducing a molecule into a species.
  • the method can include: attaching a removable receptacle to any device made according to the seventh aspect of the invention; providing a fluid sample, wherein the fluid sample includes the species to be electroporated; applying an electrical potential to the removable receptacle, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species; providing molecules in the fluid sample, wherein the molecules are introduced into the electroporated species; removing the removable receptacle from the device made according to the seventh aspect of the invention; and optionally, repetition of each of these steps to repeat the process defined by the procedure.
  • the invention in a ninth aspect, relates to a multi channel pipette tip that includes: a plurality of receptacles having at least one proximal opening having a size suitable for receiving a distal end of at least one shaft and a plurality of distal tip openings of a size suitable for picking up and dispensing a plurality of fluid samples from the multiple channel pipette tip; a plurality of electrode assemblies, wherein each electrode assembly includes two or more electrodes, the electrodes being spaced one from the other to form a cavity wherein the fluid may be transiently retained; and at least one electrode contact assembly including two or more electrode contacts, wherein the electrode contacts are conductively connected to the electrodes.
  • the invention in a tenth aspect, relates to a device for performing electroporation of a plurality of samples.
  • the device can include: a receptacle support, wherein the receptacle support is adapted to support a multi channel pipette tip comprising at least one electrode contact assembly consisting of a first electrode contact and a second electrode contact; at least one actuator, whereby the device can provide a relative negative pressure or a relative positive pressure to at least one volume of the multi channel pipette tip; at least one electrical potential contact assembly consisting of at least a first electrical potential contact for conductively contacting the first electrode contact and at least a second electrical potential contact for conductively contacting the second electrode contact; and a controller unit for executing a procedure on the multi channel pipette tip, wherein the procedure includes the application of a the relative negative pressure or the relative positive pressure to at least one volume of the multi channel pipette tip.
  • the invention in an eleventh aspect, relates to a device implemented method for electroporating a species.
  • the method can include the steps of: attaching a multi channel pipette tip to any device made according to the tenth aspect of the invention; providing at least one fluid sample, wherein the at least one fluid sample includes species to be electroporated; applying a negative pressure to the multi channel pipette tip, thereby drawing in at least one portion of the at least one fluid sample; and applying an electrical potential to the multi channel pipette tip, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species.
  • the invention in a twelfth aspect, relates to a device-implemented method for introducing a molecule into a species.
  • the method can include: attaching a multi channel pipette tip to any device made according to the tenth aspect of the invention; providing a set of fluid samples, wherein the set of fluid samples includes the species to be electroporated; applying a negative pressure to the multi channel pipette tip, thereby drawing in a portion of the fluid samples; applying an electrical potential to the multi channel pipette tip, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species; and providing molecules in the fluid samples, wherein the molecules are introduced into the electroporated species.
  • the invention in a thirteenth aspect, relates to an electroporation cuvette plate that includes: a plate with a plurality of wells, wherein the wells can transiently retain a fluid; at least one electrode assembly including two or more electrodes, the electrodes being spaced one from another to form a cavity wherein the fluid can be transiently retained; and at least one electrode contact assembly further including two or more electrode contacts, wherein at least two electrode contacts are conductively connected to the electrodes of at least one electrode assembly.
  • the invention in a fourteenth aspect, relates to a device for performing electroporation.
  • the device can include: an electroporation cuvette plate support, wherein the electroporation cuvette plate support is adapted to support any electroporation cuvette plate made according to the thirteenth aspect of the invention; at least one electrical potential contact assembly comprising a first electrical potential contact for conductively contacting the first electrode contact and a second electrical potential contact for conductively contacting the second electrode contact; and a controller unit, adapted for executing a procedure on the electroporation cuvette plate.
  • the invention in a fifteenth aspect, relates to a device-implemented method for electroporating a species.
  • the method can include the steps of: attaching an electroporation cuvette plate to any device made according to the fourteenth aspect of the invention; providing at least one fluid sample, wherein the fluid sample includes the species to be electroporated and is present in at least one well of the electroporation cuvette plate; and applying an electrical potential to the electroporation cuvette plate, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species.
  • the invention relates to a device-implemented method for introducing a molecule into a species.
  • the method can include: attaching an electroporation cuvette plate to any device made according to the fourteenth aspect of the invention; providing at least one fluid sample, wherein the fluid sample includes the species to be electroporated and molecules to be introduced into the species and wherein the fluid sample is present in at least one well of the electroporation cuvette plate; applying an electrical potential to the electroporation cuvette plate, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species; and providing molecules in the fluid sample, wherein the molecules are introduced into the electroporated species.
  • the invention relates to a computer program product for controlling a device for performing electroporation.
  • the computer program product can include a computer-readable medium on which is recorded a procedure for controlling a device for performing electroporation.
  • the procedure can include attaching any electroporation cuvette plate made according to the thirteenth aspect of the invention.
  • the invention relates to an electrode template apparatus that includes: an array of electrode assemblies, each including two or more electrodes, the electrodes being spaced one from another to form a cavity wherein the fluid can be transiently retained; and at least one electrode contact assembly further including two or more electrode contacts, wherein at least two electrode contacts are conductively connected to the electrodes of at least one electrode assembly.
  • the invention in a nineteenth aspect, relates to a device for performing electroporation.
  • the device can include: an electrode template apparatus support, wherein the electrode template apparatus support is adapted to support any electrode template apparatus made according to the eighteenth aspect of the invention; at least one electrical potential contact assembly comprising two or more electrical potential contacts, wherein the two or more electrical potential contacts include a first electrical potential contact for conductively contacting a first electrode contact and a second electrical potential contact for conductively contacting a second electrode contact; and a controller unit, adapted for executing a procedure on the electrode template apparatus.
  • the invention in a twentieth aspect, relates to a device implemented method for electroporating a plurality of species.
  • the method can include the steps of: attaching an electrode template apparatus made according to the eighteenth aspect of the invention; providing a plurality of fluid samples, wherein the fluid samples include species to be electroporated; contacting the electrode template apparatus with the plurality of fluid samples containing species to be electroporated, thereby transiently retaining a volume of the fluid samples in the cavities formed by the at least two electrodes of each of the plurality of electrode assemblies; and applying an electrical potential to the electrode template apparatus, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species.
  • the invention relates to a device-implemented method for introducing molecules into a plurality of species.
  • the method can include: attaching an electrode template apparatus to any device made according to the nineteenth aspect of the invention; providing a plurality of fluid samples, wherein the fluid samples include species to be electroporated and molecules to be introduced into the species; contacting the electrode template apparatus with the plurality of fluid samples containing species to be electroporated, thereby transiently retaining a volume of the fluid samples in the cavities formed by the at least two electrodes of each of the plurality of electrode assemblies; applying an electrical potential to the electrode template apparatus, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species; and providing molecules in the plurality of fluid samples, wherein the molecules are introduced into the electroporated species.
  • the invention in a twenty-second aspect, relates to a computer program product for controlling a device for performing electroporation.
  • the computer program product can include a computer-readable medium on which is recorded a procedure for controlling a device for performing electroporation.
  • the procedure can include attachment of an electrode template apparatus made according to the eighteenth aspect of the invention.
  • FIG. 1 panels A, B, and C, depicts a cross-section view, side view and perspective view, respectively, of a standard pipette tip showing certain aspects that are required for some aspects of the current invention.
  • the upper part of the tip is adapted to receive the shaft of a pipetting device, forming an airtight seal.
  • the lower part of the tip is tapered to allow liquids to be removed from or transferred to small orifices.
  • FIG. 2 panels A, B, and C, depicts a side view, a side view and a perspective view, respectively, of certain aspects of a standard electroporation cuvette.
  • FIG. 3 panels A, B, and C, depicts a cross-section view, a side view and a perspective view, respectively, of one embodiment of an electroporation pipette tip.
  • the upper part of the tip having a proximal opening enables it to attach to a distal end of a shaft of a pipette. This attachment allows the same airtight seal as a standard tip.
  • the lower part of the tip having a distal tip opening has a size suitable for picking up and dispensing a fluid.
  • the body of the electroporation pipette tip is constructed predominantly of an electrically insulating material. Electrically conductive pieces embedded in two sides of the tip form electrode contacts. These are conductively connected to the electrodes, the two conductive pieces forming parallel planes separated by a fixed distance located on the interior of the pipette tip.
  • FIG 4 panels A, B, and C, depicts a cross-section view, a side view, and a perspective view, respectively, of another embodiment of an electroporation pipette tip.
  • This example has integral metal conduction plates that more closely approximate the shape of a standard pipette tip.
  • Figure 5 depicts a schematic view of a device for performing electroporation with an electroporation pipette tip like that shown in Figure 3.
  • the device is shown moving the pipette tip from a pipette holder / cooling station to a well plate (where the sample to be processed will be drawn from) and finally to a receptacle support.
  • the sample Upon completion of electroporation, the sample will be deposited in a new well plate or other sample container for growth and/or further processing.
  • Figure 6 is a schematic view of a device for performing electroporation with an electroporation pipette tip like that shown in Figure 4.
  • the device is shown moving the pipette tip from the pipette holder / cooling station to a well plate (where the sample to be processed will be drawn from) and finally to a receptacle support.
  • the sample Upon completion of electroporation, the sample will be deposited in a new well plate or other sample container for sample growth and further processing.
  • the receptacle support and high voltage source are designed to provide fully automated operation with commercial robotic systems through the inclusion of novel control electronics and software. All components are compatible with commercial production methods.
  • Figure 7 shows the electroporation process using both a pipette tip like that shown in Figure 3 and in Figure 4.
  • FIG. 8 panels X, Y and Z, depict a side view, a cross-section view and a perspective view, respectively, of another embodiment of the pipette tip of the invention.
  • Figure 9 shows a detailed drawing of a portion of a device for performing electroporation. Included in the drawing is a depiction of a first and second electrical potential contact for conductively contacting the first electrode contact and the second electrode contact of the cuvette shown.
  • the particular example shown is further an example having electrical potential contacts that are elongate springable shapes.
  • FIG 10 depicts a top view and a perspective view, respectively, of one aspect of the invention, the rack, that maintains the removably attachable volumetric containers at a selected temperature.
  • the temperature of the rack is equilibrated by a fluid supplied to the rack by an inlet passage and removed from the rack by another passage. Some internal volume of the rack transiently retains fluid during its passage through the block.
  • Figure 11 depicts an embodiment of a multi channel tip. In the particular embodiment shown, each volume formed in the tip has a corresponding proximal and distal opening.
  • Figure 12 depicts selected aspects of a device for performing electroporation of a plurality of samples in a multi channel tip of the invention.
  • Figure 13 depicts an electroporation cuvette.
  • the depicted object is an embodiment of the invention having a plurality of wells wherein a well can include an electrode assembly adapted for electroporating samples.
  • Figure 14 depicts selected aspects of a device for performing electroporation of a plurality of samples in an electroporation cuvette of the invention.
  • Figure 15 depicts selected aspects of an electrode template apparatus for performing electroporation on a plurality of samples in a multi container array or a multi well plate.
  • Figure 16 depicts an embodiment of the invention, an automated apparatus for electroporating samples, that comprises a pipette tip of the invention, an automated device for aspirating samples into and dispensing samples from the pipette tip, a support that includes electrode potential contacts, a PCR plate comprising samples to be electroporated, and a temperature controlled pipette tip rack.
  • Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:
  • the present invention relates to a pipette tip, such as those shown, for example, in Figures 3 and 4, that includes a receptacle 10 having a proximal opening 11 having a size suitable for receiving a distal end of a shaft and a distal tip opening 12 having a size suitable for picking up and dispensing a fluid from the pipette tip, an electrode assembly 13 including two or more electrodes 14, the electrodes being spaced one from the other to form a cavity 15 wherein fluid may be transiently retained, and an electrode contact assembly 16 including two or more electrode contacts 17, 18, wherein the electrode contacts are conductively connected to the electrodes 14.
  • a pipette tip such as those shown, for example, in Figures 3 and 4, that includes a receptacle 10 having a proximal opening 11 having a size suitable for receiving a distal end of a shaft and a distal tip opening 12 having a size suitable for picking up and dispensing a fluid from the pipette tip, an electrode assembly 13 including
  • the electrode contacts are surfaces on the exterior of the receptacle that provide a means for conductively attaching the receptacle to an electrical source.
  • the electrode contacts 17, 18 can be arranged in a number of different ways in accordance with requirements which will be recognized by those of skill in the art.
  • the electrode contacts can each have at least one surface substantially perpendicular to an axis running through the center of the proximal opening 11 and the center of the distal tip opening 12 and wherein the substantially perpendicular surfaces 17a, 18a face toward the distal end of the pipette tip. If the electrode contacts are so arranged, the substantially perpendicular surface 17a of the first electrode contact 17 can be nearer to the distal tip opening 12 than the electrodes 14.
  • a portion of the electrodes 14 can be nearer to the proximal opening 11 than the first electrode contact 17.
  • the substantially perpendicular surface of the first electrode contact 18 can be closer to the proximal opening than the electrodes.
  • a portion of the electrodes can be nearer to the distal tip opening than the first electrode contact 18.
  • the substantially perpendicular surface 18a of the second electrode contact 18 can be nearer to the distal tip opening than the electrodes.
  • At least a portion of the electrodes can be nearer to the proximal opening than the second electrode contact 18.
  • the substantially perpendicular surface of the second electrode contact 18 can be closer to the proximal opening 11 than the electrodes 14.
  • At least a portion of the electrodes 14 can be nearer to the distal tip opening 12 than the second electrode contact 18.
  • the electrode contacts 17, 18 can be arranged such that at least the electrode contacts 17, 18 have at least one surface substantially parallel to an axis running through the center of the proximal opening 11 and the center of the distal dip opening 12 as is shown in Fig. 8.
  • the electrodes can be arranged in a number of different ways in accordance with requirements, which will be recognized by those of skill in the art.
  • the cavity 15 wherein a fluid may be transiently retained can be formed between at least two electrodes by arranging the electrodes such that the distance between a first point on the surface of one electrode to the nearest point on the surface of the second electrode does not vary substantially with respect to the selection of the first point.
  • This can be accomplished in practice by the use of electrodes 14 that are flat, substantially parallel plates as shown in Figs. 3 and 4.
  • the surface area and the shape of the plates can vary as desired.
  • the invention can be constructed by using coaxially orientated cylindrical electrodes. Examples of such electrodes and electrode assemblies are known in the art.
  • the electrode contacts 17, 18 can be adapted to provide points of potential physical contact adapted for receiving a force directed along an axis parallel to that defined by a line extending between the distal tip opening 12 to the proximal opening 11 of the pipette tip 10. In this manner, a force can be applied to the contacts and can be directed toward the proximal opening 11, which can be adapted to fit onto a tapered shaft of a device, such that the force would cause retention of the tip on the device.
  • the electrode contacts can be arranged so that the first electrode contact 17 is closer to the relatively large proximal end of the tip than the second electrode contact 18 which is positioned nearer to the distal end of the tip than the first electrode contact 17.
  • the first 17, the second 18 or both of the electrode contacts can be circumferential. If the electrode contacts are circumferential, it will be recognized that the relative rotational position of the pipette tip 10, in regard to the axis running from the proximal to the distal end, need not be controlled to ensure contact with an electrical potential contact. In other embodiments, as shown in Figs.
  • the pipette tip 10 can include a cavity 15 formed by the spaced electrodes 14 and can be bounded by walls 20 that define an exterior surface of the pipette tip. Further, the pipette tip 10 can be constructed so that the first 17, the second 17, or both the first 17 and the second 18 electrode contacts are positioned on the exterior surface of the pipette tip as shown in Figs. 3, 4, and 8.
  • the pipette tip can include other features and attributes as are known to those of skill in the art. These features include, but are not limited to, those discussed in U.S. Patent Nos: 4,748,859 and 6,197,259. In addition, it is contemplated that certain pipette tips will be reusable and that certain pipette tips will be single-use only products. If the pipette tips are reusable, they can be used greater than 2, 3, 5, 10, 20 or 30 times. If the pipette tips are reusable, they can also be used less than 40, 30, 20, 10, 5, 4, or 3 times.
  • the invention in a second aspect, relates to a device for performing electroporation.
  • the device can include a receptacle support, an embodiment of which is shown in Fig. 9, wherein the receptacle support 30 is adapted to support a removably attachable receptacle 10 comprising a first electrode contact 17 and a second electrode contact 18.
  • the device can include; at least one actuator, whereby the device can provide a relative negative pressure or a relative positive pressure to the volume of the removably attached receptacle; a first electrical potential contact 34 for conductively contacting the first electrode contact 17 and a second electrical potential contact 35 for conductively contacting the second electrode contact 18; and a controller unit for executing a procedure on the removably attachable receptacle, wherein the procedure includes the application of the relative negative or the relative positive pressure to the volume of the receptacle.
  • the device can include a number of elements that can be arranged in a number of different ways in accordance with requirements which will be recognized by those of skill in the art.
  • other embodiments of the device can be made so that the receptacle support and at least one electrical potential contact of maintain a constant relative spatial orientation with respect to one another.
  • the receptacle support can include at least one or two electrical potential contacts.
  • the electrical potential contacts 34,35 can be resilient contact structures having a springable shape allowing contact and with a respective portion connected to the receptacle support and having spring-like qualities.
  • a portion of receptacle support 30 which includes the electrical potential contacts 34, 35 can be spring-like so that when contacted with the electrode contact assembly 16 of the pipette tip 10 of the invention, force between the two can be maintained by spring-like tension.
  • the resilient contact structure can include a further portion, the respective portion, which can be attached to the receptacle support 30.
  • the shape of the resilient contact structures can be elongate (such as, but not limited to, those structures shown in Figure 9) or can be helical (such as, but not limited to a coil spring). Further, the shapes can be compact or extended. Further, the necessary tension, that can be supplied by inclusion of a springable shape in the resilient contact structures, as described above, can also be provided by biasing the position of the electrical potential contacts by use of springs or other equivalent mechanisms as are known to those of skill in the art.
  • the device can include a number of elements that can be arranged in a number of different ways in accordance with requirements which will be recognized by those of skill in the art. Examples of arrangements are illustrated in Figs. 5-6 and 16.
  • the receptacle support 30 and at least one actuator 37 whereby the device can provide the relative negative pressure or the relative positive pressure.
  • the receptacle support and actuator can be arranged to maintain a constant relative spatial orientation with respect to one another. If so, the receptacle support 30 can itself include at least one or two electrical potential contacts 34, 35 as is shown in Fig. 9.
  • the device can further include containers, arrays of containers or multi well plates 60 to provide solutions to be taken up into the pipette tip 10 by action of the actuator 37.
  • the electrical potential contacts 34, 35 can be positioned on a clamping apparatus 50 that provides force to maintain contact between the electrical potential contacts and the electrode contacts when desired.
  • a power source 61 that provides electrical voltage/current to the electrodes can also be provided.
  • a controller unit 62 can comprise a power source or controls its function along with movement of portions of the apparatus and actions of the actuator.
  • the device can be made so that the receptacle support 30 and at least one actuator maintain a constant relative spatial orientation with respect to the receptacle support and an average spatial position of the actuator.
  • the average spatial position can be defined as an average during the operation of the device or during the operation of a limited subset of operational movements of the device.
  • the procedure of the controller unit of the device can include application of negative pressure such that the negative pressure draws a fluid sample into a removably attachable receptacle, if a fluid sample and a removably attachable receptacle are provided. Further, the procedure can further include application of positive pressure such that the positive pressure expels a fluid sample from a removably attachable receptacle, if a fluid sample and removably attachable receptacle are provided. Further, the procedure can include the application of an electrical potential.
  • the controller unit of the device can be a controller unit that can be programmed to execute a predetermined procedure. If the controller unit can be preprogrammed, the predetermined procedure includes the steps of: attaching a removably attachable receptacle comprising a first electrode contact and a second electrode contact; applying a relative negative pressure to the volume of the removably attached receptacle, whereby a volume of solution containing a species to be electroporated may be aspirated into the removably attached receptacle; contacting the removably attached receptacle with at least a first and second electrical potential contact, wherein between the first and second electrical potential contacts, an electrical potential can be applied; applying the electrical potential to the removably attached receptacle; applying a relative positive pressure to the volume of the removably attached receptacle, whereby a volume of solution containing a species that has been electroporated may be dispensed from the re
  • the controller unit can be programmed or adapted to accept input data from sensors. If the unit can be programmed or adapted to accept input data, the data can be selected from the group consisting of the voltage of an electrical potential applied during the procedure, the time required for the applied electrical potential to dissipate, the conductivity of any sample volume present in the receptacle, the resistance of any sample volume present in the receptacle, the temperature of the sample before applying the electrical potential, the temperature of the sample during application of the electrical potential, and the temperature of the sample after applying the electrical potential. If data is accepted, it can be compared to a repository of normal values, thereby measuring variances between the input data and the normal values and wherein variances greater than an unacceptable value generate an alarm signal. Design and construction of such devices will be recognized as straightforward processes by those of skill in the art. Also, the selection of "normal" values and the limits to variances required to generate alarm signals, halt the procedure being undertaken or otherwise generate a signal is within the skill on one of the art.
  • the controller unit of the device can be adapted to maximize the ease of use.
  • Certain aspects of the controller unit can include: the ability to adjust parameters in real time or near real time using either direct control or immediate update of input information and/or control directions by the user. Further, the controller unit can display information gathered as the data is collected. The controller unit can input data into a program for executing a control procedure such that the procedure executed by the present invention can be modified or optimized during the execution of the procedure to fulfill goals as can be determined by those of skill in the art.
  • the predetermined procedure can include an automated test procedure to generate a sparse matrix screen based on user input values such as ranges of temperatures, ranges of time constants or ranges of voltages.
  • a sparse matrix screen can include 1, 2, 3, 5, 10, 20 or 50 different values for variables known or believed by those of skill in the art to be important for the outcome generated (e.g., transformation efficiencies, heating, etc.). Examples of variables include, but are not limited to, temperature of the sample, time constants, and applied voltages.
  • the sparse matrix screen generated can further include collected data relating to the result/effects of the test conditions. Design and construction of such devices, including the programming, will be recognized as straightforward by those of skill in the art.
  • the device can also include a removably attachable receptacle, wherein the receptacle includes an electrode assembly of two or more electrodes, the electrodes being spaced one from the other to form a cavity wherein a fluid can be transiently retained, and an electrode contact assembly including two or more electrode contacts, wherein the electrode contacts are conductively connected to the electrodes.
  • the included removably attachable receptacle can be an electroporation cuvette or it can be a pipette tip like that described earlier.
  • the included removably attachable receptacle can include: a receptacle having a proximal opening having a size suitable for receiving a distal end of a shaft and a distal tip opening having a size suitable for picking up and dispensing a fluid from the pipette tip; an electrode assembly including two or more electrodes, the electrodes being spaced one from the other to form a cavity wherein the fluid may be transiently retained; and an electrode contact assembly including two or more electrode contacts, wherein the electrode contacts are conductively connected to the electrodes.
  • the device can be adapted to operate in conjunction with known robotic manipulation systems or can comprise known robotic manipulation systems or portions thereof.
  • Such systems include, but are not limited to, those manufactured by Beckman-Coulter and Qiagen.
  • adaptations of or assemblies that include Biomek 2000 and Biomek FX devices as are well- known to those of skill in the art, or other related types of devices can be used (see www.beckmancoulter.com/products/instrument/austomatedsolutions/biomek/biome k2000 inst dcr.asp or www.beckmancoulter.com/piOducts/instrument/ automatedsolutions/biomek/biomekfx inst dcr.asp) and the BioRobot 8000, 3000, 9604 or 9600 (see www.qiageninstruments.com/instruments/).
  • the invention in a third aspect, relates to a device implemented method for electroporating a species.
  • One embodiment includes the steps of: attaching a removably attachable receptacle to any device 30 or 50 made according to the second aspect of the invention; providing a fluid sample, wherein the fluid sample includes the species to be electroporated; applying a negative pressure to the removably attachable receptacle 10, thereby drawing in a portion of the fluid sample; and applying an electrical potential to the removably attachable receptacle 10, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species.
  • the device-implemented method can optionally include applying a positive pressure to the removably attachable receptacle, thereby dispensing a portion of the fluid sample, or detaching the removably attachable receptacle from the device. If the receptacle is removed from the device, it can be placed in a rack adapted for retaining the removably attachable receptacle or it can be placed in a waste container.
  • the device-implemented method can be used to electroporate many different species.
  • the species can include, but are not limited to, cells, organelles, micelles and liposomes. If the species are cells, they can be, but are not limited to, gram- positive bacteria, gram-negative bacteria, acid-fast bacteria, animal cells, plant cells, fungus cells and protozoan cells.
  • the cells can be germ cells (i.e., sperm or ova).
  • the cells can be healthy or diseased.
  • Example of diseased cells include, but are not limited to cancer cells, cells deficient in expression of genes normally expressed in healthy cells or cells deficient in expression of genes not normally expressed, but which, if expressed, would ameliorate or compensate for the expression of other genes.
  • the cells can be cells derived from cell culture or from tissue samples. If the species to be electroporated are organelles, they can include, but are not limited to, nuclei, chloroplasts, mitochondria and vesicles. If the species to be electroporated are liposomes, the liposomes can be, but are not limited to, low-density liposomes, high-density liposomes, red blood cells, white blood cells and platelets.
  • the device-implemented method to electroporate species can further include contacting the electroporated species with effector agents thereby facilitating uptake of the effector agent into the electroporated species.
  • the effector agent can be, but is not limited to, oligonucleotides, RNA, DNA, nucleotides, polypeptides, peptides, hormones, antibodies, small molecules, analogs, combinations or derivatives thereof.
  • the RNA can be interfering RNA such as siRNA, that can be used in suitable cells.
  • Suitable cells include those such as, but not limited to, mammalian cells, and can be used in accordance with the methods known to those of skill in the art.
  • RNA interference and its use to silence gene expression are incorporated herein for their teachings, particularly their teachings of how to silence expression of genes, putative genes, or potential genes, by transfecting or transforming cells with RNA molecules; Fire et al., "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans," Nature 391: 806-811 (1998); Kennerdell and Carthew, "Use of dsRNA-mediated interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway," Cell 95: 1017-1026 (1998); Ngo et al., "Double-stranded RNA induces mRNA degradation in Tryp ⁇ nosom ⁇ brucei, " Proc.
  • RNAi as random degradative PCR, siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs
  • Sijen et al. “On the role of RNA amplification in dsRNA-triggered gene silencing,” Cell 107: 465-476 (2001); Grishok et al., “Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C.
  • the device-implemented method can introduce a molecule into a species.
  • the method can include: attaching a removably attachable receptacle to the device; providing a fluid sample, wherein the fluid sample includes the species to be electroporated and molecules to be introduced into the species; applying a negative pressure to the removably attachable receptacle, thereby drawing in a portion of the fluid sample; and applying an electrical potential to the removably attachable receptacle, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species, thereby introducing the molecules into the species therein.
  • the method can further include an addition step of applying a positive pressure to the removably attachable receptacle, thereby dispensing a portion of the fluid sample, after the molecules have been introduced into the species.
  • the method can further include detaching the removably attachable receptacle from the device. If the device-implemented method includes detaching the removably attachable receptacle, the removably attachable receptacle can be placed in a rack adapted for retaining the removably attachable receptacle or it can be placed in a waste container.
  • the invention in a fourth aspect, relates to a device-implemented method for introducing a molecule into a species.
  • This embodiment can include the steps of: attaching a removably attachable receptacle to any device made according to the second aspect of the invention; providing a fluid sample, wherein the fluid sample includes the species to be electroporated; applying a negative pressure to the removably attachable receptacle, thereby drawing in a portion of the fluid sample; applying an electrical potential to the removably attachable receptacle, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species; and providing molecules in the fluid sample, wherein the molecules are introduced into the electroporated species.
  • Providing molecules in the fluid sample can be accomplished by introduction of the molecules into the fluid sample either prior to or following the provision of the electrical potential and, consequently, the electroporation of the species.
  • the invention relates to a computer program product for controlling a device for performing electroporation.
  • the computer program product can include a computer-readable medium on which is recorded a procedure for controlling a device for performing electroporation.
  • the procedure can include application of a relative negative or a relative positive pressure to the volume of a removably attached receptacle.
  • the procedure encoded by the computer program product can further comprise the step of applying an electrical potential to the removably attached receptacle or detaching the removably attachable receptacle.
  • the procedure of the computer program product can further include the acceptance of input data from sensors, wherein consideration of the input data determines steps taken by the device for performing electroporation.
  • Input data which can be accepted includes, but is not limited to, the voltage of an electrical potential applied during the procedure, the time required for the applied electrical potential to dissipate, the conductivity of any sample volume present in the receptacle, the resistance of any sample volume present in the receptacle and the temperature of the sample. Sensors suitable for the collection of such data are known to those of skill in the art. If data is collected, the input data can be compared to a repository of normal values, thereby measuring variances between the input data and the normal values and wherein variances greater than an unacceptable value generate an alarm signal.
  • the invention in a sixth aspect, relates, as illustrated in Fig. 10, to an assembly 40 for holding a plurality of removably attachable volumetric containers.
  • the assembly can include a rack having an upper surface member defining a plurality of holes in a regular pattern. This plurality of holes 42 can be adapted to receive and hold volumetric containers.
  • the assembly can be adapted to maintain any containers held in the rack at a selected temperature.
  • Illustrative examples of removably attachable volumetric containers include pipette tips, cuvettes, and tubes. For example, turning to Fig.
  • the assembly 40 can be adapted for pipette tips that include: a receptacle having a proximal opening having a size suitable for receiving a distal end of a shaft and a distal tip opening having a size suitable for picking up and dispensing a fluid from the pipette tip; an electrode assembly including two or more electrodes, the electrodes being spaced one from the other to form a cavity wherein the fluid may be transiently retained; and an electrode contact assembly including two or more electrode contacts, wherein the electrode contacts are conductively connected to the electrodes.
  • the assembly 40 can comprise a surface defining a plurality of holes 42 as described. The assembly 40 can maintain the temperature of the containers at a selected temperature passively or actively.
  • Examples of passive temperature control would include the use of materials have high heat capacities.
  • Examples of active temperature control would include the use of heating and/or cooling elements such as peltier effect devices, actively heated or cooled water baths, etc.
  • the embodiment shown in Fig. 10 employs a temperature control system comprising an actively heated or cooled fluid that can be passed through the assembly through inlet 43 and outlet 44.
  • the devices could be used to control the temperature at less than 20°C, 15°C, 10°C, 8°C, 6°C, 5°C, 4°C, 3°C or 2°C.
  • the devices could be used to control the temperature at greater than 1°C, 2°C, 3°C, 4°C, 5°C or 6°C.
  • the invention in a seventh aspect, referring to Figs. 5, 6, 7 and 9, the invention relates to a device for performing electroporation.
  • the device can include: a receptacle support, wherein the receptacle support is adapted to support a removable receptacle comprising a first electrode contact 17 and a second electrode contact 18; a first electrical potential contact 34 for conductively contacting the first electrode contact 17 and a second electrical potential contact 35 for conductively contacting the second electrode contact 18; and a controller unit (such as that comprised in 62) for executing a procedure on the removable receptacle 10.
  • the procedure can include; transfer of the removable receptacle 10 from a storage area to the receptacle support; provision of a fluid sample in the removable receptacle 10, wherein the fluid sample includes the species to be electroporated and molecules to be introduced into the species; application of an electrical potential to the removable receptacle 10 through electrical potential contacts 34, 35, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species, thereby introducing the molecules into the species therein; transfer of the removable receptacle 10 previously transferred to the receptacle support from the receptacle support; and optionally, repetition of each of these steps to repeat the process defined by the procedure.
  • the invention relates to a device-implemented method for introducing a molecule into a species.
  • the method can include: attaching a removable receptacle to any device made according to the seventh aspect of the invention; providing a fluid sample, wherein the fluid sample includes the species to be electroporated; applying an electrical potential to the removable receptacle, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species; providing molecules in the fluid sample, wherein the molecules are introduced into the electroporated species; removing the removable receptacle from the device made according to the seventh aspect of the invention; and optionally, repetition of each of these steps to repeat the process defined by the procedure.
  • the invention in a ninth aspect, relates to a multi channel pipette tip.
  • this embodiment of that includes: a plurality of receptacles 10 having at least one proximal opening 11 having a size suitable for receiving a distal end of at least one shaft and a plurality of distal tip openings 12 of a size suitable for picking up and dispensing a plurality of fluid samples from the multiple channel pipette tip; a plurality of electrode assemblies, wherein each electrode assembly includes two or more electrodes 14, the electrodes being spaced one from the other to form a cavity wherein the fluid may be transiently retained 15; and at least one electrode contact assembly including two or more electrode contacts, wherein the electrode contacts are conductively connected to the electrodes.
  • Multi channel pipette tips according to the invention can be formed as a single unit or can be formed by attaching together more than one pipette tip of the invention to effect a multichannel pipette tip.
  • the multi channel pipette tip can include more than one electrode contact assembly. If there is more than one electrode contact assembly, each electrode contact assembly can be conductively connected to the electrodes spaced to form the cavity wherein the fluid can be transiently retained.
  • the multichannel pipette tip can include a single proximal opening 11 suitable for receiving a distal end of a shaft and a plurality of distal tip openings 12 of a size suitable for picking up and dispensing a plurality of fluid samples from the multiple channel pipette tip.
  • Each of the plurality of distal tip openings 12 can interconnected, (i.e., be in fluid communication) with the proximal opening 11, whereby application of a negative or positive pressure to the proximal opening delivers a negative or positive pressure to each of the distal tip openings 12.
  • the multichannel pipette tip can have more than one proximal opening 11 suitable for receiving a distal end of a shaft and a plurality of distal tip openings 12 of a size suitable for picking up and dispensing a plurality of fluid samples from the multiple channel pipette tip.
  • a subset of the plurality of distal tip openings can be interconnected with each proximal opening, whereby application of a negative or positive pressure to any one of the proximal opening delivers a negative or positive pressure to each of the distal tip openings in a single subset of distal tip openings.
  • each one of the distal tip openings can be connected to a separate proximal tip opening, whereby application of a negative or positive pressure to any one of the proximal openings delivers a negative or positive pressure to a single " distal tip opening.
  • proximal tip openings of the multichannel pipette tip can be varied. For example, there are 2, 3, 4, 6, 8, 12, 16, 20, 24, 28, 32, 40, 48, 56, 64, 96 or more proximal tip openings.
  • multichannel pipette tip like the arrangement of electrode contacts and arrangement of electrodes will correspond to the pipette tips made according to the first aspect of the invention. In other certain details there will be differences.
  • multi channel pipette tips can be made such that the multiple electrode assemblies conductively connected to the same electrical potential contact assembly are separated from one another by other electrode assemblies not connected to the same electrical potential contact assembly.
  • an eight channel pipette tip would have two separate circuits, each connected to a separate subset of electrode assemblies.
  • members of these electrode assemblies would be staggered such that the first, third, fifth, and seventh individual tip/electroporation cavity would be on one circuit and the second, fourth, sixth and eighth would be on the second.
  • the invention relates to a device for performing electroporation of a plurality of samples. In one embodiment, as shown in Fig.
  • the device can include: a receptacle support 30, wherein the receptacle support is adapted to support a multi channel pipette tip 10 comprising at least one electrode contact assembly consisting of a first electrode contact 17 and a second electrode contact 18; at least one actuator 37, whereby the device can provide a relative negative pressure or a relative positive pressure to at least one volume of the multi channel pipette tip; at least one electrical potential contact assembly consisting of at least a first electrical potential contact 34 for conductively contacting the first electrode contact and at least a second electrical potential contact for conductively contacting the second electrode contact; and a controller unit for executing a procedure on the multi channel pipette tip (not shown), wherein the procedure includes the application of a the relative negative pressure or the relative positive pressure to at least one volume of the multi channel pipette tip.
  • the device can include more than one electrical potential contact assembly.
  • each electrical contact assembly there can be a separate electrode contact assembly for each receptacle of the multi channel pipette tip.
  • the device can include at least one actuator that provides the relative negative pressure or the relative positive pressure to each volume of the multi channel pipette tip, multiple actuators can each provide the relative negative pressure or the relative positive pressure to a subset of the volumes of the multi channel pipette tip, or multiple actuators can each provide the relative negative pressure or the relative positive pressure to a single volume of the multi channel pipette tip.
  • the procedure executed by the device can include application of negative pressure such that the negative pressure draws at least one fluid sample into at least one of the plurality of receptacles of a multi channel pipette tip, if at least one fluid sample and a multi channel pipette tip are provided.
  • the procedure can include application of positive pressure such that the positive expels at least one fluid sample from at least one of the plurality of receptacles of a multi channel pipette tip, if at least one fluid sample and a multi channel pipette tip are provided.
  • the procedure can include application of an electrical potential. As will be recognized by one of skill in the art, typically the pressure will be applied to the proximal opening(s) of the pipette tip and fluid expelled or drawn through the distal opening(s).
  • the controller unit of the device for the multi channel pipette can also be programmed to execute a predetermined procedure.
  • the predetermined procedure can include the steps of: attaching a multi channel pipette tip comprising at least a first electrode contact and at least a second electrode contact to an apparatus that can provide a relative negative or relative positive pressure; applying a relative negative pressure to the volume of at least one receptacle of the multi channel pipette tip, whereby at least one volume of solution containing a species to be electroporated may be aspirated into the multi channel pipette tip; contacting the multi channel pipette tip with at least one electrical potential contact assembly consisting of at least a first and second electrical potential contact, wherein between the first and second electrical potential contacts, an electrical potential can be applied; applying the electrical potential to the multi channel pipette tip; applying a relative positive pressure to at least one volume of at least one receptacle of the multi channel pipette tip whereby at least one volume of solution containing
  • the controller unit of this aspect of the invention can be programmed to accept input data from sensors.
  • the input data collected can include aggregate data for the multi channel pipette tip or it can include data for individual volumes of the tip. Either the individual data or the aggregate data can be compared to repositories of normal values.
  • the normal values can be those for suitable for judging either individual or aggregate sets of data. Either can be used to generate alarm signals or functions.
  • the invention in an eleventh aspect, relates to a device implemented method for electroporating a species.
  • One embodiment of the method can include the steps of: attaching a multi channel pipette tip to any device made according to the tenth aspect of the invention; providing at least one fluid sample, wherein the at least one fluid sample includes species to be electroporated; applying a negative pressure to the multi channel pipette tip, thereby drawing in at least one portion of the at least one fluid sample; and applying an electrical potential to the multi channel pipette tip, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species.
  • the method can further include applying a positive pressure to the multi channel pipette tip, thereby dispensing a portion of the fluid sample.
  • a positive pressure to the multi channel pipette tip, thereby dispensing a portion of the fluid sample.
  • the method can dispense any number of the fluid samples retained in the multi channel pipette tip.
  • the method can further include detaching the multi channel pipette tip from the device.
  • the multi channel pipette tip can be placed in a rack adapted for the tip or can be placed in a waste container.
  • devices can be adapted to use multiple pipette tips according to the present invention or electroporation cuvettes to electroporate multiple samples in parallel.
  • the following method for introducing a molecule into a species can similarly be adapted.
  • the invention in a twelfth aspect, relates to a device-implemented method for introducing a molecule into a species.
  • An embodiment of this method can include: attaching a multi channel pipette tip to any device made according to the tenth aspect of the invention; providing a set of fluid samples, wherein the set of fluid samples includes the species to be electroporated; applying a negative pressure to at least one receptacle (i.e., volume) of the multi channel pipette tip, thereby drawing in a portion of the fluid samples; applying an electrical potential to the multi channel pipette tip, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species; and providing molecules in the fluid samples, wherein the molecules are introduced into the electroporated species.
  • the invention relates to an electroporation cuvette plate that includes: a plate 81 with a plurality of wells 82, wherein the wells can transiently retain a fluid; at least one electrode assembly including two or more electrodes, the electrodes being spaced one from another to form a cavity 15 wherein the fluid can be transiently retained; and at least one electrode contact assembly further including two or more electrode contacts 17, 18, wherein at least two electrode contacts are conductively connected to the electrodes 14 of at least one electrode assembly.
  • the electroporation cuvette plate can have wells that are uncovered, can have predominantly covered wells, or can further include a cover plate or cover plates that cover one or more wells.
  • the electroporation cuvette plate can include a number of separate wells.
  • a plate can have 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 72, 80, 88, 96, or more wells.
  • Each electrode assembly can have a corresponding and separate electrode contact assembly.
  • more than one electrode contact assembly can be conductively connected to each electrode contact assembly. If so, the electrode contact assemblies and electrode assemblies can be arranged so that no electrode assembly sharing the same first electrode contact assembly is directly adjacent to another electrode assembly sharing the same first electrode contact assembly.
  • each cavity in the wells of the electroporation plate can be arranged in a number of different ways in accordance with requirements which will be recognized by those of skill in the art.
  • the cavity wherein the fluid may be transiently retained can be formed between at least two electrodes by arranging the electrodes such that the distance between a first point on the surface of one electrode to the nearest point on the surface of the second electrode does not vary substantially with respect to the selection of the first point.
  • this can be accomplished in practice by the use of electrodes that are flat, substantially parallel plates.
  • it can be accomplished by using coaxially orientated cylindrical electrodes.
  • design of electrode assemblies can include substantial regions where the distance between electrodes is greater than that of the distance between the parallel regions or the coaxial regions that effect a region having similar distance between electrode surfaces. As will be recognized by those of skill in the art, such an arrangement still prevents a localized minimum region where the resistance to current flow is too small relative to the electrical potential thereby preventing "arcing" between electrodes under suitable conditions for effecting electroporation.
  • an embodiment of the device can include: an electroporation cuvette plate support 85, wherein the electroporation cuvette plate support 85 is adapted to support any electroporation cuvette plate 81 made according to the thirteenth aspect of the invention; at least one electrical potential contact assembly comprising a first electrical potential contact 34 for conductively contacting the first electrode contact 17 and a second electrical potential contact 35 for conductively contacting the second electrode contact 35; and a controller unit (not shown), adapted for executing a procedure on the electroporation cuvette plate 81.
  • the controller can be programmed to execute a predetermined procedure.
  • the predetermined procedure can include: attaching an electroporation cuvette plate; contacting the electroporation cuvette plate with at least at least one electrical potential contact assembly having a first and second electrical potential contact, to which an electrical potential can be applied; applying the electrical potential to the electroporation cuvette plate; detaching the removably attachable receptacle; and optionally, repeating the steps thereby repeating the process.
  • the process can include the addition of at least one sample fluid to at least one well of the electroporation cuvette plate following attachment of the electroporation cuvette plate. Also, the process can include collection of at least one sample fluid from at least one well of the electroporation cuvette plate following application of the electrical potential.
  • the controller unit of the device can be programmed to accept input data from sensors.
  • the data can be collected as aggregate data for the entire plate or as individual data pertaining to specific wells of the plates.
  • the input data can include the voltage of an electrical potential applied during the procedure, the time required for the applied electrical potential to dissipate, the conductivity of any sample volume present in the electroporation cuvette plate, the resistance of any sample volume present in the electroporation cuvette plate, the temperature of the sample before applying the electrical potential, the temperature of the sample during application of the electrical potential, and the temperature of the sample after applying the electrical potential.
  • the input data can be compared to a repository of normal values, thereby measuring variances between the input data and the normal values and wherein variances greater than an unacceptable value generate an alarm signal.
  • the invention in a fifteenth aspect, relates to a device-implemented method for electroporating a species.
  • the method can include the steps of: attaching an electroporation cuvette plate to any device made according to the fourteenth aspect of the invention; providing at least one fluid sample, wherein the fluid sample includes the species to be electroporated and is present in at least one well of the electroporation cuvette plate; and applying an electrical potential to the electroporation cuvette plate, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species.
  • the invention relates to a device-implemented method for introducing a molecule into a species.
  • the method can include: attaching an electroporation cuvette plate to any device made according to the fourteenth aspect of the invention; providing at least one fluid sample, wherein the fluid sample includes the species to be electroporated and molecules to be introduced into the species and wherein the fluid sample is present in at least one well of the electroporation cuvette plate; applying an electrical potential to the electroporation cuvette plate, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species; and providing molecules in the fluid sample, wherein the molecules are introduced into the electroporated species.
  • Providing molecules in the fluid can be accomplished by introduction of the molecules into the fluid sample prior to or subsequent to the application of the electrical potential.
  • the invention relates to a computer program product for controlling a device for performing electroporation.
  • the computer program product can include a computer-readable medium on which is recorded a procedure for controlling a device for performing electroporation.
  • the procedure can include attaching any electroporation cuvette plate made according to the thirteenth aspect of the invention.
  • the computer procedure can include applying an electrical potential to the electroporation cuvette plate or detaching the electroporation cuvette plate.
  • the procedure can include acceptance of input data from sensors, wherein consideration of the input data determines steps taken by the device for performing electroporation.
  • Input data can be selected from the group consisting of the voltage of an electrical potential applied during the procedure, the time required for the applied electrical potential to dissipate, the conductivity of any sample volume present in the receptacle, the resistance of any sample volume present in the receptacle and the temperature of the sample before, during or after electroporation.
  • the input data can be compared to a repository of normal values, thereby measuring variances between the input data and the normal values and wherein variances greater than an unacceptable value generate an alarm signal.
  • the invention relates to an electrode template apparatus, such as that shown in Fig. 15, that includes: an array of electrode assemblies 91, each including two or more electrodes 92, the electrodes being spaced one from another to form a cavity 93 wherein the fluid can be transiently retained; and at least one electrode contact assembly further including two or more electrode contacts 94, 95, wherein at least two electrode contacts are conductively connected to the electrodes of at least one electrode assembly.
  • the electrodes of each electrode assembly that form the cavity of each can be arranged such that the distance between a first point on the surface of one electrode to the nearest point on the surface of the second electrode does not vary substantially with respect to the selection of the first point.
  • the electrodes of each electrode assembly can be flat, substantially parallel plates.
  • the two electrodes of each electrode assembly can be coaxially orientated cylindrical electrodes.
  • the electrode template apparatus can be lowered into a multi well plate 96 or another similar array of containers that can contain a fluid.
  • the electrode template apparatus can include 2, 3, 4, 5, 6, 8, 10, 12, 16, 20,
  • Each electrode assembly can have a corresponding and separate electrode contact assembly. Alternatively, more than one electrode contact assembly can be conductively connected to each electrode contact assembly. If so, then the electrode contact assemblies and electrode assemblies can be arranged so that no electrode assembly sharing the same first electrode contact assembly is directly adjacent to another electrode assembly sharing the same first electrode contact assembly.
  • the electrode contacts of an electrode template of the invention will be recognized by those of skill in the art to be analogous to the electrode contacts of the first, and other, aspects of the invention. Similarly, the electrical potential contacts used with the electrode contacts will similarly be analogous to those of the first, and other, aspects of the invention, but will be adapted for the particular use with the electrode template assembly described herein.
  • the invention in a nineteenth aspect, relates to a device for performing electroporation.
  • the device can include: an electrode template apparatus support, wherein the electrode template apparatus support is adapted to support any electrode template apparatus 91 made according to the eighteenth aspect of the invention; at least one electrical potential contact assembly comprising a first electrical potential contact for conductively contacting the first electrode contact 94 and a second electrical potential contact for conductively contacting the second electrode contact 95; and a controller unit, adapted for executing a procedure on the electrode template apparatus.
  • the electrode contacts of an electrode template of the invention will be recognized by those of skill in the art to be analogous to the electrode contacts of the first, and other, aspects of the invention.
  • the electrical potential contacts used with the electrode contacts will similarly be analogous to those of the first, and other, aspects of the invention, but will be adapted for the particular use with the electrode template assembly described herein.
  • the controller unit of the device can be programmed to execute a predetermined procedure.
  • the procedure can include the steps of: attaching an electrode template apparatus having; an array of electrode assemblies, each including two or more electrodes, the electrodes being spaced one from another to form a cavity wherein the fluid can be transiently retained and an electrode contact assembly further including two or more electrode contacts, wherein at least two electrode contacts are conductively connected to the electrodes of at least one electrode assembly; contacting the electrode template apparatus with at least at least one electrical potential contact assembly having a first and second electrical potential contact, to which an electrical potential can be applied; applying the electrical potential to the electrode template apparatus; detaching the electrode template apparatus; and optionally, repeating the process by repeating the steps.
  • the process can include contacting the electrode template apparatus with at least one sample fluid prior to applying the electrical potential.
  • the process can include withdrawing the electrode template apparatus from contact with at least one sample prior to detaching the electrode template apparatus from the device made according to the nineteenth aspect of the invention.
  • the device can also be adapted to contact a plurality of fluid sample that are provided in an array format to receive the array of electrode assemblies.
  • the device can be adapted so that each fluid sample provided in such an array is contacted by a single electrode assembly.
  • the predetermined procedure of the device can include acceptance of input data from sensors.
  • This data can be collected as aggregate data or as data pertaining to individual electrode assemblies.
  • the input data can include the voltage of an electrical potential applied during the procedure, the time required for the applied electrical potential to dissipate, the conductivity of any sample volume contacted by the electrode template apparatus, the resistance of any sample volume contacted by the electrode template apparatus, the temperature of the sample before applying the electrical potential, the temperature of the sample during application of the electrical potential, and the temperature of the sample after applying the electrical potential.
  • the input data can be compared to a repository of normal values, thereby measuring variances between the input data and the normal values and wherein variances greater than an unacceptable value generate an alarm signal.
  • the invention in a twentieth aspect, relates to a device implemented method for electroporating a plurality of species.
  • the method can include the steps of: attaching an electrode template apparatus made according to the eighteenth aspect of the invention; providing a plurality of fluid samples, wherein the fluid samples include species to be electroporated; contacting the electrode template apparatus with the plurality of fluid samples containing species to be electroporated, thereby transiently retaining a volume of the fluid samples in the cavities formed by the at least two electrodes of each of the plurality of electrode assemblies; and applying an electrical potential to the electrode template apparatus, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species.
  • the invention relates to a device-implemented method for introducing molecules into a plurality of species.
  • the method can include: attaching an electrode template apparatus to any device made according to the nineteenth aspect of the invention; providing a plurality of fluid samples, wherein the fluid samples include species to be electroporated and molecules to be introduced into the species; contacting the electrode template apparatus with the plurality of fluid samples containing species to be electroporated, thereby transiently retaining a volume of the fluid samples in the cavities formed by the at least two electrodes of each of the plurality of electrode assemblies; applying an electrical potential to the electrode template apparatus, thereby providing an electrical potential to the species to be electroporated, wherein the provision of the electrical potential to the species electroporates the species; and providing molecules in the plurality of fluid samples, wherein the molecules are introduced into the electroporated species.
  • the molecules can be provided to the plurality of fluid samples prior to or following the application of the electrical potential to the electrode template apparatus.
  • the invention in a twenty-second aspect, relates to a computer program product for controlling a device for performing electroporation.
  • the computer program product can include a computer-readable medium on which is recorded a procedure for controlling a device for performing electroporation.
  • the procedure can include attachment of an electrode template apparatus made according to the eighteenth aspect of the invention.
  • the procedure can include applying an electrical potential to the electrode template apparatus.
  • the procedure can include detaching the electrode template apparatus.
  • the procedure can include acceptance of input data from sensors, wherein consideration of the input data determines steps taken by the device for performing electroporation.
  • data can include the voltage of an electrical potential applied during the procedure, the time required for the applied electrical potential to dissipate, the conductivity of any sample volume contacted, the resistance of any sample volume contacted and the temperature of any sample before, during or after electroporation.
  • the input data accepted can be compared to a repository of normal values, thereby measuring variances between the input data and the normal values and wherein variances greater than an unacceptable value generate an alarm signal.
  • a pipette tip of the invention similar to that shown in Figure 3 was used to take up volumes which were treated in accordance with established protocols for electroporating cells.
  • a syringe was attached to the proximal opening and operated manually to fill the volume between the electrodes with solution and, once the electroporation procedure had been completed, operated manually to dispense the electroporated cells.
  • the volumes so treated included cells only (Experiment 1) and cells + 50 pg pUC19 plasmid +100 mg/L ampicillin (Experiments 2 and 3). Experiments 4, 5 and 6 were additional controls.
  • Experiment 4 was an electroporation conducted in a standard electroporation cuvette with cells + 50 pg pUC19 plasmid + 100 mg/L ampicillin.
  • Experiment 5 was cells w/o plasmid and w/o ampicillin.
  • Experiment 5 was cells w/o plasmid and with 100 mg/L ampicillin.
  • Experiments 1 and 2 were subjected to 1.6 kV.
  • Experiments 3 and 4 were subjected to 2.0 kV.
  • Experiments 1 and 5 resulted in lawns of cells.
  • Experiments 2 and 3 electroporation using the pipette tip of the invention, resulted in 2 and 8 colonies, respectively.
  • the electroporation in the standard cuvette of the art, experiment 4 resulted in 8 colonies.
  • the negative control, where no DNA was added and ampillicin was present resulted in no colonies.
  • FIG. 16 Automated electroporation of cells using the apparatus shown in Figure 16 was used to transfect the cells and to demonstrate aspects of the present invention.
  • the liquid handling tool (an example of a device that can be attached to the receptacle of the invention and can apply a relative negative and a relative positive pressure) attached an electrotip from the temperature controlled assembly, moved the electrotip to a specified volume of the PCR plate containing solutions; took up the specified volume from the PCR plate; moved to electrotip to the apparatus adapted for supporting the electrotip that included electrical potential contacts; applied the electrical potential, thereby electroporating the cells, delivered the electroporated cells to a specified container, detached the used electrotip; and repeated the process as indicated by the programmed instructions.

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Abstract

La présente invention concerne des dispositifs (10, 81, 91) permettant l'électroporation d'objets sélectionnés, qui sont particulièrement conçus pour être mis en oeuvre par des systèmes automatisés ou semi-automatisés. Cette invention concerne notamment ces dispositifs, des systèmes automatisés conçus pour mettre en oeuvre ces dispositifs, ainsi que les utilisations de ces dispositifs eux-mêmes.
PCT/US2003/000343 2002-01-07 2003-01-07 Pointes de pipette de cuvette d'electroporation, reseaux de cuvettes a plusieurs puits, appareil gabarit d'electrode concu pour une automatisation et utilisations de ceux-ci WO2003057819A1 (fr)

Priority Applications (2)

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AU2003206415A AU2003206415A1 (en) 2002-01-07 2003-01-07 Electroporation cuvette-pipette tips, multi-well cuvette arrays, and electrode template apparatus adapted for automation and uses thereof
CA002472686A CA2472686A1 (fr) 2002-01-07 2003-01-07 Pointes de pipette de cuvette d'electroporation, reseaux de cuvettes a plusieurs puits, appareil gabarit d'electrode concu pour une automatisation et utilisations de ceux-ci

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US34680602P 2002-01-07 2002-01-07
US60/346,806 2002-01-07

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