Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/02—Details
  • A61N1/04—Electrodes
  • A61N1/0404—Electrodes for external use
  • A61N1/0408—Use-related aspects
  • A61N1/0412—Specially adapted for transcutaneous electroporation, e.g. including drug reservoirs
  • A61N1/0416—Anode and cathode
  • A61N1/042—Material of the electrode
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/02—Details
  • A61N1/04—Electrodes
  • A61N1/0404—Electrodes for external use
  • A61N1/0408—Use-related aspects
  • A61N1/0412—Specially adapted for transcutaneous electroporation, e.g. including drug reservoirs
  • A61N1/0416—Anode and cathode
  • A61N1/0424—Shape of the electrode
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/18—Applying electric currents by contact electrodes
  • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
  • A61N1/327—Applying electric currents by contact electrodes alternating or intermittent currents for enhancing the absorption properties of tissue, e.g. by electroporation
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
• • H—ELECTRICITY
  • H05—ELECTRIC TECHNIQUES NOT OTHERWISE PROVIDED FOR
  • H05H—PLASMA TECHNIQUE; PRODUCTION OF ACCELERATED ELECTRICALLY-CHARGED PARTICLES OR OF NEUTRONS; PRODUCTION OR ACCELERATION OF NEUTRAL MOLECULAR OR ATOMIC BEAMS
  • H05H1/00—Generating plasma; Handling plasma
  • H05H1/24—Generating plasma
  • H05H1/2406—Generating plasma using dielectric barrier discharges, i.e. with a dielectric interposed between the electrodes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44704—Details; Accessories
  • G01N27/44713—Particularly adapted electric power supply
• • H—ELECTRICITY
  • H05—ELECTRIC TECHNIQUES NOT OTHERWISE PROVIDED FOR
  • H05H—PLASMA TECHNIQUE; PRODUCTION OF ACCELERATED ELECTRICALLY-CHARGED PARTICLES OR OF NEUTRONS; PRODUCTION OR ACCELERATION OF NEUTRAL MOLECULAR OR ATOMIC BEAMS
  • H05H1/00—Generating plasma; Handling plasma
  • H05H1/24—Generating plasma
  • H05H1/2406—Generating plasma using dielectric barrier discharges, i.e. with a dielectric interposed between the electrodes
  • H05H1/2443—Generating plasma using dielectric barrier discharges, i.e. with a dielectric interposed between the electrodes the plasma fluid flowing through a dielectric tube
  • H05H1/246—Generating plasma using dielectric barrier discharges, i.e. with a dielectric interposed between the electrodes the plasma fluid flowing through a dielectric tube the plasma being activated using external electrodes

Definitions

• the embodiments of the present invention relate to a system for applying time variant, time invariant, or pulsed voltage potential to a series capacitive network to set up or otherwise generate uniform or non-uniform electric fields, which in turn, are used for numerous applications. More particularly, the embodiments employ dielectric materials with particular electrical properties, arranged or otherwise configured to form series capacitance networks that divide or otherwise distribute the applied potential between opposing electrodes, resulting in a concentration of the E- field in (or otherwise through) a subject material.
• Static, time variant, and pulsed electric fields with or without a magnetic field component, whether considered weak or intense fora particular application, are used in a variety of industries for a broad range of applications.
• charge carriers may be moving relative to the lab frame so a magnetic B-field component accompanies the electric E-field, however, with the embodiments of the present invention, only the E-field is pertinent. Examples of E-field applications include, but are not limited to the following:
• electrophoresis both gel and capillary type employ an electrical current through suspension media, the resistive load, thus setting up an electric field used to separate, differentiate and fractionate DNA, proteins, and other molecules
• electroporation aka electropermeabilization: intense electric fields, often pulsed with various waveforms and pulse rates, are used to cause the dielectric breakdown of living cellular membranes, thus affecting reversible and nonreversible poration and/or permeabilization for the purpose of transfection, pasteurization or sterilization; and
• electric field flow fractionation (FFF, aka EFFF, ⁇ -EFF, CyEFF, and others): employ an electric field orthogonal to a fluid flow in order to separate, fractionate, and differentiate large molecules and/or small particles from a subject liquid.
• a process or effect driven, supported or facilitated by the action of an E-field can be accelerated or otherwise improved by either increasing the field intensity for a given applied voltage, or conversely, by reducing the applied voltage for a given field intensity.
• This is due to the relationship between the material properties of permittivity, volume resistivity, and maximum allowable field stress, and the effect these parameters have on the diacritical circuit elements of field intensity, dielectric breakdown, field geometry, current flow, and energy consumption.
• Applications proceeding under the influence or direct action of an E-field are often limited by the undesirable effects of ohmic heating, electrochemistry (faradaic charge transfer), field shielding by electrolytic double layer formation, electrode polarization, and energy consumption.
• Electric current is a limiting factor for the applied filed intensity in electrophoretic, electroporation, and field flow fractionation devices due to ohmic heating of the working media (usually a liquid or gel for such applications), and undesirable electrochemistry at the media/electrode interface(s) (faradaic charge transfer).
• working media usually a liquid or gel for such applications
• undesirable electrochemistry at the media/electrode interface(s) farnesoid charge transfer
• one embodiment of the present invention includes a high impedance system for generating an electric field and comprises: a pair of electrodes comprising a dielectric material wherein each electrode has at least one surface coated with a conductive material; and wherein such dielectric material forms a barrier separating the conductive coating from the subject fluid under treatment; a fluid pathway or space formed between the pair of electrodes such that the conductive material is on an electrode surface not in contact with the fluid in the pathway or space; a time variant, time invariant, or pulsed voltage source applied across the electrodes; and a housing containing said pair of electrodes, said housing configured to maintain a subject static or dynamic fluid in said pathway or space.
• One method embodiment of the present invention includes a method of subjecting a fluid to an electric field comprising: forming a fluid pathway between a pair of electrodes wherein said electrodes comprise a dielectric material and wherein each electrode has at least one surface coated with a conductive material, said conductive material placed on an electrode surface not in the fluid pathway; housing said electrodes such that a subject static or dynamic fluid is maintained in said pathway; applying a time variant, time invariant or pulsed voltage source to each surface of the electrodes coated with the conductive material thereby creating the electric field; and causing a fluid to enter the fluid pathway such that said fluid is subjected to the electric field.
• the system and method for generating the E-field utilizes high impedance dielectric materials having a collection of three imperative material properties: high permittivity ( ⁇ ), high volume resistivity (p) and high maximum allowable E-field stress ( ⁇ ) and physical geometries that take advantage of the manner in which E-fields are divided or distributed in series capacitance networks.
• Fig. 1 illustrates a first dielectric configuration of the present invention having a parallel geometry
• Fig.2 illustrates a second dielectric configuration of the present invention having a cylindrical geometry
• Fig. 3 illustrates three dielectric sections arranged to form a series capacitance network
• Fig. 4 illustrates a circuit equivalent of the dielectric geometry of Fig. 3;
• Fig. 5 illustrates two titanate ceramic slabs coated on one side with a thin film of silver metal forming a conductive electrode surface on each;
• Fig.6 illustrates a perspective view of the electrodes of Fig.5 affixed to a polycarbonate beam support with a space between forming a fluid pathway;
• Fig. 7 illustrates a top view of the electrodes of Fig. 6;
• Fig. 8 illustrates an opposing beam support affixed to the other side of the arrangement illustrated in Figs. 6 and 7;
• Fig.9 illustrates one configuration of a high-impedance E-field device of the present invention
• Fig. 10 illustrates an equivalent circuit diagram for the parallel plate geometry of Figs. 6 and 7;
• Figs. 11 and 12 illustrate one exemplary system configuration according to the embodiments of the present invention.
• Fig. 13 illustrates an equivalent circuit diagram for the configuration illustrated in Figs. 11 and 12.
• impressed electric fields can cause either reversible or irreversible pore formation, as well as other structural defects in lipid membranes, including the membranes of bacteria, fungi, spores, virus, and mammalian (soma) cells.
• reversible electroporation the phenomena is marked by a transient increase in membrane diffusion permeability, which has been used for decades for transfection of DNA, drugs, dyes, proteins, peptides, and other molecules.
• Electroporation devices and methods for clinical and laboratory use have been available for decades, and can be readily purchased for transfection, pasteurization, and sterilization of small batch volumes (typically 1 ⁇ l to 100 ml). Much work has been expended over the past two decades to adapt these clinical and laboratory methods to commercial applications that require continuous high flow rates instead of small batch volumes. Many devices and methods have been proposed, implemented, and patented, however, common to all the current and prior art is the use of low impedance electrically conductive electrodes coupled
• HIE High Impedance Electroporation by the inventors
• Figs. 1 and 2 show a parallel dielectric configuration 100 and a cylindrical dielectric configuration 150, respectively, that may be used to facilitate the method embodiments of the present invention.
• Each configuration shows a dielectric material 105, 155, electrical conductive coating 110, 160 and material 115, 165 under test or E-field influence.
• Fig.3 considers three dielectric sections 170, 175 and 180 arranged to form a series capacitance network 125. E-fields 130, 135 through each are identified.
• Fig. 4 shows a circuit diagram 140 representing the network 125 of Fig. 3.
• Fig. 5 two high impedance dielectric electrodes 200 fabricated of lead-magnesium-lead titanate ceramic are shown. Other materials, such as a high permittivity particulate/epoxy composite or materials having similar properties can also be used.
• the two titanate ceramic slabs 200 were coated on one side with a thin film of silver metal forming a conductive surface 210.
• the electrodes measured 10 mm thick by 10 mm wide by 100 mm long and the dielectric ceramic had the following electric material properties:
• volume resistivity « 10 12 [ ⁇ -cm]; and • maximum allowable field stress: 9.0e+06 [Vm "1 ].
• the electrodes 200 were affixed to a polycarbonate beam support 220 with a space there between forming a fluid pathway channel 230 measuring 1 mm deep by 10 mm wide by 100 mm long forming a fluid pathway or space volume of 1000mm 3 or 1 ml.
• a fluid port 225 permits the inflow or outflow of subject fluid. Since the force of attraction imparted by the field between the electrodes 200 is daunting when the system is charged, beam supports 220 were provided as structural strain relief for the ceramic electrodes 200. Note that the electrodes 200 are arranged to form a series capacitance network coupled to the test liquid being treated. Directional arrow A shown in Fig.
• FIG. 7 depicts the direction of the generated E-field.
• Fig. 8 shows an opposing beam support 250 completing a liquid-tight fluid pathway.
• the electrodes 200 and beam supports 220 were then fitted into a housing 260 (e.g., a 1-1/4" PVC pipe), which was filled with a high voltage dielectric epoxy 265.
• Positive and negative high voltage cable guides 270, 275, respectively, and fluid tube fittings 280, 285 were also affixed to form the final HIE device 300 (termed an abatis by the inventors hereof).
• a liquid/bacteria suspension being treated was passed through the fluid pathway by conveyance tubes connected to the inlet tube fitting 280 and outlet tube fitting 285 as shown.
• Positive and negative high voltage wires 290, 295, respectively, (shown in Fig. 11 ) were fed through the positive and negative cable guides 270, 275 and positioned in direct contact with the silver conductive surface 210 of each respective electrode 200 forming an electrical connection for charging the system.
• Fig. 10 shows an equivalent circuit diagram 310, and legend, for such a parallel plate geometry.
• Figs 11 and 12 show one complete system configuration 350.
• the HIE device 300 was mounted on a high voltage dielectric bulkhead (e.g., Y 4 " polycarbonate plastic sheet), and wired to a 120 kVDC power supply 310.
• a first 600 ml fluid supply beaker 320 was mounted vertically above the HIE device 300 such that, by means of gravity, the inoculated liquid would drain to the fluid inlet 280 and through the HIE device 300.
• a second 600 ml beaker 325 was
• Fig. 13 shows an equivalent circuit diagram 360 for the power supply 310 and HIE device 300.
• the HlE device 300 Prior to operating the system with a test liquid inoculated with bacteria, the HlE device 300 was filled with sterile Tryptic Soy Both and its electrical properties were measured. Table #1 shows predicted and measured values:
• the maximum E-field through the liquid under test was on the order of 8.5e+07 Vm "1 , which equates to 850 kV/cm, representing a daunting electric field.
• the electric current through the HIE device 300 was on the order of 4.5e-07 amperes representing an extremely low current given the intense electric field generated (this figure does not include transient displacement current).
• the measured values represent an average of three (3) separate tests. The power supply was turned off and the HIE device 300 fully discharged between each measurement.
• the system 350 for the electroporation test was operated at an applied voltage ( ⁇ a ) of 10kV.
• the electric field Ei presented to the inoculated liquid under treatment at this voltage was 7.82e+06 Vm "1 or about 78 kV/cm.
• Electric conduction current (l a ) through the HIE device 300 during the test was approximately 4.6e-08 A (0.046 microamperes).
• the total energy dissipation U t was on the order of 2.2e-01 J (221 millijoules), putting the specific energy consumption U 3 at 1.75e-03 kJ/IR
• the cumulative exposure time (t x ) was -800 ms, an average, against flow rate over the 480 second total process time.
• the bacterial load in the test liquid was reduced by 38% (kill fraction in terms of cfu/ml), or a log reduction of approximately -0.21 logi 0 .
• the kill fraction stated is an average of seven intercepted samples taken at equal intervals during the test period. Although a - 0.21 log reduction is not significant for the purpose of commercial pasteurization, it is significant for the purpose of demonstrating the effectiveness of the embodiments of the present invention as applied to live cell electroporation. No attempt was made to detect or measure the extent of reversible electroporation (i.e. a transient increase in membrane permeability), but given the large kill fraction, electropermeabilization effects in the cohabitant survival fraction are immutable.
• the embodiments of the present invention comprise a series capacitance network.
• the following table presents an overview of critical electrical parameters for the operating electroporation devices and systems surveyed (the range of some values is broad, but still instructive):
• the data collected from the study encompass a wide range of both reversible (transient permeabilization) and irreversible (death) electroporation effects, a number of different bacteria types, and includes systems with coaxial (cylindrical) and parallel fluid pathway geometries, as well as both batch type and continuous flow schemes.
• the specific energy U s required by the device or method to affect the kill fraction attained was seldom cited (last column in Table #2).
• the survey conducted by the inventors often requiring calculations extramural to the reports under study, revealed the magnitude of energy consumption versus kill fraction to be quite high as shown.
• the HIE electroporation device 300 fabricated for the instant demonstration expended a specific energy (U s ) on the order of 1.75e-03 kJ/IR
• the acoustic energy can be applied via any desired means.
• the acoustic energy can be applied 1) by action of a piezoelectric transducer attached to the HIE device 300 in such a manner and in such a configuration as to impart the acoustic energy to the fluid in the pathway 230 or space concurrent with, concatenated between, and/or consequent to the applied electric field pulse or pulses; or, 2) by action of a piezoelectric response of the dielectric electrode 200 itself wherein the material chosen as the barrier material has suitable dielectric properties and is also a piezoelectric material.
• the acoustic energy is imparted to the fluid under treatment or test concurrently (in both space and time) with the application of the applied potential.
• the acoustic energy has the same pulse duration time and pulse interval as the applied field. It is also possible that both means of applying the acoustic energy can be employed n a single system. That is, the dielectric electrode formed of a piezoelectric material acts to impart the electric field and the acoustic energy to the fluid under treatment simultaneously, while a second dedicated electroacoustic or mechanoacoustic
• M 126653681V1 999971.565159 ⁇ 3 transducer is positioned and configured to impart acoustic energy to the fluid under treatment either concurrently with, concatenated between, and/or consequent to the applied field.
• the application of a pulsed acoustic longitudinal wave front to a biological cell during or immediately after being electroporated presents a periodic radiation pressure to the cell membrane. Since electroporation causes open pores to develop through the cellular membrane, the action of the radiation pressure, presented as a force perpendicular to the polar axis of the cell membrane, causes the cell to deform. Given the mass inertia of the cell and the viscosity of the supernatant, the cell flattens at the pole under the influence of the radiation force.
• the incident angle between the radiation vector and portions of the membrane radial to the polar axis progressively decrease, that is, become more perpendicular to the force vector, thus advancing the flattening process.
• This process of flattening continues until the radiation force is overcome by the counter inertial and viscous forces at play and the cell begins to move.
• the flattening period either, or both, of two things occur relative to the cell's geometry: 1) the cell's internal volume decreases or 2) the membrane area increases (by stretching).
• the force resulting from the normal plan-wave longitudinal acoustic radiation pressure is a steady state phenomenon, hence the flattening action only occurs once, after which, the cell's shape tends to recover, and then the entire cell tends to move with the wave front. If the acoustic energy is pulsed however, the flattening/recovery action proceeds at the pulse rate, provided the pulse rate is presented at a lower frequency than the mechanical shape recovery relaxation time of the cell. Because the deformation periodically increases and decreases the internal volume of the cell, the result is a "pumping" action.
• the pumping action causes cytoplasmic fluid to be pumped out of the cell, as well as extra-cellular fluid (supernatant) to be pumped into the cell. The action serves to hasten or accelerate cellular paralysis leading to death and/or metabolic inactivation of the organism thus improving the efficiency and/or kill fraction for a

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