WO2007000247A1 - Cellule de mesure et procede execute avec ladite cellule pour determiner le degre de desagregation de cellules biologiques induit par electroporation - Google Patents

Cellule de mesure et procede execute avec ladite cellule pour determiner le degre de desagregation de cellules biologiques induit par electroporation Download PDF

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Publication number
WO2007000247A1
WO2007000247A1 PCT/EP2006/005712 EP2006005712W WO2007000247A1 WO 2007000247 A1 WO2007000247 A1 WO 2007000247A1 EP 2006005712 W EP2006005712 W EP 2006005712W WO 2007000247 A1 WO2007000247 A1 WO 2007000247A1
Authority
WO
WIPO (PCT)
Prior art keywords
measuring
measurement
measuring cell
electroporation
cell
Prior art date
Application number
PCT/EP2006/005712
Other languages
German (de)
English (en)
Inventor
Martin Sack
Hansjoachim Bluhm
Original Assignee
Forschungszentrum Karlsruhe Gmbh
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 Forschungszentrum Karlsruhe Gmbh filed Critical Forschungszentrum Karlsruhe Gmbh
Priority to EP06754357A priority Critical patent/EP1893998A1/fr
Publication of WO2007000247A1 publication Critical patent/WO2007000247A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/46—Treatment of water, waste water, or sewage by electrochemical methods
    • C02F1/4608—Treatment of water, waste water, or sewage by electrochemical methods using electrical discharges
    • 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
    • G01N27/023—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance where the material is placed in the field of a coil
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/32—Nature of the water, waste water, sewage or sludge to be treated from the food or foodstuff industry, e.g. brewery waste waters

Definitions

  • the invention relates to a measuring cell and a method that can be carried out therewith for determining the degree of digestion of biological cells by electroporation by measuring the electrical conductivity of a mass flow of biological cells / cell assemblies guided in a tube / channel in a transport liquid.
  • / 1 / a method for measuring the degree of electroporation is described, in which the change in the ohmic resistance in the lower frequency range is evaluated in relation to the ohmic resistance in the upper frequency range.
  • the measurement is carried out via a four-electrode system, which is inserted into the cell tissue to be measured.
  • the piercing method is only suitable for sampling and is therefore unsuitable for continuously supplying measured values for a plant control.
  • the measurement at two different frequencies including amplitude determination is circuitry more complex than the determination of the phase angle at only one frequency.
  • a time measurement between the zero crossings of current and voltage is sufficient, while a current and voltage measurement is required for the amplitude determination.
  • the upper frequency range is a few megahertz, the frequency of maximum phase shift is about 50 kHz. At this frequency, the lead inductances at moderate line lengths do not significantly affect the measurement result, with an impedance correction appearing necessary in the megahertz range.
  • the object of the invention is to provide an industrial-scale measuring cell and a process suitable for industrial use with which the degree of digestion of large mass flows caused by electroporation of biological cells or cell assemblies guided in a transport fluid is reached and from this a control and / or regulating signal for optimal energetic guiding of an electroporation system can be derived.
  • the object is achieved by a measuring cell according to the features of claim 1 and a measuring method which can be carried out in accordance with the method steps of claim 7.
  • the measuring cell for determining the degree of digestion of biological cells consists according to claim 1 of a structure which can be flowed through for measuring at least a partial flow of the mass flow.
  • the measuring cell is constructed either for a resistive / capacitive measurement and then consists of two oppositely positioned electrodes, the pair of measuring electrodes, between which flows at least part of the mass flow. Or it is constructed for an inductive measuring and then consists of at least one coil in a simple solenoidal form, which at least partially comprises the cross-section of the mass flow. In the case of several coils, the coils of the measuring cell are arranged coaxially with each other.
  • the degree of digestion of the cells is determined from the decrease in the measured phase angle compared to a comparison measurement on untreated cells. Therefore, the method for determining the digestion degree caused by electroporation is more biological Cells via the measurement of the electrical conductivity of a mass flow of biological cells / cell aggregates guided in a tube / channel in a transport liquid according to claim 7 from the following steps:
  • the meter is set by a signal generator under a time-varying current and a time-varying voltage. This can be done either by means of a voltage source or a power source.
  • the current through the measuring cell and the voltage across the measuring cell are measured at a selected frequency or a selected frequency range and the phase angle or the course of the phase angle in the frequency range between the current through the measuring cell and the voltage across the measuring cell with an electronic signal processing device determined ,
  • the phase angle or its frequency-dependent profile is displayed and documented as a measure of the degree of digestion of the cells, but also derived therefrom a signal for controlling and regulating the device for electroporation.
  • the fundamental measurement error due to a proportion of current flowing past the cells through the transport liquid surrounding the cells is compensated for by calculating this current component from the degree of filling, the conductivity of the transport liquid and the geometry data of the measurement path in a computer of the signal processing device and subtracting it from the measurement current in the correct phase becomes.
  • the measuring cell is connected to a signal generator to be operated with a time-varying current and a time-varying voltage.
  • the measuring cell is manageable.
  • the two measuring electrodes, between which at least a part of the mass flow flows without obstruction when measuring mounted in a dielectric frame / tube in which they face each other in a defined position with respect to the longitudinal axis.
  • This construction will held for example via a mounted on the frame / tube lever in the mass flow, so that the cross-section of the measuring cell in the flow cross-section of the mass flow is.
  • the measuring cell is also of resistive / capacitive design and forms a section of the transport tube / channel.
  • the two measuring electrodes are installed / recessed in such a way that they do not form a flow resistance for the passing mass flow.
  • the two measuring electrodes face each other with their forehead in mirror image.
  • the two measuring electrodes may be pin-shaped or plate-shaped and follow with their exposed surface of the channel contour steadily or smoothly.
  • the measuring cell is also described as manageable and constructed for inductive measuring.
  • it consists of a coil or two coaxial coils, an excitation and a measuring coil, which can be held in the mass flow or can, so that the cross section of the measuring cell is located in the flow cross-section.
  • the stationary installation of the inductive measuring cell is described in claim 6.
  • the inductive measuring cell forms a portion of the transport tube / channel and consists for this purpose of a coil or of two mutually coaxial coils, an excitation and a measuring coil.
  • the cross section of the measuring cell then covers the flow cross section of the mass flow in any case.
  • the measured value of the degree of digestion for controlling and regulating the processing time and / or the required energy is included and the operating parameters determining the degree of digestion during electroporation, such as electric field strength, pulse length, number of pulses per volume element, temperature, number of passes through the cell disruption reactor, storage time of the cell suspension between two electroporation passages or between electroporation and extraction, according to a characteristic field based on the measurement of the Digestion set.
  • the measurement, continuous or intermittent, takes place over at most the cross section of the mass flow, the cell suspension of biological cells or such cell aggregates and the carrier liquid takes place (claim 9).
  • a periodic waveform of the current / voltage from the signal generator is applied to the measuring device.
  • a sine wave involved in the waveform with at least strong occurrence of the phase shift is evaluated for measurement.
  • a pulse-shaped, aperiodically or periodically damped curve profile is used according to claim 11.
  • a frequency with the strongest occurrence of the phase shift or a narrowband frequency range with a strong occurrence of the phase shift is evaluated for measurement.
  • the electroporation pulse itself can also be used for the measurement.
  • this pulse must have a sufficiently large frequency components in the frequency range sensitive to the phase measurement.
  • the operating point of the system can be adapted to the present before the electroporation passage degree of digestion of the cells.
  • the adaptation is done by means of a characteristic field, as described in claim 8.
  • a method for correcting a parallel resistance of the suspension water by means of additional conductivity and filling degree measurement is specified. To minimize energy, a waiting period between electroporation and extraction is maintained. The control is performed on the basis of a characteristic field.
  • the proposed electroporation meter is a conductivity meter which measures the complex conductivity of a cell suspension at the frequency at which the phase shift between current and voltage is ideally greatest and, as described above, evaluates this phase shift. With the continuous measurement of the degree of electroporation in large mass flows the energy-optimized system control is possible. By installing the measuring device before the
  • Electroporation distance can be concluded from the initial degree of digestion of the cells. For example, the cells of frosted sugar beets are already open-minded. The minimum energy consumption is given by waiting time and setting of the optimal operating point according to the characteristic field.
  • Figures show, on the one hand, the schematic structure of the measuring instrument and, on the other hand, experimental results at the end. It shows:
  • FIG. 1 shows the resistive / capacitive measuring cell
  • FIG. 2 shows the inductive transformer measuring cell
  • FIG. 3 shows the inductive measuring cell
  • FIG. 4 shows the equivalent circuit diagram of the cell tissue
  • FIG. 5 shows the course of the complex impedance
  • FIG. 6 shows the complex impedance at 50 kHz
  • FIG. 8a R s / R p as a function of the waiting time 12 min;
  • FIG. 1 schematically shows the situation for the resistive / capacitive measurement.
  • the mass flow is indicated by the arrow as the direction of flow in the pipe 7.
  • the two measuring electrodes 1 are exposed so that they do not form a flow obstacle, but directly touch the passing cell suspension. This can be embedded in the pipe wall plates or rods.
  • the two electrodes 1 are connected to the voltage source 3, the signal generator.
  • the complex impedance 2 is representative of that of the electrical measuring circuit.
  • the electrodes are connected to the evaluation unit 4.
  • the current measurement takes place indirectly at the shunt 5.
  • FIG. 2 shows in the same schematic way the inductive type of measurement with only one coil 6, the transformer coupling which comprises the tube 7 with the mass flow flowing therein and thus does not form a flow obstacle.
  • the complex impedance of an association of intact biological cells shows a capacitive component in the middle frequency range, while at low and high frequencies the impedance 2 is almost ohmic.
  • a simplistic equivalent circuit diagram of the biological cell tissue is used (FIG. 4).
  • the capacitance C represents the effective capacity of the cell membranes
  • the parallel resistance R P the effective ohmic resistance of the cell membranes
  • the series resistance R s the effective resistance of the cell interior.
  • the sum of R 5 and R P acts in the lower frequency range and only Rp in the upper frequency range.
  • FIG. 5 confirms the informative value of the detection of the phase shift for the degree of electroporation.
  • and the phase angle ⁇ are plotted against the frequency f before and after the electroporation by the equivalent circuit diagram (FIG. 4). The largest phase angle occurs at about 50 kHz.
  • Tab. 1 shows the pressing results in comparison to the phase angle at the beginning of the pressing. Since, due to the preparatory work for the pressing, the phase angle could only be measured up to approx. 5 min before the pressing, the expected phase angle was extrapolated on the basis of the curves. The degree of digestion determined by pressing correlates with the decrease of the phase angle within the scope of natural scattering and measurement uncertainties.
  • the test results also show that the waiting time between electroporation and pressing off plays a decisive role in the level of digestion.
  • the diagrams Fign. 7 and fig. 8 show the decrease of the resistance R P and the decrease of the phase angle after single pulses. For this purpose, waiting times of about 2 minutes (figure 7) and about 12 minutes (figure 8) were recorded between the individual pulses. held. Clearly the pulse-dependent decrease of R P and ⁇ can be recognized on the second pulse. Only after each 1st pulse was no decrease in these parameters visible, sometimes a slight increase in Rp.

Abstract

L'invention concerne une cellule de mesure destinée à déterminer le degré de désagrégation de cellules biologiques induit par électroporation, laquelle cellule est conçue pour une mesure résistive/capacitive ou pour une mesure inductive. Le procédé mis en oeuvre avec ladite cellule de mesure consiste à appliquer un courant et une tension variables dans le temps à la cellule de mesure par l'intermédiaire d'un générateur de signaux, à mesurer le courant traversant la cellule de mesure et la tension appliquée sur la cellule de mesure à une fréquence sélectionnée ou une gamme de fréquences sélectionnée puis à déterminer l'angle de phase entre le courant et la tension mesurés avec un dispositif de traitement de signal électronique.
PCT/EP2006/005712 2005-06-24 2006-06-14 Cellule de mesure et procede execute avec ladite cellule pour determiner le degre de desagregation de cellules biologiques induit par electroporation WO2007000247A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP06754357A EP1893998A1 (fr) 2005-06-24 2006-06-14 Cellule de mesure et procede execute avec ladite cellule pour determiner le degre de desagregation de cellules biologiques induit par electroporation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE200510029414 DE102005029414B4 (de) 2005-06-24 2005-06-24 Verfahren zur Bestimmung des durch Elektroporation bewirkenden Aufschlussgrades biologischer Zellen
DE102005029414.6 2005-06-24

Publications (1)

Publication Number Publication Date
WO2007000247A1 true WO2007000247A1 (fr) 2007-01-04

Family

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PCT/EP2006/005712 WO2007000247A1 (fr) 2005-06-24 2006-06-14 Cellule de mesure et procede execute avec ladite cellule pour determiner le degre de desagregation de cellules biologiques induit par electroporation

Country Status (3)

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EP (1) EP1893998A1 (fr)
DE (1) DE102005029414B4 (fr)
WO (1) WO2007000247A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2500216A (en) * 2012-03-13 2013-09-18 Ugcs University Of Glamorgan Commercial Services Ltd Method and apparatus for magnetic induction spectroscopy

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SI1980158T1 (sl) * 2007-04-05 2010-09-30 Intersnack Knabber Geback Gmbh Postopek za odstranjevanje celičnih sestavin kitvorijo akrilamid in ali melanoidin iz škrobnatega rastlinskega materiala in rastlinski materialz zmanjšano vsebnostjo akrilamida in ali melanoidinov
DE202013009847U1 (de) * 2013-12-06 2015-03-09 Hugo Vogelsang Maschinenbau Gmbh Vorrichtung zur elektrischen Desintegration mit mehreren Wirkzonen

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4764473A (en) * 1983-05-13 1988-08-16 Kerforshungsanlage Julich Chamber for the treatment of cells in an electrical field
US5676646A (en) * 1992-04-08 1997-10-14 Genetronics, Inc. Flow through electroporation apparatus
WO2001080946A1 (fr) * 2000-04-21 2001-11-01 Igea S.R.L. Dispositif d'electroporation avec mesure des proprietes electriques
US20030148524A1 (en) * 2002-01-21 2003-08-07 Ulrich Zimmermann Method and device for electroporation of biological cells
WO2004031353A2 (fr) * 2002-09-30 2004-04-15 Maxcyte, Inc. Appareil et procede d'electroporation non statique
US20050118705A1 (en) * 2003-11-07 2005-06-02 Rabbitt Richard D. Electrical detectors for microanalysis
WO2005056788A1 (fr) * 2003-12-08 2005-06-23 Excellin Life Sciences, Inc. Dispositif et procede d'electroporation et d'apports moleculaires regules a des cellules et des tissus

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US5150062A (en) * 1991-01-02 1992-09-22 Nissan Motor Co., Ltd. Electrostatic capacitance sensing circuit
DE4116468A1 (de) * 1991-05-21 1992-11-26 Knick Elekt Messgeraete Gmbh Induktive leitfaehigkeits-messzelle

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4764473A (en) * 1983-05-13 1988-08-16 Kerforshungsanlage Julich Chamber for the treatment of cells in an electrical field
US5676646A (en) * 1992-04-08 1997-10-14 Genetronics, Inc. Flow through electroporation apparatus
WO2001080946A1 (fr) * 2000-04-21 2001-11-01 Igea S.R.L. Dispositif d'electroporation avec mesure des proprietes electriques
US20030148524A1 (en) * 2002-01-21 2003-08-07 Ulrich Zimmermann Method and device for electroporation of biological cells
WO2004031353A2 (fr) * 2002-09-30 2004-04-15 Maxcyte, Inc. Appareil et procede d'electroporation non statique
US20050118705A1 (en) * 2003-11-07 2005-06-02 Rabbitt Richard D. Electrical detectors for microanalysis
WO2005056788A1 (fr) * 2003-12-08 2005-06-23 Excellin Life Sciences, Inc. Dispositif et procede d'electroporation et d'apports moleculaires regules a des cellules et des tissus

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2500216A (en) * 2012-03-13 2013-09-18 Ugcs University Of Glamorgan Commercial Services Ltd Method and apparatus for magnetic induction spectroscopy
GB2500216B (en) * 2012-03-13 2017-07-26 Usw Commercial Services Ltd Method and apparatus for magnetic induction spectroscopy

Also Published As

Publication number Publication date
DE102005029414B4 (de) 2009-09-03
DE102005029414A1 (de) 2006-12-28
EP1893998A1 (fr) 2008-03-05

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