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WO2001052997A1 - Procede et appareil de separation de particules - Google Patents

Procede et appareil de separation de particules Download PDF

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Publication number
WO2001052997A1
WO2001052997A1 PCT/GB2001/000239 GB0100239W WO0152997A1 WO 2001052997 A1 WO2001052997 A1 WO 2001052997A1 GB 0100239 W GB0100239 W GB 0100239W WO 0152997 A1 WO0152997 A1 WO 0152997A1
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WO
WIPO (PCT)
Prior art keywords
electrodes
frequency
voltage
particle
particles
Prior art date
Application number
PCT/GB2001/000239
Other languages
English (en)
Inventor
Walter Bernard Betts
Keith Richard Milner
Andrew Paul Brown
Original Assignee
Cell Analysis Limited
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 Cell Analysis Limited filed Critical Cell Analysis Limited
Priority to AU2001228636A priority Critical patent/AU2001228636A1/en
Publication of WO2001052997A1 publication Critical patent/WO2001052997A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/02—Separators
    • B03C5/022—Non-uniform field separators
    • B03C5/026—Non-uniform field separators using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C3/00—Separating dispersed particles from gases or vapour, e.g. air, by electrostatic effect
    • B03C3/34—Constructional details or accessories or operation thereof
    • B03C3/88—Cleaning-out collected particles
    • B03C3/885—Cleaning-out collected particles by travelling or oscillating electric fields, e.g. electric field curtains

Definitions

  • the present invention relates to a method and apparatus for collecting and separating abiotic and/or biotic particles, such as latex beads, mammalian cells or microbial cells, viruses, prions and chemicals or biochemicals using dielectrophoresis. It also relates to a method and apparatus for enumerating a particular particle present in a test sample.
  • abiotic and/or biotic particles such as latex beads, mammalian cells or microbial cells, viruses, prions and chemicals or biochemicals using dielectrophoresis.
  • WO 98/04355 disclosed an apparatus and method for rapidly determining the dielectrophoretic spectrum of a particle suspension, characterising the properties of specified particle types within a fluid.
  • This invention described an array of 20 electrodes separated from a common ground electrode, whereby a separate and distinct frequency of voltage could be applied between each electrode in the array and the common ground.
  • This enabled a complete dielectrophoresis spectrum to be obtained in a single experiment by applying electric fields of the same voltage but differing frequency to respective electrodes in the array.
  • the disadvantage of this arrangement is that it is less convenient for separating different particle types and reducing the amount of background material.
  • Dielectrophoresis This AC electrokinetic technique, known as Dielectrophoresis (DEP), has been shown to be useful for particle and cell characterisation and also for the separation of a particle type from a mixed suspension (Hagedorn et al., 1992; Huang et al., 1993; Gascoyne et al., 1992; Gascoyne et al., 1994; Huang et al., 1992).
  • Cells or particles become polarised by the action of AC electric fields and will experience a dielectrophoretic force when these fields are non- uniform.
  • the dielectrophoretic force is a function of frequency, determined by the electrical properties of the cell, reflecting cell structure and morphology.
  • the frequency of the electric field and the dielectric constant and electrical conductivity of the suspending medium is selected to produce positive and negative dielectrophoretic forces, where the positive dielectrophoretic force acts upon some only of the particles in the suspension (to attract particles to electrode surfaces where the field gradient is high), and the negative dielectrophoretic force acts upon a different population of particles in the suspension (repelling these particles to a spatially separate region of low, normally zero, electric field gradient) (Pethig et al., 1992). Markx et al.
  • WO 91/11262 disclosed the application of electrical fields of different characteristics to several separate arrays of electrodes, energised independently, for the purposes of spatially separating particle and cell types from a mixture on the basis of dielectrophoretic properties.
  • WO 94/22583 discloses a further separation device based upon the same localised spatial separation using dielectrophoretic action described in WO 91/11262.
  • a mixed suspension of two particle types is introduced into the centre of an electrode array and exposed to dielectrophoretic forces. The conditions are selected to cause positive DEP action upon one of the particle types, and negative DEP action upon the other particle type, resulting in a spatial separation.
  • the two components are separated and the individual components can be removed from either end of the electrode chamber.
  • GB 2,266,153 described a column array of interdigitating electrodes which could be energised to selectively retard cell populations within a mixture for subsequent elution of separated components, acting as a dielectrophoretic chromatographic column.
  • a similar invention described by Markx et al. (1997) is that of field flow fractionation (FFF), whereby dielectrophoretic levitation of particles is used to displace particles into different regions of a parabolic flow profile travelling at different velocities.
  • Patent WO 97/27933 discloses the combined use of field flow fractionation and phase shifted travelling wave DEP to bring about particle separations.
  • a method of separating different particles present in a liquid or gaseous sample comprising the steps of passing or circulating the sample through a region of non-uniform electric field density produced by at least one pair of electrodes, energising said electrodes with a first voltage having a first predetermined frequency selected to attract a first predetermined variety of particle in the sample to said electrodes, superimposing on said electrodes a second voltage having a second predetermined frequency selected to attract a second predetermined variety of particle in the sample, switching off either the first or the second voltage thereby releasing either the first or second variety of particle for subsequent collection and/or enumeration.
  • More than two different voltages having different predetermined frequencies may be superimposed on and applied to the electrodes in order to attract all the particles in the liquid sample to them. This can be applied to any number of cell or particle types within a mixture which may have wide variations in their DEP frequency spectrum.
  • the particles can then be subsequently released en masse by switching off all of the voltages, thus permitting a total particle count to be determined.
  • the particles may be released from the electrodes individually by type by switching off a selected voltage or voltages, thus facilitating separation of the particles for subsequent collection, identification and/or enumeration. Separation by this method may be enhanced by elution with specific suspending media of variable conductivity, pH or other physical characteristic.
  • the method may be used for separating different biotic particles such as microorganisms and/or different cell types and cell organelles including plasmids.
  • micro-organism is intended to embrace bacteria, viruses, yeasts, algae, protozoa, fungi and prions.
  • Abiotic particles which may be separated include for example latex beads, metal particles or any inorganic or organic material, chemical or biochemical species can also be separated.
  • an apparatus for separating particles present in a liquid sample comprising a support defining a fluid flow channel through a region of non-uniform electric field density produced by at least one pair of spaced electrodes, circulating means for passing or circulating said sample containing said particles through said channel, a first AC source for applying a first voltage at a first frequency to said electrodes, said frequency being selected to cause a first predetermined type of particle to be attracted to said electrodes, a second AC source for applying a second voltage at a second frequency to said electrodes, said second frequency being selected to cause a second predetermined type of particle to be attracted to said electrodes and means for determining the quantity of either the first or second predetermined type of particle when either the first voltage or second voltage is not applied.
  • the use of multiple frequencies may be simultaneously applied to any design of electrodes, with the electrodes being shaped to generate maximum DEP effect.
  • the electrodes may be castellated or they may comprise an electrode array which may or may not define more than one fluid flow channel. Several pairs of electrodes may be used thus forming a set of electrodes which may be linked together.
  • Figure 1 is an electrical and fluid circuit of an apparatus in accordance with the invention.
  • Figure 2 is a perspective fragmentary section taken through the collection block of the apparatus of Figure 1;
  • Figure 3 is a block diagram for the manufacture of a multiple frequency sinewave generator which can be used as an alternative to inductively coupled signal generators 28 and 30 of Figure 1;
  • Figure 4 is a graph showing dielectrophoretic frequency spectra of polystyrene latex spheres and E.coli bacteria demonstrating the differences in frequency of collection
  • Figure 5 is a graph showing the number of released latex beads counted over a period of time when different frequencies or voltage are applied individually and simultaneously using the multiple frequency sinewave generator;
  • Figure 6 is a graph showing the number of released E.coli counted over a period of time when different frequencies or voltage are applied individually and simultaneously using the multiple frequency sinewave generator;
  • Figure 7 is a graph showing the number of released latex beads and E.coli counted over a period of time when a 10 kHz voltage is applied;
  • Figure 8 is a graph showing the number of released latex beads and E.coli counted over a period of time when a 1 MHz voltage is applied
  • Figure 9 is a graph showing the number of released latex beads and E.coli counted over a period of time when both 10 kHz and 1 MHz voltages are applied.
  • Figure 10 is a graph showing the number of released latex beads and E.coli counted over a period of time when both 10 kHz and 1 MHz voltages are applied and then disabled sequentially.
  • the apparatus shown in Figure 1 comprises a collection block 2 in which particles can be collected.
  • the collection block 2 contains a pair of spaced electrodes 4 and 6 lying in a common plane and a fluid flow channel 8 positioned to cause a liquid to flow across the upper faces of the two electrodes.
  • the structure can be more clearly seen in Figure 2.
  • the structure includes an electrically insulating substrate 10 on which two elongate electrodes 4 and 6 have been deposited in parallel but spaced relationship with each other.
  • Two electrically insulating side walls 12 and 14 are attached to the substrate 10 and cover both the electrodes 4 and 6.
  • the side walls can be composed of a polyimide.
  • the side walls 12 and 14 define the channel 8 which extends at right angles to the electrodes 4 and 6.
  • a further electrically insulating layer (not shown) extends over the side walls 12 and 14 and the channel 8 to form the roof of the channel 8.
  • the exposed face of each electrode may be covered with a thin electrically insulating layer as required.
  • a reservoir 20, for containing a sample of liquid to be analysed is connected by a duct 22 to the upstream end of the channel 8.
  • a duct 24 connected to the downstream end of the channel 8 feeds liquid from the channel 8 through a pump 26 back to the reservoir 20.
  • the pump 26 is advantageously a peristaltic pump to prevent any contamination or damage to the sample liquid and particles therein.
  • the liquid in the reservoir 20 may be agitated by bubbling air or other gas therethrough to keep the particles in suspension.
  • a multiple frequency sinewave generator may alternatively be used according to the schematic shown in Figure 3.
  • the multiple frequency generator comprises a set of fixed frequency oscillators which can be energised to output a specific frequency sinewave. The outputs from these are mixed and outputted to energise electrodes 4 and 6 of Figure 1.
  • Five fixed frequency oscillators are shown in the figure (at frequencies 1kHz, 10kHz, 100kHz, 1MHz and 10MHz) each providing an 8N peak output voltage. Thus it is possible to simultaneously superimpose voltages of all five of these frequencies on to electrodes 4 and 6.
  • any number of signal generators may be inductively coupled to apply more frequencies of voltage to the pair of electrodes or superimposing using other methods.
  • the multiple frequency sinewave generator can be manufactured with any number of frequency oscillators.
  • By using an appropriate number of frequencies it should be possible to collect every type of particle in a suspension even with widely differing frequency spectrum characteristics, such as those of E.coli and polystyrene latex as demonstrated in Figure 4. Following this it would be possible to determine the number of particles collected with the image analysis technique or another method. This would then be a measure of the total number of particles in the test suspension.
  • Impedance matching may be used to make the isolation transformers and the electrode structure match the 50 ⁇ impedance of any coaxial cables used to connect the system. This will create a constant voltage across the electrode gap within the range of frequencies to be used.
  • the apparatus according to the invention may be computer controlled to enable rapid, easy control of the signal generators or multiple frequency generator. This will be of particular benefit if all particle types are to be collected from a sample and released individually by changing the applied frequencies.
  • the liquid sample is placed in the reservoir 20 and pumped by pump 26 via duct 22 through the channel 8 over electrodes 4 and 6.
  • Latex beads are known to collect well on the electrodes when an output frequency of 10 kHz is applied to the electrodes 4 and 6. Signal generator 30 is therefore set to have an output frequency of 10 kHz (or the 10kHz oscillator on the multiple frequency sinewave generator is applied). Latex beads do not collect well at 1 MHz. In contrast, E.coli bacteria collect well at both frequencies as shown in Figure 4. Signal generator 28 is set to have an output frequency of 1 MHz (or the 1MHz oscillator on the multiple frequency sinewave generator is applied).
  • one or both of the voltage inputs were applied for a period of time, while the image analysis program counted particles in order to determine the background level. The voltage was then turned off. If any particles had collected at the electrodes they would then be released and pass beneath the microscope, leading to an increase in the number of particles counted as the released packet of particles flowed by. The number of counted particles would then drop back to the background level. Changing the phase/contrast of the microscope allowed either the latex beads or the E.coli to be labelled and counted separately.
  • the latex beads collected well at 10 kHz, as there was a significant increase in the number counted once the voltage was disabled and the particles were released, before the count dropped back to a background level. At 1 MHz there was very poor collection, with the count remaining at a background level throughout the experiments.
  • Application of the individual frequencies produced a frequency spectral profile very similar to that shown in Figure 5. The application of all voltages simultaneously resulted in good collection of the latex.
  • the E.coli collected well at both 10 kHz and 1 MHz, but there was an increase in collection with all voltages applied.
  • spectrophotometric including fluorescence
  • laser impedance analysis
  • radiometric may be used to count the number of particles collected at or released from the electrodes.
  • the method and apparatus defined above may be used to separate a single species from the test suspension by either collecting all cell types bar the desired one, or by only collecting the desired species. Another option may be to collect all of the cells in the suspension and then release them one at a time by either turning off one frequency or by changing one or more of the frequencies such that one cell type no longer collects. This method could be used to separate any number of species in the suspension, by collecting them all and selectively releasing them individually.
  • any number of the species from a large suspension onto the electrodes by appropriate choice of applied frequencies. Following the collection it will be possible to elute the collected cells into a much smaller volume suspension, effectively concentrating the suspension. In addition, the collected cells could be eluted into a different suspending medium as a further form of sample preparation.

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  • Engineering & Computer Science (AREA)
  • Microelectronics & Electronic Packaging (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Electrostatic Separation (AREA)

Abstract

L'invention concerne un procédé et un appareil permettant de collecter et de séparer des particules abiotiques et/ou biotiques, et/ou des substances chimiques dans un échantillon liquide ou gazeux. L'appareil comprend un substrat (10) supportant au moins une paire d'électrodes (4, 6), et définissant un canal fluidique (8) sur lesdites électrodes. Ces électrodes (4, 6) sont excitées par au moins deux tensions possédant des fréquences prédéterminées différentes qui attirent les particules, par diélectrophorèse, en direction desdites électrodes. Il est possible de libérer sélectivement un type de particule destiné à une collecte et/ou une énumération subséquente, par suppression d'au moins une tension.
PCT/GB2001/000239 2000-01-22 2001-01-22 Procede et appareil de separation de particules WO2001052997A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001228636A AU2001228636A1 (en) 2000-01-22 2001-01-22 Method and apparatus for the separation of particles

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0001376.3 2000-01-22
GB0001376A GB2358361B (en) 2000-01-22 2000-01-22 Method and apparatus for the separation of particles

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WO2001052997A1 true WO2001052997A1 (fr) 2001-07-26

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AU (1) AU2001228636A1 (fr)
GB (1) GB2358361B (fr)
WO (1) WO2001052997A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0812999D0 (en) * 2008-07-16 2008-08-20 Blood Analysis Ltd Detection of microorganisms
CN104174492B (zh) * 2014-08-22 2017-03-29 成都代代吉前瞻科技股份有限公司 一种高效静电‑介电电泳除尘器

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991011262A1 (fr) * 1990-01-30 1991-08-08 P & B (Sciences) Limited Manipulation de substances solides, semi-solides ou liquides
US5795457A (en) * 1990-01-30 1998-08-18 British Technology Group Ltd. Manipulation of solid, semi-solid or liquid materials
US5814200A (en) * 1993-03-31 1998-09-29 British Technology Group Limited Apparatus for separating by dielectrophoresis

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991011262A1 (fr) * 1990-01-30 1991-08-08 P & B (Sciences) Limited Manipulation de substances solides, semi-solides ou liquides
US5795457A (en) * 1990-01-30 1998-08-18 British Technology Group Ltd. Manipulation of solid, semi-solid or liquid materials
US5814200A (en) * 1993-03-31 1998-09-29 British Technology Group Limited Apparatus for separating by dielectrophoresis

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GB2358361A (en) 2001-07-25
GB2358361B (en) 2003-04-23
GB0001376D0 (en) 2000-03-08
AU2001228636A1 (en) 2001-07-31

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