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WO2001052996A1 - Procedes et appareil de detection de corps microscopiques - Google Patents

Procedes et appareil de detection de corps microscopiques Download PDF

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Publication number
WO2001052996A1
WO2001052996A1 PCT/GB2001/000226 GB0100226W WO0152996A1 WO 2001052996 A1 WO2001052996 A1 WO 2001052996A1 GB 0100226 W GB0100226 W GB 0100226W WO 0152996 A1 WO0152996 A1 WO 0152996A1
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WO
WIPO (PCT)
Prior art keywords
voltage
frequency
sample
bodies
electrode structure
Prior art date
Application number
PCT/GB2001/000226
Other languages
English (en)
Inventor
Keith Richard Milner
Walter Bernard Betts
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 AU2001226948A priority Critical patent/AU2001226948A1/en
Publication of WO2001052996A1 publication Critical patent/WO2001052996A1/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]

Definitions

  • the present invention relates to methods and apparatus for detecting microscopic bodies.
  • Dielectrophoresis has been defined as the motion of a polarised but uncharged particle situated within a region of non-uniform electric field. When a particle is placed within a region of electric field, a dipole may be induced within that particle in addition to any permanent dipole that may be present within that particle.
  • the particle is within a region of non-uniform electric field there may be an imbalance in the forces exerted upon either end of the net dipole within the particle owing to one end of the dipole being situated in a region of higher electric field than the other end, even though the particle remains electrically neutral and has no excess charge.
  • the imbalance of forces exerted upon the particle may lead to a net attractive force being exerted upon the particle inducing a translational motion towards areas of highest electric field non- uniformity (referred to as positive dielectrophoresis), or may lead to a net repulsive force being exerted upon the particle inducing a translational motion towards areas of lowest electric field non-uniformity (referred to as negative dielectrophoresis). If the net force exerted upon the particle is zero, then no induced motion will occur. In addition, rotational motions may be induced upon the particle when the electric field acts to align the dipole moment. This phenomenon is referred to as electrorotation (W M Arnold et al; J Electrostat; 21, 2-3, 151- 191, 1988).
  • the electrodes respond to different frequencies in a non-uniform electric field, the electrodes are supplied with a voltage having a frequency corresponding to a selected type and these particles are then attracted to one or other of the electrodes under positive dielectrophoresis (X B Wang et al; J Phys D; 26, 8, 1278-1285, 1993).
  • the liquid is cycled for a period sufficiently long for substantially all the selected organisms to be collected at the electrodes. Discontinuing the voltage releases the particles back into the circulating liquid.
  • a microscope over the electrodes or downstream of the electrodes can be used to count the number of passing particles in a representative cross-section of the flow and from this the total number of particles can be determined.
  • the problem is solved by determining the concentration of particles by measuring the effect that they have on the electrical characteristics of the electrodes, namely the amount by which they change the impedance of the electrodes.
  • a comparison can be effected between the change in voltage across a pair of electrodes supplied with the sample liquid and a pair of reference electrodes supplied with a reference liquid, to determine the net change in impedance caused by the aggregation of particles at the pair of electrodes supplied with the sample liquid.
  • the change in voltage across a pair of electrodes supplied with the sample liquid can be monitored over time to determine the change in impedance caused by the aggregation of particles at the electrodes over time relative to impedance of the electrodes prior to the aggregation of particles at the electrodes.
  • the electrodes in either case may be configured as a number of pairs or in another fashion designed to manipulate the particles in the suspension in to a region where impedance measurements may be most effectively taken or particle aggregation is most efficient or designed for an other purpose.
  • body is defined as non-exclusively including abiotic or biotic particles, biological cells, viruses, viroids, prions, subcellular organelles, chemical and biological molecules.
  • a method of determining the quantity of bodies present in a fluid sample comprising the steps of circulating the fluid sample through a region of non-uniform electric field density produced by an electrode structure, circulating a reference fluid having similar electrical characteristics to the carrier fluid of the sample through a region of non-uniform electric field density produced by a second electrode structure having a similar configuration to said electrode structure, energising both electrode structures with a first voltage having a predetermined frequency and for a sufficient period selected to attract a predetermined variety of bodies in the sample to the first electrode structure, superimposing on both electrode structures a second voltage having a second frequency selected so as to impose substantially no translational force on said predetermined bodies in said sample, measuring the voltages at said second frequency across said two electrode structures, processing said measurements to determine the differences in capacitance and conductance between said two electrode structures directly related to the quantity of bodies built up on said electrodes.
  • an apparatus for determining the quantity of bodies present in a sample comprising a support defining first and second similar fluid flow channels through similar regions of non-uniform electric field density respectively produced by first and second electrode structures of similar configuration, first circulation means for circulating a sample containing said bodies through said first channel and second circulation means for circulating a fluid of similar electrical characteristics to the fluid of said sample but without said bodies through the second channel, circuit means connecting said first electrode structure in series with a first capacitor, said second electrode structure in series with a second capacitor and both said circuit series in parallel with each other, a first AC source for supplying a first voltage at a first frequency across the parallel circuit, said frequency being selected to cause a predetermined variety of bodies to be attracted to said first electrode structure, a second AC source for supplying a second voltage at a second frequency across said parallel circuit, said second frequency being selected so that it imposes substantially no force on said predetermined bodies in said sample, means for measuring said second voltage, a third voltage at said second frequency appearing across said
  • a second method of determining the quantity of bodies present in a fluid sample comprising the steps of circulating the fluid sample through a region of non-uniform electric field density produced by an electrode structure, energising said electrode structure with a first voltage having a predetermined frequency selected so as to impose substantially no translational force on said predetermined bodies in said sample, measuring the voltages at said frequency across said electrode structure, then superimposing on the electrode structure a second voltage having a second predetermined frequency for a sufficient period selected to attract a predetermined variety of bodies in the sample to the electrode structure, measuring the voltages at said first frequency across said two electrode structures, processing said measurements to determine the variation with time of capacitance and conductance of said electrode structure, directly related to the quantity of bodies that builds up on said electrodes.
  • a second set of apparatus for determining the quantity of bodies present in a sample comprising a support defining a fluid flow channel through a region of non-uniform electric field density respectively produced by an electrode structure, circulation means for circulating a sample containing said bodies through said channel, circuit means connecting said electrode structure in series with a capacitor, a first AC source for supplying a first voltage at a first frequency across the circuit, said frequency being selected so that it imposes substantially no force on said predetermined bodies in said sample, a second AC source for supplying a second voltage at a second frequency across said circuit, said second frequency being selected to cause a predetermined variety of bodies to be attracted to said electrode structure, means for measuring said first voltage and a third voltage at said first frequency appearing across said electrode structure and the relative phase between the third voltage and the first voltage, performing said measurements both with the second voltage disabled and at predetermined time intervals with said second voltage enabled, and means for processing said measurements to determine the difference in the capacitive and conductive elements of the imped
  • Figure 1 is an electrical and fluid circuit of one embodiment of the apparatus
  • Figure 2 is a perspective fragmentary section taken through the collection block of the apparatus of Figure 1 ;
  • Figure 3 is a graph showing variation in capacitance change recorded with drive voltage source frequency for a variety of sample liquids consisting of differing concentrations of 2 ⁇ m diameter abiotic latex beads suspended in deionised water;
  • Figure 4 is a graph showing variation in capacitance change recorded with concentration of 2 ⁇ m diameter abiotic latex bead sample liquids for a variety of drive voltage source frequencies;
  • Figure 5 is a graph showing variation in capacitance change recorded with drive voltage source frequency for a variety of sample liquids containing differing concentrations of 100 ran diameter abiotic latex beads suspended in deionised water
  • Figure 6 is a graph variation in capacitance change recorded with concentration of 100 nm diameter abiotic latex bead sample liquids for a variety of drive voltage source frequencies.
  • the apparatus shown in Figure 1 comprises a collection block 2 in which micron or submicron sized organisms can be collected and measurements taken.
  • the collection block 2 contains a pair of spaced electrodes 4 and 6 lying in common plane and a fluid flow channel 8 positioned to cause 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 the two elongate electrodes 4 and 6 have been deposited in parallel but spaced relationship with each other.
  • the resulting slot 16 between the two strips 12 and 14 defines the flow channel 8 across the two electrodes 4 and 6.
  • a further electrically insulating layer (not shown) extends over the strips 12 and 14 and the slot 16 to form the roof of the channel 8.
  • the exposed face of each electrode may be covered with a thin electrically insulating layer, or non-stick or other coating 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 damage, injury or contamination to the sample liquid.
  • the liquid in the reservoir 20 may be agitated by bubbling air or other gas therethrough or by using a magnetic stirrer or other device to keep the microorganisms in suspension.
  • the reservoir 20A contains a reference liquid selected as will be described in more detail hereinafter and the channel 8 A and the electrodes 4A and 6 A share the same substrate 10.
  • the capacitor formed by the electrodes 4 and 6 is connected, in series, with a capacitor 30 to form a potential divider.
  • the output voltage V A is obtained from a centre tap terminal 32 between the two capacitors.
  • the capacitor formed by the electrodes 4 A and 6 A is connected in series with a capacitor 34 to form another potential divider.
  • An output voltage V ⁇ is obtained from a centre tap terminal 36.
  • the two potential dividers are connected in parallel with each other and an impedance matching resistor 38 across a pair of input terminals 40 and 42.
  • a terminal 41 provides a voltage V JN appearing across the terminals 40 and 42.
  • An AC probe voltage source 44 supplies a voltage to the primary winding of a transformer 46.
  • An AC drive voltage source 48 is connected in series with the secondary winding of the transformer 46 across the terminals 40 and 42.
  • a first impedance matching resistor 50 is connected in parallel with the source 48 and a second impedance matching resistor 51 is connected in parallel with the secondary winding of the transformer 46.
  • the drive voltage source 48 is a variable frequency source having an output voltage selected to cause microorganisms to migrate to one or other of the two electrodes 4 and 6.
  • the voltage may be of the order of 12 volts and the frequency varied in the range of from l0 3 to l0 7 Hz.
  • the probe voltage source 44 is a fixed frequency source having a voltage sufficiently low as to have little or no effect in attracting microorganisms to one or other of the two electrodes 4 and 6.
  • the voltage may be of the order of 0.5 volts and the frequency around 810 Hz.
  • a lock-in amplifier (not shown) is used to monitor the relative changes in magnitude and relative phase of the voltages V A , V B and V IN -
  • the liquid contained in the reservoir 20A is selected to have similar electrical characteristics to the carrier liquid for the microorganisms.
  • both pumps 26 and 26A are operated to cycle respective liquids. Then, with the drive source 48 de-energised and probe source 44 energised, the magnitudes and relative phases of the voltages V ⁇ M , V A and V B are determined.
  • the drive source 48 is then energised at a frequency selected to attract a specific organism and, after a period of sufficient length to allow all those organisms to be attracted to one of the electrodes 4 and 6, the magnitude and relative phases of the voltages V IN , V A and V B , are again determined.
  • V IN , V A and V B can be represented as complex numbers V IN *, V A * and V B *.
  • Z * M the impedance of the capacitor 30
  • Z * E the impedance of the electrodes 4 and 6
  • an AC probe voltage source 44 supplies a voltage to the primary winding of a transformer 46.
  • An AC drive voltage source 48 is connected in series with the secondary winding of the transformer 46 across the terminals 40 and 42.
  • a first impedance matching resistor 50 is connected in parallel with the source 48 and a second impedance matching resistor 51 is connected in parallel with the secondary winding of the transformer 46.
  • the drive voltage source 48 is a variable frequency source having an output voltage selected to cause microorganisms to migrate to one or other of the two electrodes 4 and 6.
  • the voltage may be of the order of 12 volts and the frequency varied in the range of from l0 3 to l0 7 Hz.
  • the probe voltage source 44 is a fixed frequency source having a voltage sufficiently low as to have little or no effect in attracting microorganisms to one or other of the two electrodes 4 and 6.
  • the voltage may be of the order of 0.5 volts and the frequency around 7 kHz.
  • a lock-in amplifier (not shown) is used to monitor the relative changes in magnitude and relative phase of the voltages V A and V IN -
  • pump 26 is operated to cycle the sample liquid. Then, with the drive source 48 de-energised and probe source 44 energised, the magnitudes and relative phases of the voltages V IN and V A are determined.
  • the drive source 48 is then energised at a frequency selected to attract a specific organism and the magnitude and relative phases of the voltages V I and V A (or of V A alone) are repetitively determined at predetermined time intervals, until a period of sufficient length has elapsed to allow all those organisms to be attracted to one of the electrodes 4 and 6.
  • the mathematical process described earlier can be used to determine the values C and G of the electrodes prior to particle attraction and at predetermined time intervals while particle attraction is occurring, whereby
  • the impedance spectrum of particles held at a pair of electrodes supplied with a sample liquid is monitored.
  • This method may be used with either of the apparatus layouts described earlier, that is with a single collection block supplied with a sample liquid, in which micron or submicron sized organisms can be collected and measurements taken, along with associated pump and duct configurations and appropriate electrical power supplied and components; alternatively with two collection blocks, one supplied with a sample liquid, in which micron or submicron sized organisms can be collected and measurements taken and the other supplied with a reference liquid selected to have similar electrical characteristics to the carrier liquid for the microorganisms, along with associated pump and duct configurations and appropriate electrical power supplied and components.
  • pumps are operated to cycle the liquids.
  • the drive voltage source is then energised at a frequency preselected to attract a specific organism to the electrodes. With the organisms held at the electrodes, the capacitive and resistive components of the electrodes across a frequency range are then determined using the mathematical process described earlier, by measuring the magnitudes and phases of voltages Vj j and V A (and V B if a pair of electrodes supplied with a reference liquid is used) for a variety of probe voltage source frequencies.
  • dielectric spectroscopy H P Schwan in 'Electrical Properties of Tissue and Cell Suspensions, Vol 5, Advances in Medical and Biological Physics', S H Lawrence and C A Tobias, Academic Press, New York, 1957
  • a comparison may be effected between the dielectric spectrum of the pair of electrodes supplied with a sample liquid and the pair of reference electrodes supplied with a reference liquid,
  • the resultant ⁇ C and ⁇ G spectra were found to be both characteristic of the organisms in the liquid sample and also directly related to the quantity of organisms built up on the electrodes.
  • a number of cell characterisation spectra were obtained using the above experimental technique whereby a comparison was effected between a pair of electrodes supplied with a sample liquid and a pair of reference electrodes supplied with a reference liquid.
  • a variety of sample liquids consisting of differing concentrations of optically resolvable 2 ⁇ m diameter abiotic latex beads suspended in deionised water were used in conjunction with a similar number of reference liquids comprising deionised water alone.
  • Each characterisation spectrum was obtained by varying the frequency of the drive voltage source and recording the resulting variations in voltages developed across the measurement capacitors at the frequency of the probe voltage source with a lock-in amplifier.
  • Figure 3 illustrates the resulting capacitance change spectra.
  • concentration of a sample liquid containing an unknown number of any particle type could be determined by measuring the capacitance change value recorded at a discrete drive voltage source frequency and referring a similar calibration curve developed for the particle type in question. It will also be apparent to those skilled in the art that many modifications and changes could be made to this specific apparatus without departing from the scope of the invention. For example, other electrode configurations could be used to produce the non-uniform electric field, other fluid flow channels could be constructed over said electrodes, or other frequencies or magnitudes of the probe and drive voltage sources could be employed.

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  • Engineering & Computer Science (AREA)
  • Microelectronics & Electronic Packaging (AREA)
  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

On décrit un procédé et un appareil qui permettent de déterminer la quantité de micro-organismes dans un échantillon. L'appareil comprend un substrat (10) qui supporte deux paires d'électrodes (4, 6; 4A, 6A) de configuration similaire et définit une paire de passages (8; 8A) pour le fluide au-dessus des électrodes. La paire d'électrodes (4, 6) est excitée par une source ayant une fréquence particulière qui, par diélectrophorèse, attire des micro-organismes spécifiques en direction de la paire d'électrodes. On a remarqué qu'un changement de la capacitance est directement lié à la quantité de micro-organismes accumulés sur les électrodes. Lorsqu'on utilise la deuxième paire d'électrodes (4A, 6A) en tant que référence, le changement de l'impédance dû à la diélectrophorèse peut être déterminé. L'élément capacitif du changement d'impédance peut ensuite être déterminé lorsqu'on mesure la tension au niveau des deux paires d'électrodes (4, 6; 4A, 6A) ainsi que le changement de phase par rapport à la source (44).
PCT/GB2001/000226 2000-01-22 2001-01-22 Procedes et appareil de detection de corps microscopiques WO2001052996A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001226948A AU2001226948A1 (en) 2000-01-22 2001-01-22 Methods and apparatus for detecting microscopic bodies

Applications Claiming Priority (2)

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GB0001374.8 2000-01-22
GB0001374A GB2358473B (en) 2000-01-22 2000-01-22 Methods and apparatus for detecting microscopic bodies

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005088287A1 (fr) * 2004-03-17 2005-09-22 National Research Council Of Canada Procede et dispositif servant a detecter des micro-organismes
CN107615041A (zh) * 2015-10-07 2018-01-19 Afi技术公司 检查装置、检查系统以及检查方法

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7306924B2 (en) * 2000-04-17 2007-12-11 Purdue Research Foundation Biosensor and related method
DE10203636B4 (de) * 2002-01-30 2004-02-12 Testo Gmbh & Co Vorrichtung zum Nachweis von Partikeln in einem Fluid
WO2010092773A1 (fr) 2009-02-10 2010-08-19 パナソニック株式会社 Dispositif et procédé de mesure de fines particules

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2238619A (en) * 1989-11-27 1991-06-05 Nat Res Dev Dielectrophoretic characterisation of micro-organisms and other particles
WO1991011262A1 (fr) * 1990-01-30 1991-08-08 P & B (Sciences) Limited Manipulation de substances solides, semi-solides ou liquides
WO1998004355A1 (fr) * 1996-07-26 1998-02-05 Btg International Limited Appareil et procede de test de particules par dielectrophorese

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Publication number Priority date Publication date Assignee Title
GB9208357D0 (en) * 1992-04-16 1992-06-03 British Tech Group Apparatus for separating a mixture

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2238619A (en) * 1989-11-27 1991-06-05 Nat Res Dev Dielectrophoretic characterisation of micro-organisms and other particles
WO1991011262A1 (fr) * 1990-01-30 1991-08-08 P & B (Sciences) Limited Manipulation de substances solides, semi-solides ou liquides
WO1998004355A1 (fr) * 1996-07-26 1998-02-05 Btg International Limited Appareil et procede de test de particules par dielectrophorese

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MILNER K R ET AL: "DIELECTROPHORETIC CLASSIFICATION OF BACTERIA USIN DIFFERENTIAL IMPEDANCE MEASUREMENTS", ELECTRONICS LETTERS,IEE STEVENAGE,GB, vol. 34, no. 1, 8 January 1998 (1998-01-08), pages 66 - 68, XP000773611, ISSN: 0013-5194 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005088287A1 (fr) * 2004-03-17 2005-09-22 National Research Council Of Canada Procede et dispositif servant a detecter des micro-organismes
CN107615041A (zh) * 2015-10-07 2018-01-19 Afi技术公司 检查装置、检查系统以及检查方法
US20200064274A1 (en) * 2015-10-07 2020-02-27 Afi Corporation Inspection device, inspection system, and inspection method
CN107615041B (zh) * 2015-10-07 2020-12-11 Afi技术公司 检查装置、检查系统以及检查方法
US10876974B2 (en) * 2015-10-07 2020-12-29 Afi Corporation Inspection device, inspection system, and inspection method

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GB0001374D0 (en) 2000-03-08
AU2001226948A1 (en) 2001-07-31
GB2358473B (en) 2003-10-08
GB2358473A (en) 2001-07-25

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