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WO2010007350A1 - Détections de microorganismes par diélectrophorèse - Google Patents

Détections de microorganismes par diélectrophorèse Download PDF

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Publication number
WO2010007350A1
WO2010007350A1 PCT/GB2009/001698 GB2009001698W WO2010007350A1 WO 2010007350 A1 WO2010007350 A1 WO 2010007350A1 GB 2009001698 W GB2009001698 W GB 2009001698W WO 2010007350 A1 WO2010007350 A1 WO 2010007350A1
Authority
WO
WIPO (PCT)
Prior art keywords
fluid
microorganisms
microelectrode
sample
dielectrophoresis
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/GB2009/001698
Other languages
English (en)
Inventor
Peter Salmon
Robert Stewart Anthony
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BLOOD ANALYSIS Ltd
Original Assignee
BLOOD ANALYSIS Ltd
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 BLOOD ANALYSIS Ltd filed Critical BLOOD ANALYSIS Ltd
Priority to EP09784661A priority Critical patent/EP2310133A1/fr
Publication of WO2010007350A1 publication Critical patent/WO2010007350A1/fr
Priority to US12/981,345 priority patent/US20110123979A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • B03C5/005—Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength
    • 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 a method and apparatus for collecting, detecting and enumerating microorganism in fluids.
  • it relates to the rapid detection and enumeration of microorganisms in mammalian fluids such as blood or blood products.
  • a previously used method for detecting and enumerating microbial contamination of blood or blood products involved culruring a sample of blood or blood product. However, such a method was too slow, requiring several hours/days incubation. It was also too insensitive to be of any practical use. Elder, A F et al found that up to 50% of bacterially contaminated platelets may escape detection by culture at 24 hours (see Transfusion, (2007), 47, 1134).
  • a solid phase laser scanner has been used to enumerate bacteria in water.
  • Broadway, S C et al in Appl Environ Microbial. (2003), 69(7), 4272-4273 described the rapid staining and enumeration of small numbers of bacteria in water using solid-phase laser cytometry.
  • a sample of water was filtered through a black polycarbonate membrane and then an overlay of SYBR Green I dye was applied to the filter. After incubation, and removal of the stain, the membrane was dried and chilled prior to laser scanning.
  • This method suffers from the disadvantage that interaction of the dye with the membrane filter produces non-specific spots of stain.
  • microscopic examination of the membrane is usually necessary to identify possible non-specific stains. Other particulates in the water may take up the dye and become trapped on the membrane resulting in false positive counts.
  • Dielectrophoresis which is the motion of electrically neutral particles or cells in response to a non-uniform electric field and can occur equally well in both DC and AC electric fields, has been used to quantify the number of particles in a liquid sample. Allsopp, D W E et al in J Phvs D: App Phvs. (1999), 32, 1066-1074 described an impedance technique for measuring dielectrophoretic collection of microbiological particles. The authors showed that measurement of the impedance change resulting from the collection of microbiological particles at coplanar electrodes enabled them to quantify the concentration of particles collected under positive dielectrophoretic force.
  • the disadvantages of this method are a) low sensitivity in that at least 10 5 ml bacteria are required to incur a measurable impedance change, b) inflexible sample conditions in that the bacteria must be suspended in a buffer with an extremely low conductivity. Furthermore the change in impedance does not correlate with an accurate bacterial count. The size and cell wall characteristics influence the magnitude of the impedance change.
  • a method of collecting and detecting microorganisms in a fluid comprising the steps of subjecting a sample of said fluid to dielectrophoresis and collecting the microorganisms onto a microelectrode, scanning the microelectrode using a scanning laser and determining the number of microorganisms present on the microelectrode.
  • the method may be used for detecting microorganisms such as bacteria, viruses, yeasts, algae, protozoa and fungi. .
  • the fluid may be any mammalian fluid such as urine or cerebrospinal fluid, however, the method is particularly useful for detecting microorganisms in blood or blood products, such as platelets.
  • a lysis solution is preferably added in order to lyse any mammalian cells present.
  • the contaminating microorganism may then be separated using centrifugation, after which they are stained or labelled so that they will fluoresce when subjected to the scanning laser.
  • Suitable stains or labels may include non-specific nuclear dyes such as SYBR Green I and acridine orange, metabolic substrates that become fluorescent through enzymatic activity, antibodies, including monoclonal antibodies, to microbial proteins, or molecular probes which can hybridise to microbial genetic material or a combination of these.
  • Figure 1 shows the concept of DEP
  • Figure 2 shows an electrode placed in a laser scanner
  • Figure 3 shows an electrode in a well.
  • the labelled contaminating microorganisms suspended in a fluid are then loaded onto a microelectrode.
  • the microelectrode comprises at least one pair of adjacent co-planar electrodes of micron dimensions for electrode width or gap size, and is supported on a substrate.
  • the microelectrodes can be manufactured in various metals, including gold and aluminium.
  • the substrate material is transparent and preferably of low autofluorescence, for example glass or plastic.
  • the microelectrode may be manufactured by micro- fabrication technology employing photolithography or by printing metal ink technology or by printing and electrode plating technology or by a combination of these methods.
  • the microelectrode may be placed in the bottom of a well, which may form part of a 96-well plate.
  • the fluid remains static during DEP collection of the microorganism.
  • the microelectrode may be placed in a flow through chamber where the suspending fluid containing the microorganisms is passed over the microelectrode on one side of the chamber in order to facilitate DEP collection from a larger sample.
  • the microelectrode structure in these examples would normally be of a co- planar type.
  • the microelectrode may be of a grid construction where a series of insulated grids are aligned to allow the passage of fluid.
  • Dielectrophoretic forces are produced across the microelectrode by an alternating current of fixed amplitude and wavelength. This may vary depending upon the conductivity of the suspending medium and the type of microorganisms to be collected.
  • the signal may be a sine wave or a square wave and may or may not have a direct current offset depending upon the conductivity of the sample and the type of microorganism to be collected.
  • DEP collects and concentrates any microorganisms suspended in the fluid onto the edge of the microelectrode. . .
  • the current is switched off and the microelectrode is placed in the laser scanner.
  • Scanning lasers and photomultiplier tube (PMT) detectors scan the area surrounding the microelectrode to excite the fluorescently labelled microorganisms and detect the emitted light.
  • the wavelength used for laser scanning may vary depending on the fluorochrome used in the marker dye. In systems where two or more marker dyes of different excitation wavelengths are used, more than one source of laser light will be used.
  • the fluorescence intensity is collected at regular intervals by the PMTs and thresholding algorithms identify all the fluorescence intensities above background levels.
  • Object intensity profiles enable the calculation of a range of morphological and fluorescent parameters to identify microorganisms collected onto the electrodes from the fluid sample.
  • Lysis solution (0.2 ml of 5% Triton, phosphate buffered saline (PBS) containing 10 9 polyethylene imide (PEI) coated paramagnetic beads (10mm diameter)) was added to ⁇ human blood platelets (1 ml) in a microfuge tube (2 ml). The tube was centrifuged using Eppendorf centrifuge 5424 at 20,00 xg for 2 min and then placed in a magnetic particle separator (mps). The beads and contaminating bacteria were allowed to collect. on the wall of the tube and the lysate supernatant was poured off. The tube was removed from the mps and staining solution (50 ⁇ l of SYBR green 1: 1,000 in 2 micro Si quarter strength Ringers solution) was added.
  • PBS Triton, phosphate buffered saline
  • PEI polyethylene imide
  • the tube was incubated at room temperature for 5 minutes in the dark.
  • the stained sample was the pipetted into a well in a dielectrophoretic microelectrode chip and connected to an alternating current (AC) signal of 100 KHz, 10 V amplitude for 10 minutes:
  • the AC signal source was disconnected, the microelectrode chip was inverted in the scanning holder and loaded into a Bac-Detect laser scanner (Bac-Detect is available from Blood Analysis Ltd, PO BOX 71, Slough SL2 3SE).
  • Laser scanning of the DEP collected bacterial was initiated.
  • the results were displayed by the software as pass or fail depending on the level of bacterial contamination detected in the platelet sample. Alternatively they can be displayed as an exact bacterial count and the bacteria visualised by an image of then scanning surface.
  • Figure 1 shows DEP schematically.
  • a sample A is passed across a substrate 1 having printed upon it, or otherwise formed upon it, a microelectrode structure comprising interdigitated electrodes 2 and 3.
  • AC current 4 is generated and applied to the electrode via connections 5 and 6 to the respective electrodes 2 and 3.
  • the electrodes are of micron dimensions and are energised with the voltage of a predetermined frequency using AC generator 4.
  • the relevant particles collect on the electrode array and then, after the deposition process, the substrate can be analysed by visual inspection using microscopes or otherwise to count the number of particles and therefore information about the type and/or concentration of particles can be determined.
  • Figure 2 shows, again very schematically, a scanning laser 6 and focusing optics 7 focussing a laser beam 8 onto a microelectrode 9.
  • the optics may be an integral part of the laser, or separate.
  • Scanning means, for causing the beam to scan relative to the sample may be included.
  • DEP is used to collect microorganisms onto the edge of the electrode as discussed and the laser scans to detect these.
  • Figure 3 shows a microelectrode 9a within a well 10.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Microelectronics & Electronic Packaging (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention porte sur un procédé de collecte, de détection et de dénombrement de microorganismes dans un fluide. Ce procédé consiste à soumettre un échantillon du fluide à une diélectrophorèse et à recueillir les microorganismes sur une microélectrode, à balayer la microélectrode à l'aide d'un laser à balayage et à déterminer le nombre de microorganismes présents sur la microélectrode.
PCT/GB2009/001698 2008-07-16 2009-07-09 Détections de microorganismes par diélectrophorèse Ceased WO2010007350A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP09784661A EP2310133A1 (fr) 2008-07-16 2009-07-09 Détections de microorganismes par diélectrophorèse
US12/981,345 US20110123979A1 (en) 2008-07-16 2010-12-29 Detection of microorganisms

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0812999A GB0812999D0 (en) 2008-07-16 2008-07-16 Detection of microorganisms
GB0812999.1 2008-07-16

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/981,345 Continuation-In-Part US20110123979A1 (en) 2008-07-16 2010-12-29 Detection of microorganisms

Publications (1)

Publication Number Publication Date
WO2010007350A1 true WO2010007350A1 (fr) 2010-01-21

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2009/001698 Ceased WO2010007350A1 (fr) 2008-07-16 2009-07-09 Détections de microorganismes par diélectrophorèse

Country Status (3)

Country Link
EP (1) EP2310133A1 (fr)
GB (1) GB0812999D0 (fr)
WO (1) WO2010007350A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013522590A (ja) * 2010-03-12 2013-06-13 独立行政法人理化学研究所 生物材料用透明化試薬、及びその利用
US20160266016A1 (en) 2013-08-14 2016-09-15 Riken Composition for preparing biomaterial with excellent light-transmitting property, and use thereof
US10274492B2 (en) 2015-04-10 2019-04-30 The Curators Of The University Of Missouri High sensitivity impedance sensor
CN109722378A (zh) * 2018-12-20 2019-05-07 安徽中青检验检测有限公司 一种食品细菌含量检测装置
US10444124B2 (en) 2011-05-20 2019-10-15 Riken Clarifying reagent for biological materials and use thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002009836A2 (fr) * 2000-08-01 2002-02-07 Surromed, Inc. Procedes de nanoextraction et de desorption liquide-solide
US20060252054A1 (en) * 2001-10-11 2006-11-09 Ping Lin Methods and compositions for detecting non-hematopoietic cells from a blood sample
WO2007067733A2 (fr) * 2005-12-09 2007-06-14 Massachusetts Institute Of Technology Compositions et procédés pour suivre l'absorption d'arn par des cellules
WO2008008515A2 (fr) * 2006-07-14 2008-01-17 Aviva Biosciences Corporation Procédés et compositions servant à détecter des cellules rares dans un échantillon biologique

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2358361B (en) * 2000-01-22 2003-04-23 Cell Analysis Ltd Method and apparatus for the separation of particles
US7341841B2 (en) * 2003-07-12 2008-03-11 Accelr8 Technology Corporation Rapid microbial detection and antimicrobial susceptibility testing

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002009836A2 (fr) * 2000-08-01 2002-02-07 Surromed, Inc. Procedes de nanoextraction et de desorption liquide-solide
US20060252054A1 (en) * 2001-10-11 2006-11-09 Ping Lin Methods and compositions for detecting non-hematopoietic cells from a blood sample
WO2007067733A2 (fr) * 2005-12-09 2007-06-14 Massachusetts Institute Of Technology Compositions et procédés pour suivre l'absorption d'arn par des cellules
WO2008008515A2 (fr) * 2006-07-14 2008-01-17 Aviva Biosciences Corporation Procédés et compositions servant à détecter des cellules rares dans un échantillon biologique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP2310133A1 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013522590A (ja) * 2010-03-12 2013-06-13 独立行政法人理化学研究所 生物材料用透明化試薬、及びその利用
US10444124B2 (en) 2011-05-20 2019-10-15 Riken Clarifying reagent for biological materials and use thereof
US20160266016A1 (en) 2013-08-14 2016-09-15 Riken Composition for preparing biomaterial with excellent light-transmitting property, and use thereof
US10267714B2 (en) 2013-08-14 2019-04-23 Riken Composition for preparing biomaterial with excellent light-transmitting property, and use thereof
US10274492B2 (en) 2015-04-10 2019-04-30 The Curators Of The University Of Missouri High sensitivity impedance sensor
US11422134B2 (en) 2015-04-10 2022-08-23 The Curators Of The University Of Missouri High sensitivity impedance sensor
CN109722378A (zh) * 2018-12-20 2019-05-07 安徽中青检验检测有限公司 一种食品细菌含量检测装置

Also Published As

Publication number Publication date
EP2310133A1 (fr) 2011-04-20
GB0812999D0 (en) 2008-08-20

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