DE19906352A1 - Apparatus to identify and count biological microparticles - Google Patents

Apparatus to identify and count biological microparticles

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Publication number
DE19906352A1
DE19906352A1 DE19906352A DE19906352A DE19906352A1 DE 19906352 A1 DE19906352 A1 DE 19906352A1 DE 19906352 A DE19906352 A DE 19906352A DE 19906352 A DE19906352 A DE 19906352A DE 19906352 A1 DE19906352 A1 DE 19906352A1
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DE
Germany
Prior art keywords
particles
capillary
ferromagnetic
metal coil
antibodies
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.)
Withdrawn
Application number
DE19906352A
Other languages
German (de)
Inventor
Kilian Dr Hennes
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.)
Individual
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Individual
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 Individual filed Critical Individual
Priority to DE19906352A priority Critical patent/DE19906352A1/en
Publication of DE19906352A1 publication Critical patent/DE19906352A1/en
Priority to DE19939208A priority patent/DE19939208C2/en
Priority to DE19946656A priority patent/DE19946656A1/en
Priority to AU41020/00A priority patent/AU4102000A/en
Priority to PCT/EP2000/001214 priority patent/WO2000049407A2/en
Priority to EP00920436A priority patent/EP1198712A2/en
Priority to CA002370745A priority patent/CA2370745A1/en
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/04—Investigating sedimentation of particle suspensions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/0656—Investigating concentration of particle suspensions using electric, e.g. electrostatic methods or magnetic methods
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/019—Biological contaminants; Fouling
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Dispersion Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analyzing Materials By The Use Of Magnetic Means (AREA)

Abstract

Apparatus to identify and count suspended biological microparticles, in a liquid sample, uses a sample where the microparticles are bonded to particles immunologically, pathalogically or microbiologically. The current flow from a metal coil gives them inductive changes which can be measured and they can be counted. The bonding particles, which can be changed by induction, are ferromagnetic and are held by an electromagnet (4) in a plastics capillary (3) before the metal coil current flow. The sample is passed through the capillary, where the biological microparticles bond with the ferromagnetic particles, and the liquid of the sample flows out of the capillary. The metal coil (5) is part of an electronic oscillation circuit. An Independent claim is included for an operation to give biologically activated ferromagnetic particles. Monovalent primary antibodies are mixed with ferromagnetic particles in a multiple surplus, which have been coated with secondary antibodies. Using a centrifuge for partial sedimentation, particles are gathered and separated into particles of a monovalent primary antibody and ferromagnetic particles coated with antibodies. Preferred Features: Viruses can be used instead of the primary antibodies, with the secondary antibody directed against their protein shrouding.

Description

Die Zählung von Bakterien, Blutzellen oder Zellbestandteilen in wäßrigen Lösungen erfolgt bisher mittels Durchflußzytometer oder Coultercounter. Hier werden die entsprechenden Partikel gefärbt und anhand von optischen Signalen identifiziert oder durch kapazitive Messungen gezählt.Bacteria, blood cells or cell components in aqueous solutions have so far been counted using a flow cytometer or Coulter counter. Here the corresponding particles are colored and identified on the basis of optical signals or counted by capacitive measurements.

Um den apparativen Aufwand bei der optischen Messung zu umgehen und eine höhere Spezifität gegenüber der kapazitiven Messung zu erreichen, wird für den Nachweis des einzelnen Partikels ein anderes Meßprinzip eingesetzt: Die Messung der Induktivitätsänderung einer Mikrospule aus Metall. Da biologische Partikel aber eine Permeabilitätskonstante µ von annähernd 1 haben, müssen diese zum Nachweis und zur Zählung mittels Spule zuvor mit induktivitätsändernden Substanzen markiert werden. Diese Markierung geschieht durch die immunologische, phagologische oder molekularbiologische Ankopplung von ferromagnetischen Partikeln, welche monovalent entweder mit Antikörpern, mit Virus-Andockmolekülen oder mit Gensonden an Spacermolekülen verbunden sind.In order to avoid the expenditure on equipment for optical measurement and a higher specificity compared to the capacitive measurement, is used for the detection of the individual particle Another measuring principle used: The measurement of the change in inductance of a metal micro coil. Since biological particles have a permeability constant µ of approximately 1, they have to previously marked with inductivity-changing substances for detection and counting by means of a coil become. This marking happens through the immunological, phagological or molecular biological coupling of ferromagnetic particles which are monovalent with either Antibodies are connected with virus docking molecules or with gene probes on spacer molecules.

Die Kopplung der ferromagnetischen Marker erfolgt in einer Vorrichtung, welche gleichzeitig eine Anreicherung der zu zählenden Partikel ermöglicht: Die Marker werden in einer Teflonkapillare mittels einem Elektromagnet als Sorptions-Schicht festgehalten, bis die gesamte Probe in die Kapillare gepumpt wurde und gleichzeitig die überschüssige Probe aus der Kapillare herausgelaufen ist. Hierauf wird der Magnet ausgeschaltet, damit die Marker frei diffundieren und die Oberfläche der biologischen Partikel sättigen können. Darauf wird der Kapillaren-Inhalt mit einer piezoelektrischen Pumpe durch eine Metallspule gepumpt, die als Spirale auf eine Leiterplatte geätzt wurde und mit Kondensator und Widerstand als Schwingkreis geschaltet ist. Der Schwingkreis wird mit einer Frequenz angeregt die derjenigen Eigenschwingfrequenz entspricht, welche entsteht, wenn sich ein durchschnittlich markierter biologischer Mikropartikel in der Spule befindet. Dadurch entsteht im Schwingkreis immer dann eine Resonanzschwingung, wenn ein entsprechender Mikropartikel durch die Spule tritt.The coupling of the ferromagnetic markers takes place in a device which is also a Enrichment of the particles to be counted enables: The markers are placed in a Teflon capillary an electromagnet as a sorption layer until the entire sample is held in the capillary was pumped and at the same time the excess sample ran out of the capillary. The magnet is then switched off so that the markers diffuse freely and the surface of the can saturate biological particles. The capillary content is then covered with a piezoelectric Pump pumped through a metal coil, which was etched as a spiral on a printed circuit board and with Capacitor and resistor is connected as a resonant circuit. The resonant circuit is with a Frequency excited which corresponds to the natural vibration frequency which arises when a average labeled biological microparticles in the coil. This creates in The resonant circuit always resonates when a corresponding microparticle passes through the coil kicks.

Ein Beispiel für die Anwendung dieses Verfahrens ist der Nachweis von Kolibakterien in Wasserproben (siehe schematische Zeichnung, Vermerk 1). Hierzu werden monovalente primäre E.-coli-spezifische Antikörper mit an magnetische Beads gekoppelten sekundären Antikörpern konjugiert. Die Suspension dieser Konjugate (2) wird in die Teflon-Kapillare (3) gepumpt und mittels Elektromagnet (4) dort fixiert. Beim Durchströmen der Kapillare mit der zu untersuchenden Wasserprobe werden Kolibakterien über die primären Antikörper an den Konjugaten festgehalten. Nach dem Abschalten des Magnetes kann die Suspension von magnetisch markierten Kolibakterien durch die Meßspule (5) gepumpt werden. Die Anzahl der Resonanz-Ereignisse im angeschlossenen Schwingkreises entspricht der Anzahl der Kolibakterien in der ursprünglichen Wasserprobe. Durch den Einsatz dieses Gerätes und der entsprechenden Konjugate ist es möglich, ohne den aufwendigen Einsatz der Durchflußzytometrie Bakterien automatisch zu zählen. Des weiteren ist es möglich, mit dieser Meßmethode eine Miniaturisierung des Nachweisgerätes zu erreichen.An example of the application of this method is the detection of coli bacteria in water samples (see schematic drawing, note 1 ). For this purpose, monovalent primary E. coli-specific antibodies are conjugated with secondary antibodies coupled to magnetic beads. The suspension of these conjugates ( 2 ) is pumped into the Teflon capillary ( 3 ) and fixed there by means of an electromagnet ( 4 ). When the water sample to be examined flows through the capillary, coli bacteria are retained on the conjugates via the primary antibodies. After the magnet has been switched off, the suspension of magnetically marked coli bacteria can be pumped through the measuring coil ( 5 ). The number of resonance events in the connected resonant circuit corresponds to the number of coli bacteria in the original water sample. By using this device and the corresponding conjugates, it is possible to automatically count bacteria without the complex use of flow cytometry. It is also possible to use this measuring method to miniaturize the detection device.

Claims (5)

1. Nachweis- und Zählgerät für suspendierte biologische Mikropartikel in flüssigen Proben, dadurch gekennzeichnet, daß die biologischen Partikel immunologisch, phagologisch oder molekularbiologisch mit Partikeln verbunden werden, welche beim anschließenden Durchströmen einer Metallspule meßbare und zählbare Induktivitätsänderungen bewirken.1. Detection and counting device for suspended biological microparticles in liquid samples, characterized in that the biological particles are connected immunologically, phagologically or molecular biologically with particles which cause measurable and countable changes in inductance when subsequently flowing through a metal coil. 2. Zählgerät nach Patentanspruch 1, dadurch gekennzeichnet, daß induktivitätsändernde, ferromagnetische Partikel vor dem Durchströmen der Metallspule mittels Elektromagnet in einer Kunststoffkapillare festgehalten werden und dort mit den in die Kapillare einströmenden biologischen Mikropartikeln verbunden werden, während die Probe, in welcher diese enthalten waren, aus der Kapillare herausfließt.2. Counter according to claim 1, characterized, that inductivity-changing, ferromagnetic particles before flowing through the metal coil be held in a plastic capillary by means of an electromagnet and there with the in the Capillary inflowing biological microparticles are connected while the sample is in which contained these, flows out of the capillary. 3. Zählgerät nach Patentanspruch 1, dadurch gekennzeichnet, daß die Metallspule als Teil eines elektronischen Schwingkreises beim Durchströmen der induktivitätsändernden Partikel, Änderungen der Eigenschwingfrequenz bewirkt, die gezählt werden.3. Counter according to claim 1, characterized, that the metal coil as part of an electronic resonant circuit when flowing through the inductance-changing particles, changes in the natural oscillation frequency causes that counted become. 4. Verfahren für die Herstellung von biologisch aktivierten ferromagnetischen Partikeln, dadurch gekennzeichnet, daß monovalente primäre Antikörper mit ferromagnetischen Partikeln im mehrfachen Überschuß gemischt werden, welche mit sekundären Antikörpern beschichtet sind, und anschließend mittels partieller Sedimentation in einer Zentrifuge aggregierte Partikel aus einem monovalenten primären Antikörper und antikörper-beschichteten ferromagnetischen Teilpartikeln abgetrennt werden.4. Process for the production of biologically activated ferromagnetic particles, characterized, that monovalent primary antibodies with ferromagnetic particles in multiple excess are mixed, which are coated with secondary antibodies, and then by means of partial sedimentation in a centrifuge aggregated particles from a monovalent primary Antibodies and antibody-coated ferromagnetic partial particles are separated. 5. Verfahren nach Patentanspruch 4, dadurch gekennzeichnet, daß anstelle primärer Antikörper Viren verwendet werden, gegen deren Hüllproteine die sekundären Antikörper gerichtet sind.5. The method according to claim 4, characterized, that viruses are used instead of primary antibodies, against whose coat proteins the secondary antibodies are directed.
DE19906352A 1999-02-17 1999-02-17 Apparatus to identify and count biological microparticles Withdrawn DE19906352A1 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
DE19906352A DE19906352A1 (en) 1999-02-17 1999-02-17 Apparatus to identify and count biological microparticles
DE19939208A DE19939208C2 (en) 1999-02-17 1999-08-18 Process for displaying biologically activated inductivity-changing particles for their detection and counting and device therefor
DE19946656A DE19946656A1 (en) 1999-02-17 1999-09-29 Production of biologically activated magnetic particles, useful for detection and quantification of e.g. bacteria, by attachment of specific antibody
AU41020/00A AU4102000A (en) 1999-02-17 2000-02-15 Method for representing biologically activated inductance-altering particles anddevice for carrying out the method
PCT/EP2000/001214 WO2000049407A2 (en) 1999-02-17 2000-02-15 Method for representing biologically activated inductance-altering particles and device for carrying out the method
EP00920436A EP1198712A2 (en) 1999-02-17 2000-02-15 Method for representing biologically activated inductance-altering particles and device for carrying out the method
CA002370745A CA2370745A1 (en) 1999-02-17 2000-02-15 Method for representing biologically activated inductance-altering particles and device for carrying out the method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
DE19906352A DE19906352A1 (en) 1999-02-17 1999-02-17 Apparatus to identify and count biological microparticles

Publications (1)

Publication Number Publication Date
DE19906352A1 true DE19906352A1 (en) 1999-07-22

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Application Number Title Priority Date Filing Date
DE19906352A Withdrawn DE19906352A1 (en) 1999-02-17 1999-02-17 Apparatus to identify and count biological microparticles
DE19939208A Expired - Fee Related DE19939208C2 (en) 1999-02-17 1999-08-18 Process for displaying biologically activated inductivity-changing particles for their detection and counting and device therefor
DE19946656A Withdrawn DE19946656A1 (en) 1999-02-17 1999-09-29 Production of biologically activated magnetic particles, useful for detection and quantification of e.g. bacteria, by attachment of specific antibody

Family Applications After (2)

Application Number Title Priority Date Filing Date
DE19939208A Expired - Fee Related DE19939208C2 (en) 1999-02-17 1999-08-18 Process for displaying biologically activated inductivity-changing particles for their detection and counting and device therefor
DE19946656A Withdrawn DE19946656A1 (en) 1999-02-17 1999-09-29 Production of biologically activated magnetic particles, useful for detection and quantification of e.g. bacteria, by attachment of specific antibody

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000049407A2 (en) * 1999-02-17 2000-08-24 Kilian Hennes Method for representing biologically activated inductance-altering particles and device for carrying out the method
DE10129815A1 (en) * 2001-06-24 2003-01-09 Profos Ag Process for the purification of bacterial cells and cell components
CN108169480A (en) * 2018-02-07 2018-06-15 上海澜澈生物科技有限公司 A kind of detection method of molecular amounts of biomarker and system, chip

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002029430A1 (en) * 2000-10-04 2002-04-11 Evotec Technologies Gmbh Method and device for examining biological and/or chemical samples by means of giant magneto-impedance (gmi)
DE10111520B4 (en) * 2001-03-09 2004-01-15 Chemagen Biopolymer-Technologie Aktiengesellschaft Process for the purification of biomolecules using magnetic particles
AT501194A1 (en) * 2004-12-30 2006-07-15 Thomas Dr Schlederer METHOD FOR ISOLATING CELLS AND VIRUSES
US20110263044A1 (en) * 2008-07-31 2011-10-27 Eads Deutschland Gmbh Device and method for the automatic detection of biological particles
DE102013109467A1 (en) 2013-08-30 2015-03-05 MRB Forschungszentrum für Magnet - Resonanz - Bayern e.V. Method and device for analyzing a sample volume comprising magnetic particles

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Publication number Priority date Publication date Assignee Title
SU907484A1 (en) * 1980-06-30 1982-02-23 Рижский Краснознаменный Институт Инженеров Гражданской Авиации Им.Ленинского Комсомола Device for checking ferromagnetic particle content in liquid
US4913883A (en) * 1987-07-20 1990-04-03 Hitachi, Ltd. Particle agglutination immunoassay apparatus
DE68919565T2 (en) * 1988-07-20 1995-06-29 Olympus Optical Co Immunoassay method using magnetic marker particles.
US5385707A (en) * 1988-12-28 1995-01-31 Stefan Miltenyi Metal matrices for use in high gradient magnetic separation of biological materials and method for coating the same
WO1996005326A1 (en) * 1994-08-17 1996-02-22 Fox John S Method and apparatus for magnetically detecting proteins and nucleic acids
FR2732116B1 (en) * 1995-03-21 1997-05-09 Bio Merieux METHOD AND DEVICE FOR THE QUALITATIVE AND / OR QUANTITATIVE DETERMINATION OF AN ANALYTE, IN PARTICULAR OF A BACTERIA, IN A SAMPLE, BY MAGNETIC WAY

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000049407A2 (en) * 1999-02-17 2000-08-24 Kilian Hennes Method for representing biologically activated inductance-altering particles and device for carrying out the method
WO2000049407A3 (en) * 1999-02-17 2002-02-07 Kilian Hennes Method for representing biologically activated inductance-altering particles and device for carrying out the method
DE10129815A1 (en) * 2001-06-24 2003-01-09 Profos Ag Process for the purification of bacterial cells and cell components
US7579142B2 (en) 2001-06-24 2009-08-25 Profos Ag Methods for purification of bacterial cells and components
US8932580B2 (en) 2001-06-24 2015-01-13 Hyglos Invest Gmbh Methods for purification of bacterial cells and cell components
CN108169480A (en) * 2018-02-07 2018-06-15 上海澜澈生物科技有限公司 A kind of detection method of molecular amounts of biomarker and system, chip
CN108169480B (en) * 2018-02-07 2024-03-08 上海澜澈生物科技有限公司 Method, system and chip for detecting molecular number of biomarker

Also Published As

Publication number Publication date
DE19939208A1 (en) 2000-09-07
DE19946656A1 (en) 2000-08-24
DE19939208C2 (en) 2001-05-17

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