EP1198712A2 - Method for representing biologically activated inductance-altering particles and device for carrying out the method - Google Patents

Method for representing biologically activated inductance-altering particles and device for carrying out the method

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Publication number
EP1198712A2
EP1198712A2 EP00920436A EP00920436A EP1198712A2 EP 1198712 A2 EP1198712 A2 EP 1198712A2 EP 00920436 A EP00920436 A EP 00920436A EP 00920436 A EP00920436 A EP 00920436A EP 1198712 A2 EP1198712 A2 EP 1198712A2
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EP
European Patent Office
Prior art keywords
particles
inductivity
changing
measuring
metal coil
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Withdrawn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
EP00920436A
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German (de)
French (fr)
Inventor
Kilian Hennes
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Individual
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Individual
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Publication date
Priority claimed from DE19906352A external-priority patent/DE19906352A1/en
Application filed by Individual filed Critical Individual
Publication of EP1198712A2 publication Critical patent/EP1198712A2/en
Withdrawn legal-status Critical Current

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    • 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 thereof, e.g. conductivity or capacity
    • 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
    • G01N2015/019—

Definitions

  • the invention relates to a method for the display of biologically activated inductivity-changing — especially ferromagnetic or superparamagnetic
  • the invention also relates to a device for the detection and counting of suspended biological microparticles in liquid samples, in particular for carrying out the method mentioned.
  • Bacteria, blood cells or cell components in aqueous solutions have so far been counted using a flow cytometer or Coulter counter.
  • the corresponding particles are colored and identified using optical signals or paid by capacitive measurements.
  • monovalent primary antibodies are mixed with inductivity-changing, especially ferromagnetic or superparamagnetic, particles in multiple excess, which are coated with secondary antibodies; then, by means of partial sedimentation in a centrifuge, aggregated particles are separated, which consist of a monovalent primary antibody and antibody coated ferromagnetic partial particles exist.
  • inductivity-changing especially ferromagnetic or superparamagnetic, particles in multiple excess, which are coated with secondary antibodies; then, by means of partial sedimentation in a centrifuge, aggregated particles are separated, which consist of a monovalent primary antibody and antibody coated ferromagnetic partial particles exist.
  • viruses or gene probes can also be used, against whose hull proteins or spacer molecules the secondary antibodies are directed.
  • the biological particles for detection or counting can be linked immunologically, phagologically or molecular biologically with aggregated particles which can be measured when subsequently flowing through a metal coil - in particular the gap in a C-shaped metal coil with a ferromagnetic core and trigger affordable changes in inductance.
  • the metal coil as part of an electronic resonant circuit is intended to generate payable changes in the natural oscillation frequency.
  • a different measuring principle is used for the detection of the individual particle: the measurement of the change in inductance of a metal micro coil.
  • biological particles have a permeability constant ⁇ of approximately 1, they have to be marked with inductivity-changing substances beforehand for detection and payment by means of a coil.
  • This labeling takes place through the immunological, phagological or molecular biological coupling of ferromagnetic or superparamagnetic particles which are monovalently linked either to antibodies, to virus docking molecules or to gene probes on spacer molecules.
  • a device of the type mentioned at the outset with a feed line for a sample to be measured, which is surrounded as a measuring line by a metal coil as a measuring coil, which in turn is connected to a device for exciting the vibration and measuring resonance events.
  • this metal coil is placed around an approximately C-shaped core, the ends of which delimit a gap; the measuring line is laid through this gap.
  • the delivery line is connected to a device with capillaries - in particular with Teflon capillaries; the latter are assigned to an electromagnet and can be arranged in a space surrounded by a pole piece.
  • a branch line for excess samples is advantageously provided between the electromagnets and a valve of the delivery line.
  • a resistance and a capacitor can be arranged upstream of that device for exciting the vibration and measuring resonance events towards the metal coil.
  • the measuring coil, a piezo pump arranged upstream of it and a downstream resistor or capacitor are intended to be parts of a microsystem unit.
  • the coupling of the ferromagnetic markers thus takes place in the device, which at the same time enables the particles to be paid to be enriched: the markers are held in that Teflon capillary by means of an electromagnet as a sorption layer until the entire sample has been pumped into the capillary and at the same time the excess sample has run out of the capillary. The magnet is then switched off so that the markers diffuse freely and the surface of the biological particles can sate. Then the capillary content is pumped through the metal coil with the above-mentioned piezoelectric pump, in particular through the gap of the C-shaped metal coil with ferromagnetic core.
  • the metal coil was etched onto a printed circuit board as a spiral and is connected as a resonant circuit with a capacitor and resistor.
  • the oscillating circuit is excited with a frequency that corresponds to the natural oscillation frequency that is generated when an average marked biological microparticle is in the coil or in the gap. This creates a resonance oscillation in the resonant circuit whenever a corresponding microparticle passes through the coil.
  • An example of the application of this method is the detection of coli bacteria in water samples.
  • monovalent primary E. -coli-specific antibodies conjugated to secondary antibodies coupled to megnetic beads The suspension of these conjugates is pumped into the Teflon capillary and fixed there by means of an electromagnet.
  • coli bacteria are retained on the conjugates via the primary antibodies.
  • the suspension of magnetically marked coli bacteria can be pumped through the measuring coil or the gap in the metal coil.
  • the number of resonance events in the connected resonant circuit corresponds to the number of coli bacteria in the original water sample.
  • the detection device uses this measurement method.
  • particles such as bacteria, cells or cell components in aqueous solutions are detected and counted.
  • This technique enables miniaturization of the automatic particle counting process.
  • the particles are marked by reaction with monovalent antibody-coated or virus-coated ferromagnetic particles before the measurement.
  • the inductive measurement is based on passing the ferromagnetic particles aggregated with the biological particles through the microcoil of an electronic resonant circuit designed in the manner described. The resonance events that occur as they pass are counted.
  • the device according to the invention can be used in medicine, microbiology and hygiene, for example for counting blood cells; ecologically relevant microorganisms can be counted or pathogens detected.
  • FIGS. 1, 3 a detail of FIGS. 1, 3 in a schematic oblique view.
  • the reagent with ferromagnetic, biologically activated particles is pumped via lines 12 and 16 into a Teflon capillary 20 and fixed there by means of an electromagnet 22, the magnet coil of which is designated 24 and to which the Z-shaped wound Teflon capillary 20 in a concentric pole shoe 26 is assigned.
  • the free ends 38, 38 a of the measuring coil 36, 36 a are - after a resistor 42 and a capacitor 44 - connected to a device 46 for exciting the vibration and for measuring resonance events; there is a conversion into counts.
  • the number of resonance events in the connected resonant circuit corresponds to the number of coli bacteria in the original water sample Z.
  • a line branch 18 - containing a valve 48 - for excess sample portions Q is provided between the Teflon capillary 20 and the piezo pump 32 .

Abstract

According to the inventive method for representing biologically activated inductance-altering particles, especially ferromagnetic or superparamagnetic particles, monovalent primary antibodies, viruses or DNA probes are mixed with inductance-altering particles in excess, the latter being coated with secondary antibodies. Aggregated particles are then separated by partial sedimentation, said aggregated particles consisting of a monovalent primary antibody and antibody-coated inductance-altering partial particles. A detecting and counting device for suspended biological microparticles in liquid samples has a delivery line (16) for a sample to be measured which is configured as a measuring line (34) and surrounded by a metal coil which is configured as a measuring coil (36a). The measuring coil is connected to a device (46) for exciting oscillation and measuring resonance events. The metal coil (36a) is placed around a core (50) which is bent approximately into a C shape and which has a gap (52) through which the measuring line (34) is guided.

Description

BESCHREIBUNG DESCRIPTION
Verfahren zum Darstellen von biologisch aktivierten induktivitatsandernden Partikeln sowie Vorrichtung dafürMethod for displaying biologically activated inductivity-changing particles and device therefor
Die Erfindung betrifft ein Verfahren für die Darstellung von biologisch aktivierten induktivitatsandernden -- msbe- sondere ferromagnetischen bzw. superparamagnetischenThe invention relates to a method for the display of biologically activated inductivity-changing — especially ferromagnetic or superparamagnetic
Partikeln. Zudem betrifft die Erfindung eine Vorrichtung zum Nachweis und Zahlen von suspendierten biologischen Mikropartikeln in flussigen Proben, insbesondere zum Durchfuhren des genannten Verfahrens.Particles. The invention also relates to a device for the detection and counting of suspended biological microparticles in liquid samples, in particular for carrying out the method mentioned.
Das Zahlen von Bakterien, Blutzellen oder Zellbestandteilen in wassrigen Losungen erfolgt bisher mittels Durch- flusszytometer oder Coultercounter . Hier werden die entsprechenden Partikel gefärbt und anhand von optischen Signalen identifiziert oder durch kapazitive Messungen gezahlt .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 using optical signals or paid by capacitive measurements.
In Kenntnis dieser Gegebenheiten hat sich der Erfinder das Ziel gesetzt, derartige Messungen zu vereinfachen.Knowing these circumstances, the inventor set the goal of simplifying such measurements.
Zur Losung dieser Aufgabe fuhrt die Lehre des unabhängigen Anspruches; die Unteranspruche geben günstige Weiterbildungen an. Zudem fallen in den Rahmen der Erfindung alle Kombinationen aus zumindest zwei der in der Beschreibung, der Zeichnung und/oder den Ansprüchen offenbarten Merkmalen.The teaching of the independent claim leads to the solution of this task; the subclaims indicate favorable further training. In addition, all combinations of at least two of the features disclosed in the description, the drawing and / or the claims fall within the scope of the invention.
Erfmdungsgemaß werden monovalente primäre Antikörper mit induktivitatsandernden, vor allem ferromagnetischen bzw. superparamagnetischen, Partikeln in mehrfachem Uberschuss gemischt, welche mit sekundären Antikörpern beschichtet sind; anschließend werden mittels partieller Sedimentation in einer Zentrifuge aggregierte Partikel abgetrennt, die aus einem monovalenten primären Antikörper und antikorper- beschichteten ferromagnetischen Teilpartikeln bestehen. Anstelle primärer Antikörper können auch Viren oder Gensonden verwendet werden, gegen deren Hullproteine bzw. Spacer- molekule die sekundären Antikörper gerichtet sind.According to the invention, monovalent primary antibodies are mixed with inductivity-changing, especially ferromagnetic or superparamagnetic, particles in multiple excess, which are coated with secondary antibodies; then, by means of partial sedimentation in a centrifuge, aggregated particles are separated, which consist of a monovalent primary antibody and antibody coated ferromagnetic partial particles exist. Instead of primary antibodies, viruses or gene probes can also be used, against whose hull proteins or spacer molecules the secondary antibodies are directed.
Nach einem weiteren Merkmal der Erfindung können die biologischen Partikel zum Nachweis bzw. zum Zahlen immunologisch, phagologisch oder molekularbiologisch mit aggre- gierten Partikeln verbunden werden, die beim anschließenden Durchströmen einer Metallspule — insbesondere des Spaltes einer C-formigen Metallspule mit ferromagnetischem Kern -- messbare und zahlbare Induktivitatsanderungen auslosen.According to a further feature of the invention, the biological particles for detection or counting can be linked immunologically, phagologically or molecular biologically with aggregated particles which can be measured when subsequently flowing through a metal coil - in particular the gap in a C-shaped metal coil with a ferromagnetic core and trigger affordable changes in inductance.
Auch hat es sich als gunstig erwiesen, mduktivitatsan- dernde Partikel vor dem Durchströmen der Metallspule mittels Elektromagnet in einer Kunststoffkapillare festzuhalten und dort mit den in die Kapillare einströmenden biologischen Partikeln zu verbinden, wahrend die Probe, in welcher diese enthalten waren, aus der Kapillare herausge- fuhrt wird. Zudem sollen durch die Metallspule als Teil eines elektronischen Schwingkreises zahlbare Änderungen der Eigenschwingfrequenz erzeugt werden.It has also proven to be advantageous to hold the particles that change the activity by means of an electromagnet in a plastic capillary before flowing through the metal coil and to connect them there with the biological particles flowing into the capillary, while the sample in which they were contained comes out of the capillary - leads. In addition, the metal coil as part of an electronic resonant circuit is intended to generate payable changes in the natural oscillation frequency.
Um den apparativen Aufwand bei der optischen Messung zu um- gehen und eine höhere Spezifitat gegenüber der kapazitiven Messung zu erreichen, wird also für den Nachweis des einzelnen Partikels ein geändertes Messprinzip eingesetzt: Die Messung der Induktivitatsanderung einer Mikrospule aus Metall. Da biologische Partikel aber eine Permeabilitatskon- stante μ von annähernd 1 haben, müssen diese zum Nachweis und zur Zahlung mittels Spule zuvor mit induktivitatsandernden Substanzen markiert werden. Diese Markierung geschieht durch die immunologische, phagologische oder mole- kularbiologische Ankopplung von ferromagnetischen bzw. superparamagnetischen Partikeln, welche monovalent entweder mit Antikörpern, mit Virus-Andockmolekulen oder mit Gensonden an Spacermolekulen verbunden sind. Im Rahmen der Erfindung liegt eine Vorrichtung der eingangs genannten Art mit einer Forderleitung für eine zu messende Probe, die als Messleitung von einer Metallspule als Messspule umgeben ist, welche ihrerseits an eine Einrich- tung zum Anregen der Schwingung und Messen von Resonanzereignissen angeschlossen ist.In order to avoid the outlay in terms of apparatus for the optical measurement and to achieve a higher specificity than the capacitive measurement, a different measuring principle is used for the detection of the individual particle: the measurement of the change in inductance of a metal micro coil. However, since biological particles have a permeability constant μ of approximately 1, they have to be marked with inductivity-changing substances beforehand for detection and payment by means of a coil. This labeling takes place through the immunological, phagological or molecular biological coupling of ferromagnetic or superparamagnetic particles which are monovalently linked either to antibodies, to virus docking molecules or to gene probes on spacer molecules. Within the scope of the invention is a device of the type mentioned at the outset with a feed line for a sample to be measured, which is surrounded as a measuring line by a metal coil as a measuring coil, which in turn is connected to a device for exciting the vibration and measuring resonance events.
In einer besonderen Ausgestaltung ist diese Metallspule um einen etwa C-formig gebogenen Kern gelegt, dessen Enden einen Spalt begrenzen; durch diesen Spalt ist die Messleitung gelegt.In a special embodiment, this metal coil is placed around an approximately C-shaped core, the ends of which delimit a gap; the measuring line is laid through this gap.
Nach einem weiteren Merkmal der Erfindung ist die Forderleitung an eine Einrichtung mit Kapillaren -- insbesondere mit Teflonkapillaren -- angeschlossen; letztere sind einem Elektromagneten zugeordnet und können in einem von einem Polschuh umgebenen Raum angeordnet sein.According to a further feature of the invention, the delivery line is connected to a device with capillaries - in particular with Teflon capillaries; the latter are assigned to an electromagnet and can be arranged in a space surrounded by a pole piece.
Vorteilhafterweise ist zwischen den Elektromagneten und einem Ventil der Förderleitung eine Zweigleitung für überschüssige Proben vorgesehen. Zudem können jener Einrichtung zum Anregen der Schwingung und Messen von Resonanzereignissen zur Metallspule hin wengistens ein Widerstand sowie ein Kondensator vorgeordnet sein.A branch line for excess samples is advantageously provided between the electromagnets and a valve of the delivery line. In addition, a resistance and a capacitor can be arranged upstream of that device for exciting the vibration and measuring resonance events towards the metal coil.
Die Messspule, eine ihr vorgeordnete Piezopumpe und ein nachgeordneter Widerstand bzw. Kondensator sollen erfin- dungsgemaß Teile einer mikrosystemtechnischen Einheit sein.According to the invention, the measuring coil, a piezo pump arranged upstream of it and a downstream resistor or capacitor are intended to be parts of a microsystem unit.
Die Ankopplung der ferromagnetischen Marker geschieht also in der Vorrichtung, welche gleichzeitig eine Anreicherung der zu zahlenden Partikel ermöglicht: Die Marker werden in jener Teflonkapillare mittels eines Elektromagneten 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 sattigen können. Dann wird der Kapillaren-Inhalt mit der erwähnten piezoelektrischen Pumpe durch die Metallspule gepumpt, insbesondere durch den Spalt der C-formig gestalteten Metallspule mit ferromagnetischem Kern. Die Metall- spule wurde als Spirale auf eine Leiterplatte geatzt und ist mit Kondensator und Widerstand als Schwingkreis geschaltet. Der Schwingkreis wird mit einer Frequenz angeregt, die derjenigen Eigenschwingfrequenz entspricht, welche generiert wird, wenn sich ein durchschnittlich mar- kierter biologischer Mikropartikel in der Spule bzw. im Spalt befindet. Dadurch entsteht im Schwingkreis immer dann eine Resonanzschwingung, wenn ein entsprechender Mikropartikel durch die Spule tritt.The coupling of the ferromagnetic markers thus takes place in the device, which at the same time enables the particles to be paid to be enriched: the markers are held in that Teflon capillary by means of an electromagnet as a sorption layer until the entire sample has been pumped into the capillary and at the same time the excess sample has run out of the capillary. The magnet is then switched off so that the markers diffuse freely and the surface of the biological particles can sate. Then the capillary content is pumped through the metal coil with the above-mentioned piezoelectric pump, in particular through the gap of the C-shaped metal coil with ferromagnetic core. The metal coil was etched onto a printed circuit board as a spiral and is connected as a resonant circuit with a capacitor and resistor. The oscillating circuit is excited with a frequency that corresponds to the natural oscillation frequency that is generated when an average marked biological microparticle is in the coil or in the gap. This creates a resonance oscillation in the resonant circuit whenever a corresponding microparticle passes through the coil.
Ein Beispiel für die Anwendung dieses Verfahrens ist der Nachweis von Kolibakterien in Wasserproben. Hierzu werden monovalente primäre E . -coli-spezifische Antikörper mit an megnetische Beads gekoppelten sekundären Antikörpern konjugiert. Die Suspension dieser Konηugate wird in die Teflon- Kapillare gepumpt und mittels Elektromagnet 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 Magneten kann die Suspension von magnetisch markierten Kolibakterien durch die Messspule bzw. den Spalt der Metallspule gepumpt werden. Die Anzahl der Resonanz-Ereignisse im angeschlossenen Schwingkreis entspricht der Anzahl der Kolibakterien in der ursprünglichen Wasserprobe. Durch den Einsatz dieses Gerätes und der entsprechenden Konηugate ist es möglich, ohne den aufwendigen Einsatz der Durchflusszytometrie Bakterien automatisch zu zahlen. Des weiteren ist es möglich, mit dieser Messmethode eine Minia- turisierung des Nachweisgerates zu erreichen. Mit der beschriebenen Technik werden Partikel wie Bakterien, Zellen oder Zellbestandteile in wassrigen Losungen nachgewiesen und gezählt. Diese Technik ermöglicht eine Miniaturisierung des automatischen Partikelzählverfahrens. Dazu werden die Partikel vor der Messung durch die Reaktion mit monovalenten antikόrper- bzw. virenbeschichteten ferromagnetischen Partikeln markiert. Die induktive Messung beruht auf dem Passieren der mit den biologischen Partikeln aggregierten ferromagnetischen Partikel durch die in be- schriebener Weise gestaltete Mikrospule eines elektronischen Schwingkreises. Die beim Passieren auftretenden Resonanzereignisse werden gezählt.An example of the application of this method is the detection of coli bacteria in water samples. For this, monovalent primary E. -coli-specific antibodies conjugated to secondary antibodies coupled to megnetic beads. The suspension of these conjugates is pumped into the Teflon capillary and fixed there by means of an electromagnet. When the water sample to be examined flows through the capillary, coli bacteria are retained on the conjugates via the primary antibodies. After switching off the magnet, the suspension of magnetically marked coli bacteria can be pumped through the measuring coil or the gap in the metal coil. 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 pay for bacteria without the complex use of flow cytometry. It is also possible to miniaturize the detection device using this measurement method. With the technique described, particles such as bacteria, cells or cell components in aqueous solutions are detected and counted. This technique enables miniaturization of the automatic particle counting process. For this purpose, the particles are marked by reaction with monovalent antibody-coated or virus-coated ferromagnetic particles before the measurement. The inductive measurement is based on passing the ferromagnetic particles aggregated with the biological particles through the microcoil of an electronic resonant circuit designed in the manner described. The resonance events that occur as they pass are counted.
Die erfindungsgemäße Vorrichtung kann in der Medizin, Mi- krobiologie und Hygiene eingesetzt werden, beispielsweise zum Auszählen von Blutzellen; es können ökologisch relevante Mikroorganismen ausgezählt oder krankheitserregende Keime nachgewiesen werden. The device according to the invention can be used in medicine, microbiology and hygiene, for example for counting blood cells; ecologically relevant microorganisms can be counted or pathogens detected.
Weitere Vorteile, Merkmale und Einzelheiten der Erfindung ergeben sich aus der nachfolgenden Beschreibung eines bevorzugten Ausfuhrungsbeispieles sowie anhand der Zeichnung; diese zeigt inFurther advantages, features and details of the invention result from the following description of a preferred exemplary embodiment and from the drawing; this shows in
Fig. 1, 3: jeweils ein Schema zu einem er- findungsgemaßen Verfahren;1, 3: each show a scheme for a method according to the invention;
Fig. 2: ein Detail der Fig. 1, 3 in schemati- sierter Schragsicht.2: a detail of FIGS. 1, 3 in a schematic oblique view.
Vor einem Verfahren zum Nachweis von Kolibakterien in einer durch eine Leitung 10 zugefuhrten Wasserprobe Z werden monovalente primäre E . -coli-spezifische Antikörper mit an ma- gnetische Beads gekoppelten sekundären Antikörpern konjugiert. Die Leitung für die monovalenten magnetischen Partikel F ist mit 12 bezeichnet. Beide Leitungen 10, 12 enthalten Schlauchpumpen 14 und vereinigen sich nach diesen zu einer gemeinsamen Forderleitung 16.Before a method for the detection of coli bacteria in a water sample Z fed through a line 10, monovalent primary E. coli-specific antibodies conjugated to secondary antibodies coupled to magnetic beads. The line for the monovalent magnetic particles F is designated by 12. Both lines 10, 12 contain peristaltic pumps 14 and combine to form a common delivery line 16.
Das Reagenz mit ferromagnetischen, biologisch aktivierten Partikeln wird über die Leitungen 12 und 16 in eine Teflonkapillare 20 gepumpt und dort mittels eines Elektromagneten 22 fixiert, dessen Magnetspule mit 24 bezeichnet und dem die Z-formig aufgewickelte Teflonkapillare 20 in einem konzentrischen Polschuh 26 zugeordnet ist. Dieser begrenzt mit einem von ihm in Radialabstand umgebenen Polstift 28 einen Ringraum 30 für die Teflonkapillare.The reagent with ferromagnetic, biologically activated particles is pumped via lines 12 and 16 into a Teflon capillary 20 and fixed there by means of an electromagnet 22, the magnet coil of which is designated 24 and to which the Z-shaped wound Teflon capillary 20 in a concentric pole shoe 26 is assigned. This delimits an annular space 30 for the Teflon capillary with a pole pin 28 surrounded by it at a radial distance.
Beim Durchströmen der Kapillare 20 mit der zu untersuchenden Wasserprobe Z werden Kolibakterien als zu zahlende biologische Partikel über die primären Antikörper an den ferromagnetischen Konjugaten festgehalten. Nach dem Abschalten des Elektromagneten 22 kann die Suspension von magnetisch markierten Kolibakterien dank einer Piezopumpe 32 in einer Messleitung 34 durch eine geatzte Metallspule als Messspule 36 einer mikrosystemtechmschen Einheit 40 transportiert werden. Aus dieser werden die gezählten Partikel in Pfeilrichtung X ausgetragen.When the water sample Z to be examined flows through the capillary 20, coli bacteria as biological particles to be paid are retained on the ferromagnetic conjugates via the primary antibodies. After the electromagnet 22 has been switched off, the suspension of magnetically marked coli bacteria can be transported in a measuring line 34, thanks to a piezo pump 32, through an etched metal coil as a measuring coil 36 of a microsystem-technical unit 40 become. The counted particles are discharged from this in the direction of the arrow X.
Im Ausführungsbeispiel der Fig. 3 wird jene Suspension in der Messleitung 35 durch den Spalt 52 eines ferromagnetischen, C-förmig gebogenen Kerns 50 einer Messspule 36a transportiert .In the exemplary embodiment in FIG. 3, that suspension is transported in the measuring line 35 through the gap 52 of a ferromagnetic, C-shaped core 50 of a measuring coil 36 a .
Die freien Enden 38, 38a der Messspule 36, 36a sind -- nach einem Widerstand 42 und einem Kondensator 44 -- an eine Einrichtung 46 zum Anregen der Schwingung und zum Messen von Resonanzereignissen angeschlossen; dort erfolgt eine Umwandlung in Zählimpulse.The free ends 38, 38 a of the measuring coil 36, 36 a are - after a resistor 42 and a capacitor 44 - connected to a device 46 for exciting the vibration and for measuring resonance events; there is a conversion into counts.
Die Anzahl der Resonanzereignisse im angeschlossenen Schwingkreis entspricht der Anzahl der Kolibakterien in der ursprünglichen Wasserprobe Z.The number of resonance events in the connected resonant circuit corresponds to the number of coli bacteria in the original water sample Z.
Zwischen der Teflonkapillare 20 und der Piezopumpe 32 ist ein -- ein Ventil 48 enthaltender — Leitungsabzweig 18 für überschüssige Probeanteile Q vorgesehen, dem in der Förderleitung 16 ein Ventil 48 nachgeschaltet ist. Provided between the Teflon capillary 20 and the piezo pump 32 is a line branch 18 - containing a valve 48 - for excess sample portions Q, which is followed by a valve 48 in the delivery line 16.

Claims

PATENTANSPRÜCHE PATENT CLAIMS
1. Verfahren für die Darstellung von biologisch aktivier- ten induktivitatsandernden, insbesondere ferromagnetischen bzw. superparamagnetischen, Partikeln,1. Process for the representation of biologically activated inductivity changing, in particular ferromagnetic or superparamagnetic, particles,
dadurch gekennzeichnet,characterized,
dass monovalente primäre Antikörper mit induktivitatsandernden Partikeln im Uberschuss gemischt werden, welche mit sekundären Antikörpern beschichtet sind, und anschließend mittels partieller Sedimentation aggregierte Partikel abgetrennt werden, die aus einem monovalenten primären Antikörper und antikorper-be- schichteten induktivitatsandernden Teilpartikeln bestehen .that monovalent primary antibodies are mixed with inductivity-changing particles in excess, which are coated with secondary antibodies, and then aggregated particles are separated by means of partial sedimentation, which consist of a monovalent primary antibody and antibody-coated inductivity-changing partial particles.
2. Verfahren für die Darstellung von biologisch aktivier- ten induktivitatsandernden, insbesondere ferromagnetischen bzw. superparamagnetischen, Partikeln, dadurch gekennzeichnet, dass Viren mit induktivitatsandernden Partikeln im Uberschuss gemischt werden, welche mit gegen die Hullproteine der Viren gerichteten Anti- korpern beschichtet sind, und anschließend mittels partieller Sedimentation aggregierte Partikel abgetrennt werden, die aus einem Virus und antikorper-be- schichteten induktivitatsandernden Teilpartikeln bestehen .2. A method for the display of biologically activated inductivity-changing, in particular ferromagnetic or superparamagnetic, particles, characterized in that viruses are mixed with inductivity-changing particles in excess, which are coated with antibodies directed against the Hull proteins of the viruses, and then by means of partial sedimentation, aggregated particles are separated, which consist of a virus and antibody-coated inductivity-changing partial particles.
3. Verfahren für die Darstellung von biologisch aktivierten induktivitatsandernden, insbesondere ferromagnetischen bzw. superparamagnetischen, Partikeln, dadurch gekennzeichnet, dass spacermolekul-gekoppelte Oligo- nukleotid-Gensonden mit induktivitatsandernden Partikeln im Uberschuss gemischt werden, welche mit gegen die Spacermolekule gerichteten Antikörpern beschichtet sind, und anschließend mittels partieller Sedimenta- tion aggregierte Partikel abgetrennt werden, die aus einer Gensonde und antikorper-beschichteten induktivitatsandernden Teilpartikeln bestehen.3. A method for the display of biologically activated inductivity-changing, in particular ferromagnetic or superparamagnetic, particles, characterized in that spacer molecule-coupled oligonucleotide gene probes are mixed with inductivity-changing particles in excess, which are coated with antibodies directed against the spacer molecules, and then by means of partial sediment tion aggregated particles are separated, which consist of a gene probe and antibody-coated inductivity-changing partial particles.
4. Verfahren nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, dass biologische Partikel zum Nachweis bzw. zur Zahlung immunologisch, phagologisch oder molekularbiologisch mit den aggregierten Partikeln verbunden werden, die als Marker beim anschließenden Durchströmen einer Metallspule meßbare und zahlbare Induktivitatsanderungen auslosen.4. The method according to any one of claims 1 to 3, characterized in that biological particles for detection or payment are immunologically, phagologically or molecular biologically linked to the aggregated particles, which trigger measurable and payable inductivity changes as markers during the subsequent flow through a metal coil.
5. Verfahren nach Anspruch 4, dadurch gekennzeichnet, dass die Marker beim Durchströmen des Spaltes an einem etwa C-formig gebogenen Kern einer Metallspule messbare und zahlbare Induktivitatsanderungen auslosen.5. The method according to claim 4, characterized in that the markers trigger measurable and payable changes in inductivity when flowing through the gap on an approximately C-shaped core of a metal coil.
6. Verfahren nach Anspruch 4 oder 5, dadurch gekennzeichnet, dass mduktivitatsandernde Partikel vor dem Durchströmen der Metallspule mittels Elektromagnet in einer Kunststoffkapillare festgehalten und dort mit den in die Kapillare einströmenden biologischen Partikeln verbunden werden, wahrend die sie enthaltende Probe aus der Kapillare herausgeführt wird.6. The method according to claim 4 or 5, characterized in that mduktivitatsanderende particles before flowing through the metal coil by means of an electromagnet in a plastic capillary and there are connected to the inflowing into the capillary biological particles, while the sample containing it is led out of the capillary.
7. Verfahren nach einem der Ansprüche 4 bis 6, dadurch gekennzeichnet, dass durch die Metallspule als Teil eines elektronischen Schwingkreises beim Durchströmen der induktivitatsandernden Partikel zahlbare Anderun- gen der Eigenschwingfrequenz erzeugt werden.7. The method according to any one of claims 4 to 6, characterized in that the metal coil as part of an electronic resonant circuit when the inductivity-changing particles flow through produces numerous changes in the natural oscillation frequency.
8. Vorrichtung zum Nachweis und Zahlen für suspendierte biologische Partikel in flussigen Proben, insbesondere Vorrichtung zum Durchfuhren der Verfahren nach wenigstens einem der vorausgehenden Ansprüche, dadurch gekennzeichnet, dass eine Forderleitung (16) für eine zu messende Probe als Messleitung (34) von einer Metallspule als Messspule (36, 36a) umgeben und diese an eine Einrichtung (46) zum Anregen der Schwingung und Messen von Resonanzereignissen angeschlossen ist.8. A device for the detection and numbers of suspended biological particles in liquid samples, in particular a device for performing the method according to at least one of the preceding claims, characterized in that a feed line (16) for a sample to be measured as a measuring line (34) from a metal coil as a measuring coil (36, 36 a ) and surround it a device (46) for exciting the vibration and measuring resonance events is connected.
9. Vorrichtung nach Anspruch 8, dadurch gekennzeichnet, dass die Metallspule (36a) um einen etwa C-formig gebogenen Kern (50) gelegt und dieser einen Spalt (52) aufweist, durch den die Messleitung (34) gefuhrt ist.9. The device according to claim 8, characterized in that the metal coil (36 a ) around an approximately C-shaped bent core (50) and this has a gap (52) through which the measuring line (34) is guided.
10. Vorrichtung nach Anspruch 8 oder 9, dadurch gekenn- zeichnet, dass die Forderleitung (16) an eine Einrichtung mit Kapillaren (20) , insbesondere Teflonkapilla- ren, angeschlossen ist sowie letztere einem Elektromagneten (22) zugeordnet sind.10. The device according to claim 8 or 9, characterized in that the delivery line (16) is connected to a device with capillaries (20), in particular Teflon capillaries, and the latter are assigned to an electromagnet (22).
11. Vorrichtung nach Anspruch 10, dadurch gekennzeichnet, dass die Kapillare/n (20) in einem von einem Polschuh (24) umgebenen Raum (30) angeordnet sind.11. The device according to claim 10, characterized in that the capillary (s) (20) are arranged in a space (30) surrounded by a pole piece (24).
12. Vorrichtung nach einem der Ansprüche 8 bis 11, dadurch gekennzeichnet, dass zwischen den Elektromagneten (22) und einem Ventil (48) der Forderleitung (16) eine Zweigleitung (18) für überschüssige Proben (Q) angeordnet ist.12. Device according to one of claims 8 to 11, characterized in that a branch line (18) for excess samples (Q) is arranged between the electromagnets (22) and a valve (48) of the delivery line (16).
13. Vorrichtung nach einem der Ansprüche 8 bis 12, dadurch gekennzeichnet, dass der Einrichtung (46) zum Anregen der Schwingung und Messen von Resonanzereignissen zur13. Device according to one of claims 8 to 12, characterized in that the device (46) for exciting the vibration and measuring resonance events
Metallspule (36, 36a) hin wenigstens ein WiderstandMetal coil (36, 36 a ) out at least one resistor
(42) sowie ein Kondensator (44) vorgeordnet sind. (42) and a capacitor (44) are arranged upstream.
4. Vorrichtung nach einem der Ansprüche 8 bis 13, dadurch gekennzeichnet, dass die Messspule (36, 36a) mit vorgeordneter Piezopumpe (32) und nachgeordnetem Widerstand (42) bzw. Kondensator (44) Teile einer mikro- systemtechnischen Einheit (40) sind. 4. Device according to one of claims 8 to 13, characterized in that the measuring coil (36, 36 a ) with upstream piezo pump (32) and downstream resistor (42) or capacitor (44) parts of a microsystem unit (40) are.
EP00920436A 1999-02-17 2000-02-15 Method for representing biologically activated inductance-altering particles and device for carrying out the method Withdrawn EP1198712A2 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE19906352 1999-02-17
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
DE19939208 1999-08-18
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

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