EP2726844A1 - Detecting individual analytes by means of magnetic flow measurement - Google Patents

Detecting individual analytes by means of magnetic flow measurement

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Publication number
EP2726844A1
EP2726844A1 EP12742894.4A EP12742894A EP2726844A1 EP 2726844 A1 EP2726844 A1 EP 2726844A1 EP 12742894 A EP12742894 A EP 12742894A EP 2726844 A1 EP2726844 A1 EP 2726844A1
Authority
EP
European Patent Office
Prior art keywords
magnetic
analytes
analyte
component
magnetoresistive
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Withdrawn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
EP12742894.4A
Other languages
German (de)
French (fr)
Inventor
Michael Johannes HELOU
Oliver Hayden
Sandro Francesco Tedde
Mathias Reisbeck
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.)
Siemens AG
Original Assignee
Siemens AG
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 Siemens AG filed Critical Siemens AG
Publication of EP2726844A1 publication Critical patent/EP2726844A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • 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
    • B03C1/00—Magnetic separation
    • B03C1/005—Pretreatment specially adapted for magnetic separation
    • B03C1/01—Pretreatment specially adapted for magnetic separation by addition of magnetic adjuvants
    • 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
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/28—Magnetic plugs and dipsticks
    • B03C1/288—Magnetic plugs and dipsticks disposed at the outer circumference of a recipient
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/02—Measuring direction or magnitude of magnetic fields or magnetic flux
    • G01R33/06—Measuring direction or magnitude of magnetic fields or magnetic flux using galvano-magnetic devices
    • G01R33/09—Magnetoresistive devices
    • 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
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/18—Magnetic separation whereby the particles are suspended in a liquid
    • 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
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/24—Details of magnetic or electrostatic separation for measuring or calculating parameters, efficiency, etc.

Definitions

  • the present invention relates to the magnetomotive ⁇ flow measurement of magnetically labeled analytes, particularly the magnetic flow cytometry.
  • Cell suspension in combination with an enrichment of the magnetically labeled cells increases the distance and the cells can be individually guided over the sensor, but this results in a very disadvantageous unwanted extension of the measuring time.
  • the magnetic labeling of analytes in a sample is generated.
  • a flow of the analytes is generated, which leads the analyte via a sensor arrangement, wherein the flow of the analytes is guided at least part of a magnetoresistive ⁇ part.
  • a magnetic gradient ⁇ field is generated, by means of which the labeled analytes are enriched over the magnetoresistive component, and it is generated a homogeneous magnetic field, which extends to magnetore ⁇ sistiven component, that the homogeneous magnetic field is not detected by the magnetoresistive component.
  • the magnetic marker is in this case made in the inventive process such that the labeled analytes each vor physician a stray magnetic field in the homogeneous magnetic field forth ⁇ whose detectable maxima at a distance from analog lytstoff Vietnamese are smaller than the hydrodynamic Analytradius.
  • the magnetic marker of an analyte in homo ⁇ genes magnetic stray field detected by the magneto resistive element This is caused by the magnetic marker of an analyte in homo ⁇ genes magnetic stray field detected by the magneto resistive element.
  • the x- Component of this stray field measured, the x-direction is defined as the flow direction, ie the direction of the stray field, which is parallel to the surface of the magnetoresistive component.
  • These detectable stray field maxima thus define a distance from the analyte center, which is also referred to below as the magnetic radius.
  • the magnetic labeling of analytes for example Zellana- LYTEN, or beads, these have a magnetic diameter which is less than the optical or hy- rodynamische diameter, which means that the maximum scattering ⁇ field in x-direction within the Analytgates lies. Since ⁇ by and even by detection of the x-component of this stray field, for example with a magnetoresistive construction ⁇ element that is sensitive in the horizontal x-direction, detection of two immediately alsnismfol ⁇ constricting cells than two individual events may take place separately.
  • this has the advantage, even at high Zellkonzentratio ⁇ NEN, in which the analytes are in the smallest possible distance to detect them individually, so to be able to dissolve so-called individual events.
  • suitable magnetic label that is, the stray field of a marking ⁇ th analyte is influenced in particular in a vertical external magnetic field so that a high recovery rate of the magnetic Einzelanalytdetetation is ensured.
  • the sensor arrangement may particularly at least one but also a plurality of magnetoresistive components, ie for example comprise Einzelwi ⁇ resistors.
  • the sensor arrangement has magnetoresistive individual resistances which are approximately in one
  • Wheatstone 'see measuring bridge are interconnected. As is known from Pa ⁇ application DE 10 2010 040 391.1, since ⁇ be generated by particularly advantageous characteristic waveforms.
  • the method is characterized by the magnetic marking with magnetic nanobeads, in particular superparamagnetic nanobeads. accepted.
  • the nanobeads have hydrodynamic diameters between 10 nm and 500 nm.
  • their surface area and / or number of epitopes determines which size and type of marking is particularly advantageous.
  • the small Na ⁇ nobeads between 10 nm and 500 nm diameter have the advantage that occupancy densities on the analyte surface between 10% and 90% can be achieved, which achieve a shift of the stray field maximum in the interior of the analyte.
  • an analyte eg, a cell marked so that the maximum of the x-component of the stray field is located between 50% and 90% of the cell radius from the cell center point ⁇ removed.
  • the magnetic marking is carried out with nano ⁇ beads, which have the material magnetite or Maghemit Ma ⁇ material maghemite.
  • the material content of the nanobeads is chosen in particular such that the saturation magnetization of the magnetic beads is approximately between 10 (Am 2 ) / kg and 60 (Am 2 ) / kg.
  • such a suitable magnetic marking can cause a stray field maximum in the x-direction at a distance of 4 ym from the center of the cell.
  • This is a particularly advantageous reduced magnetic radius, which ensures that the cells thus marked can be detected individually in an external vertical magnetic field, even if they flow in direct contact with each other via the sensor arrangement.
  • the individual labeled analytes are detected by means of the magnetic gradient field above the magnetorec enriched sistiven component, so that they are locally present in ho ⁇ her concentration. Based on sample concentrations between 0.1 and 10 4 analytes per microliter, the concentration is increased to between
  • the method therefore simultaneously has the advantages of a high detection rate of the measuring system, even in the case of two directly successive cells, and the advantage of being able to carry out a measurement on a suspension in which the analytes to be detected are present in very high concentration. If the magnetic marking causes the stray field maximum to be within the cell, it is possible to measure two directly consecutively labeled analytes as two individual events. In particular, in the method, the individual analytes overflow the magnetoresistive component in direct contact with one another.
  • magneto-magnetic enrichment of the magnetically-labeled analytes can additionally be carried out to enrich the magnetically-labeled analytes.
  • An advantageous magnetophore ⁇ tables enrichment is known, for example from the patent applica ⁇ application DE 10 2009 0477 801.9.
  • a system is provided for a targeted transport of magnetically labeled cells in a flowing medium for magneti ⁇ rule flow cytometry.
  • the flow speed is provided a ⁇ in the method that the analytes are performed at constant speed over the magneto resistive element.
  • Insbesonde- re the flow rate is adjusted so that the analytes that are in particular cells, roll over the magnetic ⁇ toresistiven component. They are in particular in contact with the channel wall, on or in which the magneto resistive element is disposed, preferably, is comparable to rotate and roll off on the wall and thus above the magnetic tore ⁇ sistiven component.
  • the magnetoresistive component or, for example, the plurality of magnetoresistive bridge elements are in particular GMR sensors (giant magneto resistance).
  • Figure 1 shows a side view of the magnetic unit 22 for generating the gradient field and the homogeneous Mag ⁇ netfeldes 220, which is located with arrows perpendicular to the magnetic unit 22.
  • the magnetic marking of the analyte 1 causes a magnetic stray field of the analyte 24, the magnetic field line around the analyte 1 is shown around.
  • the analyte 1 is shown in cross section as a circle Darge ⁇ .
  • the arrow 40 pointing from left to right in FIG. 1, indicates the flow direction of the analyte 1.
  • Which may ⁇ genetic unit 22 is, for example, below a flow channel for an analyte, which is playing, a cell sample at ⁇ .
  • the double function of the magnetic unit 22 can be described, for example, as follows:
  • the gradient field generated by the external magnet 22 attracts the superparamagnetically labeled cells 1 to the sensor surface 20.
  • the cells 1 are stochastically distributed.
  • the cells 1 are magnotophore-marked, for example, by means of nickel strips.
  • table over the magnetoresistive sensors 20 out.
  • a substantially homogeneous field 220 is generated, which, as shown in FIG. 1, extends only in the z direction.
  • the sensor 20 does not see such a vertical field 220, because it is sensitive only in the x-direction.
  • FIG. 2 shows a diagram with a distribution function N and measuring points with a square line. It was measured how many analytes 1, which are cells, for example, have a stray field 24 whose maximum in the x direction, which is detected by the sensors, a certain distance ⁇ from the center of the analyte. This distance ⁇ is given in ym.
  • FIG. 3 once again shows the representation of the permanent magnet 22 and of the homogeneous magnetic field 220 generated by the permanent magnet 22.
  • the cell 1 has an optical or hydrodynamic diameter r opt , but also a so-called magnetic diameter r mag , which is in particular smaller than the optical diameter r opt , ie which lies within the cell 1.
  • This smaller through ⁇ knife is that the maximum leakage field component in the x-direction, which is detected by the magnetic sensors 20, is located at a position of the cell which is located within the cell. 1
  • FIG. 1 shows the representation of the permanent magnet 22 and of the homogeneous magnetic field 220 generated by the permanent magnet 22.
  • the cell 1 has an optical or hydrodynamic diameter r opt , but also a so-called magnetic diameter r mag , which is in particular smaller than
  • the channel bottom 11 has at least one magnetic sensor 20 and below the channel bottom 11, the magnetic unit 22 for generating the gradient field and the homogeneous magnetic field 220 is attached.
  • the magnetic sensor 20 includes in particular ⁇ sondere to a length in the flow direction X20 40th
  • the ers ⁇ te maximum measurement rash does not happen in the Mo ⁇ ment, in which the cell 1 with its optical or hydrodyna ⁇ mix diameter r op t reaches the sensor 20, but as indicated by a dashed line, only when the in the cell 1 extending stray magnetic field 24 pushes its maximum of the x-component over the edge of the sensor 20.
  • This point marks the magnetic radius r mag , which in particular is smaller than the optical radius r op t of the cell 1. If the cell 1 has swept over the magnetic sensor 20, a second maximum measuring deflection is registered in the other magnetic field direction.
  • Figure 5 shows how the cavities recorded over a time magnetoresistive signal from a plurality of successive cells on ⁇ 1 behaves.
  • the magnetic diameter r mag coincides with the optical or actual cell diameter r op t of the cell 1
  • a first positive measurement excursion caused by the first one would sensor sponding 20 überstrei ⁇ cell 1
  • a second negative measuring rash, ver ⁇ ursacht by the end of the second cell 1 detects the ⁇ .
  • the measuring deflections which together ⁇ associated with the maximum of the x-component of the stray field 24 of a cell 1 as far separated Ati that each cell 1 causes a complete measurement signal of two measuring rashes, as shown in the lower diagram of FIG.
  • the time interval of the measurement deflections At of a cell signal correlates with the magnetic diameter 2-r mag of a magnetically marked cell 1.
  • the homogeneous magnetic field 220 is also drawn in the z-direction.
  • the distance of the cells 1 to the channel bottom 11 is marked with Z20.
  • the cells 1 sweep the magnetic sensor 20 in the flow direction 40.

Abstract

The invention relates to magnetic flow measurement, in particular flow cytometry. In the method according to the invention, individual analytes are detected in the through-flow. For this purpose, the analytes (1) to be detected, such as cells for example, are marked with magnetic labels directly in the medium surrounding the analytes and transported through the flow channel of a measuring device comprising at least one magnetic sensor (20). Using the magnetic marking of the analytes (1), the magnetic analyte diameter (rmag) is detected rather than the optical or hydrodynamic size (ropt) of the analytes, said analyte diameter being determined by the stray field maximum. The analyte diameter is smaller than the analyte size, whereby the detection of individual analytes is ensured even at high analyte concentrations.

Description

Beschreibung description
Einzelanalyterfassung mittels magnetischer Durchflussmessung Die vorliegende Erfindung betrifft die magnetische Durch¬ flussmessung von magnetisch markierten Analyten, insbesondere die magnetische Durchflusszytometrie . Einzelanalyterfassung using magnetic flow measurement, the present invention relates to the magnetomotive ¬ flow measurement of magnetically labeled analytes, particularly the magnetic flow cytometry.
Im der magnetischen Durchflusszytometrie werden bislang zwei Ansätze zur Einzelzelldetektion verfolgt, bei denen das Prob¬ lem der eindeutigen Trennung von zwei direkt aufeinanderfolgenden Zellen folgendermaßen umgangen wird: In the magnetic flow cytometry two approaches to single-cell detection are far pursued where the prob ¬ lem the clear separation of two directly successive cells is bypassed as follows:
Wie beispielsweise aus Loureiro et al . , "Journal of Applied Physics", 2011, 109, 07B311 bekannt, werden superparamagne- tisch markierte Zellanalyten von einem magnetoresistiven Sensor erfasst. Durch den Verzicht auf eine Anreicherung der markierten Zellen sind diese zwar nicht sehr hoch konzentriert, jedoch führt dies gleichermaßen zu einer sehr geringen Wiederfindungsrate, d.h. dass nur ein geringer Prozentsatz der markierten Zellen überhaupt vom magnetoresistiven Sensor erfasst werden kann. For example, from Loureiro et al. , "Journal of Applied Physics", 2011, 109, 07B311, superparamagnetically labeled cell analytes are detected by a magnetoresistive sensor. By not enriching the labeled cells, these are not very highly concentrated, but this equally results in a very low recovery rate, i. that only a small percentage of the labeled cells can be detected by the magnetoresistive sensor at all.
Alternativ wird im Stand der Technik mit verdünnten Proben gearbeitet. Mit der Verminderung der Konzentration einerAlternatively, the prior art uses dilute samples. With the reduction of the concentration of a
Zellsuspension in Kombination mit einer Anreicherung der magnetisch markierten Zellen vergrößert sich der Abstand und die Zellen können einzeln über den Sensor geführt werden, jedoch ergibt sich daraus sehr nachteilig eine unerwünschte Verlän- gerung der Messzeit. Cell suspension in combination with an enrichment of the magnetically labeled cells increases the distance and the cells can be individually guided over the sensor, but this results in a very disadvantageous unwanted extension of the measuring time.
Es ist Aufgabe der vorliegenden Erfindung eine Einzelzellde¬ tektion mit hoher Wiederfindungsrate und kurzer Messzeit an¬ zugeben . It is an object of the present invention release a Einzelzellde ¬ tektion with high recovery rate and a short measuring time ¬.
Die Aufgabe wird durch ein Verfahren gemäß Patentanspruch 1 gelöst. Vorteilhafte Ausgestaltungen der Erfindung sind Gegenstand der Unteransprüche. Das erfindungsgemäße Verfahren zur magnetischen Durchfluss¬ messung eines Analyten umfasst die folgenden Schritte: The object is achieved by a method according to claim 1. Advantageous embodiments of the invention are the subject of the dependent claims. The inventive method for magnetic flow ¬ measurement of an analyte comprising the steps of:
Zunächst erfolgt die magnetische Markierung von Analyten in einer Probe. Dann wird ein Fluss der Analyte erzeugt, welcher die Analyte über eine Sensoranordnung führt, wobei der Fluss der Analyten dabei zumindest über ein magnetoresistives Bau¬ teil geführt wird. Außerdem wird ein magnetisches Gradienten¬ feld erzeugt, mittels welchem die markierten Analyte über dem magnetoresistiven Bauteil angereichert werden, und es wird ein homogenes Magnetfeldes erzeugt, welches so zum magnetore¬ sistiven Bauteil verläuft, dass das homogene Magnetfeld nicht vom magnetoresistiven Bauteil erfasst wird. Mittels der Sensoranordnung mit dem zumindest einen magnetoresistiven Bau- teil erfolgt die Erfassung einzelner markierter Analyte. Die magnetische Markierung wird in dem erfindungsgemäßen Verfahren dabei derart vorgenommen, dass die markierten Analyte je ein magnetisches Streufeld in dem homogenen Magnetfeld her¬ vorrufen, dessen erfassbare Maxima in einem Abstand vom Ana- lytmittelpunkt liegen, der kleiner ist als der hydrodynamische Analytradius . First, the magnetic labeling of analytes in a sample. Then, a flow of the analytes is generated, which leads the analyte via a sensor arrangement, wherein the flow of the analytes is guided at least part of a magnetoresistive ¬ part. In addition, a magnetic gradient ¬ field is generated, by means of which the labeled analytes are enriched over the magnetoresistive component, and it is generated a homogeneous magnetic field, which extends to magnetore ¬ sistiven component, that the homogeneous magnetic field is not detected by the magnetoresistive component. By means of the sensor arrangement with the at least one magnetoresistive component, the detection of individual labeled analytes takes place. The magnetic marker is in this case made in the inventive process such that the labeled analytes each vorrufen a stray magnetic field in the homogeneous magnetic field forth ¬ whose detectable maxima at a distance from analog lytmittelpunkt are smaller than the hydrodynamic Analytradius.
Zur Erzeugung des magnetischen Gradientenfeldes sowie des ho¬ mogenen Magnetfeldes wird insbesondere nur eine magnetische Einheit benötigt, die eine doppelte Funktion erfüllt. In wei¬ terem Abstand von der magnetischen Einheit erzeugt diese das Gradientenfeld zur Anreicherung der magnetisch markierten Analyte. Nahe an der magnetischen Einheit verlaufen die Mag¬ netfeldlinien jedoch homogen. Die magnetische Einheit ist da- bei so zum Sensor, d.h. dem magnetoresistiven Bauteil angeordnet, dass das homogene Feld in eine Richtung verläuft, in der der Sensor nicht sensitiv ist. Das heißt zum Beispiel, dass das homogene Magnetfeld in z-Richtung verläuft, während der Sensor in x-Richtung, senkrecht dazu, sensitiv ist. To generate the magnetic gradient field and the ho ¬ magnetized magnetic field in particular only one magnetic unit is needed, which fulfills a dual function. In wei ¬ terem distance from the magnetic assembly of the latter generates the gradient field for the enrichment of the magnetically labeled analytes. Close to the magnetic unit running the Mag ¬ netfeldlinien but homogeneous. The magnetic unit is thus arranged relative to the sensor, ie the magnetoresistive component, so that the homogeneous field extends in a direction in which the sensor is not sensitive. This means, for example, that the homogeneous magnetic field runs in the z-direction, while the sensor is sensitive in the x-direction, perpendicular to it.
Das durch die magnetische Markierung eines Analyten im homo¬ genen Magnetfeld hervorgerufene Streufeld wird durch das magnetoresistive Bauteil erfasst. Insbesondere wird die x- Komponente dieses Streufeldes gemessen, wobei die x-Richtung als die Durchflussrichtung definiert ist, d.h. die Richtung des Streufeldes, die parallel zur Oberfläche des magnetore- sistiven Bauteils liegt. Diese erfassbaren Streufeldmaxima legen also einen Abstand vom Analytmittelpunkt fest, welcher im Folgenden auch magnetischer Radius genannt wird. Durch die magnetische Markierung von Analyten, beispielsweise Zellana- lyten, oder auch Beads können diese einen magnetischen Durchmesser aufweisen, der geringer ist als der optische oder hyd- rodynamische Durchmesser, was heißt, dass das maximale Streu¬ feld in x-Richtung innerhalb des Analytumfanges liegt. Da¬ durch und durch die Detektion der x-Komponente eben dieses Streufeldes, beispielsweise mit einem magnetoresistiven Bau¬ element, welches in dieser horizontalen x-Richtung sensitiv ist, kann die Detektion von zwei unmittelbar aufeinanderfol¬ genden Zellen als zwei Einzelevents getrennt erfolgen. This is caused by the magnetic marker of an analyte in homo ¬ genes magnetic stray field detected by the magneto resistive element. In particular, the x- Component of this stray field measured, the x-direction is defined as the flow direction, ie the direction of the stray field, which is parallel to the surface of the magnetoresistive component. These detectable stray field maxima thus define a distance from the analyte center, which is also referred to below as the magnetic radius. The magnetic labeling of analytes, for example Zellana- LYTEN, or beads, these have a magnetic diameter which is less than the optical or hy- rodynamische diameter, which means that the maximum scattering ¬ field in x-direction within the Analytumfanges lies. Since ¬ by and even by detection of the x-component of this stray field, for example with a magnetoresistive construction ¬ element that is sensitive in the horizontal x-direction, detection of two immediately aufeinanderfol ¬ constricting cells than two individual events may take place separately.
Dies hat also den Vorteil, auch bei hohen Zellkonzentratio¬ nen, in denen die Analyten in kleinstmöglichem Abstand vor- liegen, diese einzeln zu detektieren, also auch wirklich sogenannte Einzelevents auflösen zu können. Durch die geeignete magnetische Markierung wird also das Streufeld eines markier¬ ten Analyten insbesondere in einem vertikalen externen Magnetfeld so beeinflusst, dass eine hohe Wiederfindungsrate der magnetischen Einzelanalytdetektion gewährleistet ist. Thus, this has the advantage, even at high Zellkonzentratio ¬ NEN, in which the analytes are in the smallest possible distance to detect them individually, so to be able to dissolve so-called individual events. By suitable magnetic label that is, the stray field of a marking ¬ th analyte is influenced in particular in a vertical external magnetic field so that a high recovery rate of the magnetic Einzelanalytdetektion is ensured.
Die Sensoranordnung kann insbesondere mindestens ein aber auch mehrere magnetoresistive Bauteile, d.h. z.B. Einzelwi¬ derstände aufweisen. Vorzugsweise weist die Sensoranordnung magnetoresistive Einzelwiderstände auf, die etwa in einerThe sensor arrangement may particularly at least one but also a plurality of magnetoresistive components, ie for example comprise Einzelwi ¬ resistors. Preferably, the sensor arrangement has magnetoresistive individual resistances which are approximately in one
Wheatstone ' sehen Messbrücke verschaltet sind. Wie aus der Pa¬ tentanmeldung DE 10 2010 040 391.1 bekannt ist, können da¬ durch besonders vorteilhaft charakteristische Signalverläufe generiert werden. Wheatstone 'see measuring bridge are interconnected. As is known from Pa ¬ application DE 10 2010 040 391.1, since ¬ be generated by particularly advantageous characteristic waveforms.
In einer vorteilhaften Ausgestaltung der Erfindung wird bei dem Verfahren die magnetische Markierung mit magnetischen Na- nobeads, insbesondere superparamagnetischen Nanobeads vorge- nommen. Die Nanobeads weisen insbesondere hydrodynamische Durchmesser zwischen 10 nm und 500 nm auf. Je nach zu markierenden Analyten, beispielsweise je nach Zellsorte, bestimmt deren Oberfläche und/oder Epitopenanzahl , welche Größe und Art der Markierung besonders vorteilhaft ist. Die kleinen Na¬ nobeads zwischen 10 nm und 500 nm Durchmesser haben den Vorteil, dass damit Belegungsdichten auf der Analytoberflache zwischen 10 % und 90 % erreicht werden können, welche eine Verlagerung des Streufeldmaximums in das Innere des Analyten erzielen. Insbesondere wird ein Analyt, z.B. eine Zelle, so markiert, dass das Maximum der x-Komponente des Streufeldes sich zwischen 50 % und 90 % des Zellradius vom Zellmittel¬ punkt entfernt befindet. In einer weiteren vorteilhaften Ausgestaltung der Erfindung wird bei dem Verfahren die magnetische Markierung mit Nano¬ beads vorgenommen, welche das Material Magnetit oder das Ma¬ terial Maghemit aufweisen. Insbesondere weisen die zur Mar¬ kierung verwendeten Nanobeads ein Material auf, dessen Sätti- gungsmagnetisierung in etwa zwischen 80 und 90 emu/g beträgt. In an advantageous embodiment of the invention, the method is characterized by the magnetic marking with magnetic nanobeads, in particular superparamagnetic nanobeads. accepted. In particular, the nanobeads have hydrodynamic diameters between 10 nm and 500 nm. Depending on the analytes to be labeled, for example, depending on the type of cell, their surface area and / or number of epitopes determines which size and type of marking is particularly advantageous. The small Na ¬ nobeads between 10 nm and 500 nm diameter have the advantage that occupancy densities on the analyte surface between 10% and 90% can be achieved, which achieve a shift of the stray field maximum in the interior of the analyte. In particular, an analyte, eg, a cell marked so that the maximum of the x-component of the stray field is located between 50% and 90% of the cell radius from the cell center point ¬ removed. In a further advantageous embodiment of the invention, in the method, the magnetic marking is carried out with nano ¬ beads, which have the material magnetite or Maghemit Ma ¬ material maghemite. In particular, the nano-beads used for Mar ¬ kierung on a material whose saturation magnetization is approximately between 80 and 90 emu / g.
Der Materialanteil an den Nanobeads ist dabei insbesondere so gewählt, dass die Sättigungsmagnetisierung der Magnetbeads in etwa zwischen 10 (A-m2)/kg und 60 (A-m2)/kg beträgt. The material content of the nanobeads is chosen in particular such that the saturation magnetization of the magnetic beads is approximately between 10 (Am 2 ) / kg and 60 (Am 2 ) / kg.
Beispielsweise kann mit einer derart geeigneten magnetischen Markierung bei Zellen mit durchschnittlich 12 ym Durchmesser ein Streufeldmaximum in x-Richtung bei einem Abstand vom Zellmittelpunkt von durchschnittlich 4 ym hervorgerufen wer- den. Dies ist ein besonders vorteilhafter verringerter magnetischer Radius, der gewährleistet, dass die so markierten Zellen in einem externen vertikalen Magnetfeld einzeln er- fasst werden können, auch wenn diese in direktem Kontakt zueinander über die Sensoranordnung fließen. For example, with cells of average diameter 12 ym in diameter, such a suitable magnetic marking can cause a stray field maximum in the x-direction at a distance of 4 ym from the center of the cell. This is a particularly advantageous reduced magnetic radius, which ensures that the cells thus marked can be detected individually in an external vertical magnetic field, even if they flow in direct contact with each other via the sensor arrangement.
In einer besonders vorteilhaften Ausgestaltung der Erfindung werden in dem Verfahren die einzelnen markierten Analyte mittels des magnetischen Gradientenfeldes über dem magnetore- sistiven Bauteil angereichert, so dass sie dort lokal in ho¬ her Konzentration vorliegen. Ausgehend von Probenkonzentrationen zwischen 0,1 und 104 Analyten pro Mikroliter wird die Konzentration durch die Anreicherung auf das zwischen In a particularly advantageous embodiment of the invention, in the method, the individual labeled analytes are detected by means of the magnetic gradient field above the magnetorec enriched sistiven component, so that they are locally present in ho ¬ her concentration. Based on sample concentrations between 0.1 and 10 4 analytes per microliter, the concentration is increased to between
lOOfache bis zu lOOOOfache erhöht. Dies hat den Vorteil einer sehr hohen Widerfindungsrate, da nur ein verschwindend gerin¬ ger Anteil an zu detektierenden Analyten nicht nahe genug am Sensor vorbeigeführt wird, um von diesem erfasst zu werden. Gleichzeitig birgt die hohe Konzentration, bei der auch die einzelnen Analyte in direktem Kontakt miteinander stehen können, nicht den Nachteil, dass diese als Einzelevent gezählt werden, sondern aufgrund des verringerten magnetischen Radius der letztendlich von der magnetoresistiven Sensoranordnung erfasst wird, auch bei direktem Kontakt der Zellen noch ge- trennt werden können. Das Verfahren birgt also gleichzeitig die Vorteile einer hohen Widerfindungsrate des Messsystems, auch bei zwei direkt aufeinander folgenden Zellen und den Vorteil eine Messung an einer Suspension vornehmen zu können, in der die zu detektierenden Analyte in sehr hoher Konzentra- tion vorliegen. Bewirkt die magnetische Markierung, dass das Streufeldmaximum innerhalb der Zelle liegt, wird also eine Messung von zwei direkt aufeinanderfolgenden markierten Analyten als zwei Einzelevents möglich. Insbesondere überfließen also bei dem Verfahren die einzelnen Analyte das magnetore- sistive Bauteil in direktem Kontakt miteinander. 100 times increased up to 100,000 times. This has the advantage of very high recovery rate, since only a very clotting ¬ ger proportion of analyte to be detected is not passed close enough to the sensor to be covered by this. At the same time, the high concentration, at which the individual analytes can also be in direct contact with each other, does not have the disadvantage that they are counted as a single event, but due to the reduced magnetic radius which is ultimately detected by the magnetoresistive sensor arrangement, even if the sensor is in direct contact Cells can still be separated. The method therefore simultaneously has the advantages of a high detection rate of the measuring system, even in the case of two directly successive cells, and the advantage of being able to carry out a measurement on a suspension in which the analytes to be detected are present in very high concentration. If the magnetic marking causes the stray field maximum to be within the cell, it is possible to measure two directly consecutively labeled analytes as two individual events. In particular, in the method, the individual analytes overflow the magnetoresistive component in direct contact with one another.
Zur Anreicherung der magnetisch markierten Analyte kann neben dem magnetischen Gradientenfeld, welches insbesondere durch einen Permanentmagneten hervorgerufen werden kann, noch zu- sätzlich eine magnetophoretische Anreicherung der magnetisch markierten Analyte erfolgen. Eine vorteilhafte magnetophore¬ tische Anreicherung ist beispielsweise aus der Patentanmel¬ dung DE 10 2009 0477 801.9 bekannt. Dabei wird zur magneti¬ schen Durchflusszytometrie ein System für einen gezielten Transport magnetisch markierter Zellen in einem strömenden Medium angegeben. In einer weiteren vorteilhaften Ausgestaltung der Erfindung wird bei dem Verfahren die Durchflussgeschwindigkeit so ein¬ gestellt, dass die Analyte mit konstanter Geschwindigkeit über das magnetoresistive Bauteil geführt werden. Insbesonde- re wird die Durchflussgeschwindigkeit so eingestellt, dass die Analyte, welche insbesondere Zellen sind, über dem magne¬ toresistiven Bauteil abrollen. Dabei werden sie insbesondere bei Kontakt mit der Kanalwand, an oder in der vorzugsweise das magnetoresistive Bauteil angeordnet ist, in Rotation ver- setzt und rollen an der Wand und damit über dem magnetore¬ sistiven Bauteil ab. Bei dem magnetoresistiven Bauteil oder beispielsweise den mehreren magnetoresistiven Brückenelementen handelt es sich insbesondere um GMR-Sensoren (giant magneto resistance) . In addition to the magnetic gradient field, which can be caused, in particular, by a permanent magnet, magneto-magnetic enrichment of the magnetically-labeled analytes can additionally be carried out to enrich the magnetically-labeled analytes. An advantageous magnetophore ¬ tables enrichment is known, for example from the patent applica ¬ application DE 10 2009 0477 801.9. Here, a system is provided for a targeted transport of magnetically labeled cells in a flowing medium for magneti ¬ rule flow cytometry. In a further advantageous embodiment of the invention, the flow speed is provided a ¬ in the method that the analytes are performed at constant speed over the magneto resistive element. Insbesonde- re the flow rate is adjusted so that the analytes that are in particular cells, roll over the magnetic ¬ toresistiven component. They are in particular in contact with the channel wall, on or in which the magneto resistive element is disposed, preferably, is comparable to rotate and roll off on the wall and thus above the magnetic tore ¬ sistiven component. The magnetoresistive component or, for example, the plurality of magnetoresistive bridge elements are in particular GMR sensors (giant magneto resistance).
Ausführungsformen der vorliegenden Erfindung werden in exemplarischer Weise mit Bezug auf die Figuren 1 bis 5 der ange¬ hängten Zeichnung beschrieben. Figur 1 zeigt eine Seitenansicht der magnetischen Einheit 22 zur Erzeugung des Gradientenfeldes sowie des homogenen Mag¬ netfeldes 220, welches mit Pfeilen senkrecht zur magnetischen Einheit 22 eingezeichnet ist. Die magnetische Markierung des Analyten 1 bewirkt ein magnetisches Streufeld des Analyten 24, dessen Magnetfeldlinienverlauf um den Analyten 1 herum gezeigt ist. Der Analyt 1 ist im Querschnitt als Kreis darge¬ stellt. Der Pfeil 40, von links nach rechts in der Figur 1 weisend, zeigt die Flussrichtung des Analyten 1 an. Die mag¬ netische Einheit 22 befindet sich beispielsweise unterhalb eines Durchflusskanals für eine Analytprobe, welche bei¬ spielsweise eine Zellprobe ist. Embodiments of the present invention will be described by way of example with reference to FIGS . 1 to 5 of the attached drawing. Figure 1 shows a side view of the magnetic unit 22 for generating the gradient field and the homogeneous Mag ¬ netfeldes 220, which is located with arrows perpendicular to the magnetic unit 22. The magnetic marking of the analyte 1 causes a magnetic stray field of the analyte 24, the magnetic field line around the analyte 1 is shown around. The analyte 1 is shown in cross section as a circle Darge ¬ . The arrow 40, pointing from left to right in FIG. 1, indicates the flow direction of the analyte 1. Which may ¬ genetic unit 22 is, for example, below a flow channel for an analyte, which is playing, a cell sample at ¬.
Die doppelte Funktion der magnetischen Einheit 22 kann z.B. folgendermaßen beschrieben werden: Das Gradientenfeld, er- zeugt vom externen Magneten 22 zieht die superparamagnetisch markierten Zellen 1 an die Sensoroberfläche 20. Dort liegen die Zellen 1 stochastisch verteilt vor. Im Fluss 40 werden die Zellen 1 z.B. mit Hilfe von Nickelstreifen magnotophore- tisch über die magnetoresistiven Sensoren 20 geführt. Direkt über dem Sensor 20 wird ein im Wesentlichen homogenes Feld 220 generiert, welches, wie in Figur 1 gezeigt, nur in z- Richtung verläuft. So ein vertikales Feld 220 sieht der Sen- sor 20 nicht, denn er ist nur in x-Richtung sensitiv. In Figur 1 sieht man also beispielsweise eine superparamagnetisch markierte Zelle 1, die das Feld 220 in ihrer Umgebung ver¬ zerrt. Die x-Komponente dieses Streufeldes 24 ist das Feld, das mit dem Sensor 20 erfasst wird. In der Vorrichtung wird also die Inhomogenität des Magneten 22 ausgenutzt, der das externe Feld generiert. Dabei handelt es sich z.B. um einen NeFeB Magneten. Je nach Qualität des Magneten 22 variiert der homogenen Bereich 220 nahe am Magneten 22. Genau dieser Bereich wird unter dem Sensor 20 platziert. Das Gradientenfeld, für die Anreicherung benötigt wird ist dann gegeben durch die Inhomogenität des Magnetfeldes außerhalb des homogenen Be¬ reichs 220. The double function of the magnetic unit 22 can be described, for example, as follows: The gradient field generated by the external magnet 22 attracts the superparamagnetically labeled cells 1 to the sensor surface 20. There, the cells 1 are stochastically distributed. In the flow 40, the cells 1 are magnotophore-marked, for example, by means of nickel strips. table over the magnetoresistive sensors 20 out. Directly above the sensor 20, a substantially homogeneous field 220 is generated, which, as shown in FIG. 1, extends only in the z direction. The sensor 20 does not see such a vertical field 220, because it is sensitive only in the x-direction. In Figure 1, so you can see for example, a superparamagnetic-labeled cell 1 ver ¬ drags the field 220 in their environment. The x-component of this stray field 24 is the field detected by the sensor 20. In the device, therefore, the inhomogeneity of the magnet 22 is exploited, which generates the external field. This is, for example, a NeFeB magnet. Depending on the quality of the magnet 22, the homogeneous region 220 varies close to the magnet 22. It is precisely this region that is placed under the sensor 20. The gradient field is needed for the enrichment is then given by the inhomogeneity of the magnetic field outside of the homogeneous Be ¬ Reich 220th
Die Figur 2 zeigt ein Diagramm mit einer Verteilungsfunktion N und quadratisch eingezeichneten Messpunkten. Dabei wurde gemessen, wie viele Analyte 1, welche beispielsweise Zellen sind, ein Streufeld 24 aufweisen, dessen Maximum in x- Richtung, welche von den Sensoren detektiert wird, einen gewissen Abstand Δχ vom Zentrum des Analyten beträgt. Dieser Abstand Δχ ist in ym angegeben. FIG. 2 shows a diagram with a distribution function N and measuring points with a square line. It was measured how many analytes 1, which are cells, for example, have a stray field 24 whose maximum in the x direction, which is detected by the sensors, a certain distance Δχ from the center of the analyte. This distance Δχ is given in ym.
In der Figur 3 ist wiederum die Darstellung des Permanentmagneten 22 und des durch den Permanentmagneten 22 erzeugten homogenen Magnetfeldes 220 dargestellt. Die Zelle 1 weist einen optischen oder hydrodynamischen Durchmesser ropt auf, aber auch einen sogenannten magnetischen Durchmesser rmag, welcher insbesondere kleiner ist als der optische Durchmesser ropt , d.h. der innerhalb der Zelle 1 liegt. Dieser kleinere Durch¬ messer liegt daran, dass die maximale Streufeldkomponente in x-Richtung, die von den Magnetsensoren 20 erfasst wird, an einer Position der Zelle liegt, welche sich innerhalb der Zelle 1 befindet. D.h. auch wenn die magnetischen Marker auf der Oberfläche der Zelle 1 sitzen, ist das durch die magneti- sehe Markierung erzeugte Streufeld 24 nicht nur außerhalb, sondern auch innerhalb der Zelle 1 zu finden und sogar dessen Maximum in x-Richtung. Die Figur 4 zeigt schematisch den Messaufbau, sozusagen einen Ausschnitt aus einer Mikrofluidik mit einem Durchflusskanal. Der Kanalboden 11 weist zumindest einen Magnetsensor 20 auf und unterhalb des Kanalbodens 11 ist die magnetische Einheit 22 zur Erzeugung des Gradientenfeldes sowie des homogenen Magnetfeldes 220 angebracht. Der Magnetsensor 20 weist insbe¬ sondere eine Länge X20 in Durchflussrichtung 40 auf. Der ers¬ te maximale Messausschlag passiert jedoch nicht in dem Mo¬ ment, in dem die Zelle 1 mit ihrem optischen oder hydrodyna¬ mischen Durchmesser ropt den Sensor 20 erreicht, sondern wie durch eine gestrichelte Linie angezeigt, erst wenn das in der Zelle 1 verlaufende magnetische Streufeld 24 sein Maximum der x-Komponente über den Rand des Sensors 20 schiebt. Diese Stelle markiert den magnetischen Radius rmag, welcher insbesondere kleiner ist als der optische Radius ropt der Zelle 1. Hat die Zelle 1 den Magnetsensor 20 überstrichen, wird ein zweiter maximaler Messausschlag in die andere Magnetfeldrichtung registriert. FIG. 3 once again shows the representation of the permanent magnet 22 and of the homogeneous magnetic field 220 generated by the permanent magnet 22. The cell 1 has an optical or hydrodynamic diameter r opt , but also a so-called magnetic diameter r mag , which is in particular smaller than the optical diameter r opt , ie which lies within the cell 1. This smaller through ¬ knife is that the maximum leakage field component in the x-direction, which is detected by the magnetic sensors 20, is located at a position of the cell which is located within the cell. 1 This means that even if the magnetic markers sit on the surface of cell 1, this is due to the magnetic See mark generated stray field 24 not only outside, but also within the cell 1 and even find its maximum in the x direction. FIG. 4 schematically shows the measurement setup, as it were a section of a microfluidic system with a flow channel. The channel bottom 11 has at least one magnetic sensor 20 and below the channel bottom 11, the magnetic unit 22 for generating the gradient field and the homogeneous magnetic field 220 is attached. The magnetic sensor 20 includes in particular ¬ sondere to a length in the flow direction X20 40th However, the ers ¬ te maximum measurement rash does not happen in the Mo ¬ ment, in which the cell 1 with its optical or hydrodyna ¬ mix diameter r op t reaches the sensor 20, but as indicated by a dashed line, only when the in the cell 1 extending stray magnetic field 24 pushes its maximum of the x-component over the edge of the sensor 20. This point marks the magnetic radius r mag , which in particular is smaller than the optical radius r op t of the cell 1. If the cell 1 has swept over the magnetic sensor 20, a second maximum measuring deflection is registered in the other magnetic field direction.
Die Figur 5 schließlich zeigt wie sich das über einen Zeit- räum aufgenommene magnetoresistive Signal von mehreren auf¬ einanderfolgenden Zellen 1 verhält. Für den Fall, dass der magnetische Durchmesser rmag mit dem optischen oder tatsächlichen Zellendurchmesser ropt der Zelle 1 zusammenfällt, würde bei Überstreichen von zwei aneinanderliegenden Zellen 1 wie oben in der Figur 5 gezeigt, ein erster positiver Messausschlag, verursacht durch die erste den Sensor 20 überstrei¬ chende Zelle 1, und ein zweiter negativer Messausschlag, ver¬ ursacht durch das Ende der zweiten Zelle 1, detektiert wer¬ den. Da aber nun der magnetische Durchmesser innerhalb der Zelle 1 liegt, sind die Messausschläge, die mit dem Maximum der x-Komponente des Streufeldes 24 einer Zelle 1 zusammen¬ hängen soweit voneinander getrennt Ati, dass jede Zelle 1 ein vollständiges Messsignal von zwei Messausschlägen hervorruft, wie im unteren Diagramm der Figur 5 gezeigt. Der zeitliche Abstand der Messausschläge At eines Zellsignals korreliert mit dem magnetischen Durchmesser 2-rmag einer magnetisch markierten Zelle 1. In der Figur 5 ist auch wieder das homogene Magnetfeld 220 in z-Richtung eingezeichnet. Der Abstand der Zellen 1 zum Kanalboden 11 ist mit Z20 markiert. Die Zellen 1 überstreichen den Magnetsensor 20 in Durchflussrichtung 40. Figure 5, finally, shows how the cavities recorded over a time magnetoresistive signal from a plurality of successive cells on ¬ 1 behaves. In the event that the magnetic diameter r mag coincides with the optical or actual cell diameter r op t of the cell 1, when sweeping two adjacent cells 1 as shown above in FIG. 5, a first positive measurement excursion caused by the first one would sensor sponding 20 überstrei ¬ cell 1, and a second negative measuring rash, ver ¬ ursacht by the end of the second cell 1 detects the ¬. However, since now the magnetic diameter is within the cell 1, the measuring deflections, which together ¬ associated with the maximum of the x-component of the stray field 24 of a cell 1 as far separated Ati that each cell 1 causes a complete measurement signal of two measuring rashes, as shown in the lower diagram of FIG. The time interval of the measurement deflections At of a cell signal correlates with the magnetic diameter 2-r mag of a magnetically marked cell 1. In FIG. 5, the homogeneous magnetic field 220 is also drawn in the z-direction. The distance of the cells 1 to the channel bottom 11 is marked with Z20. The cells 1 sweep the magnetic sensor 20 in the flow direction 40.

Claims

Patentansprüche claims
Verfahren zur magnetischen Durchflussmessung eines Analy- ten, wobei das Verfahren die folgenden Schritte umfasst:A method of magnetic flow measurement of an analyte, the method comprising the steps of:
- magnetische Markierung von Analyten (1) in einer Probe,magnetic labeling of analytes (1) in a sample,
- Flusserzeugung der Analyten (1) über eine Sensoranordnung, wobei der Fluss (40) der Analyten (1) zumindest über ein magnetoresistives Bauteil (20) geführt wird,Flow generation of the analytes (1) via a sensor arrangement, the flow (40) of the analytes (1) being guided at least via a magnetoresistive component (20),
- Erzeugung eines magnetischen Gradientenfeldes, mittels welchem die markierten Analyte (1) über dem magnetore- sistiven Bauteil (20) angereichert werden, sowie eines homogenen Magnetfeldes (220), wobei das homogene Magnet feld (220) und das magnetoresistive Bauteil (20) so zu¬ einander angeordnet sind, dass das homogene Magnetfeld (220) nicht vom magnetoresistiven Bauteil (20) erfasst wird, - Generating a magnetic gradient field, by means of which the labeled analytes (1) are enriched over the magnetoresistive component (20), and a homogeneous magnetic field (220), wherein the homogeneous magnetic field (220) and the magnetoresistive component (20) are arranged to ¬ each other that the homogeneous magnetic field (220) is not detected by the magnetoresistive component (20),
- Erfassung einzelner markierter Analyte (1),  - Detection of individual labeled analytes (1),
- wobei die magnetische Markierung derart vorgenommen wird, dass die markierten Analyte (1) je ein magneti¬ sches Streufeld aufweisen, dessen durch das magnetore¬ sistive Bauteil (20) erfassbare Maxima in einem Abstand vom Analytmittelpunkt (rmag) liegen, der kleiner ist als der hydrodynamische Analytradius (ropt ) ·- Wherein the magnetic marking is carried out such that the labeled analytes (1) each have a magnetic stray ¬ strafe whose detectable by the magnetore ¬ sistive component (20) maxima are at a distance from the analyte center (r mag ), which is smaller as the hydrodynamic analyte radius (r op t) ·
2. Verfahren nach Anspruch 1, bei dem die magnetische Markierung mit magnetischen Nanobeads, insbesondere superpara- magnetischen Nanobeads, vorgenommen wird. 2. The method of claim 1, wherein the magnetic marking with magnetic nanobeads, in particular superpara- magnetic nanobeads is made.
3. Verfahren nach Anspruch 1 oder 2, bei dem die magneti- sehe Markierung mit Nanobeads vorgenommen wird, deren hydro¬ dynamischer Durchmesser zwischen 10 nm und 500 nm beträgt. 3. The method of claim 1 or 2, wherein the magnetic marker is made with nanobeads whose hydro ¬ dynamic diameter is between 10 nm and 500 nm.
4. Verfahren nach einem der vorstehenden Ansprüche, bei dem die magnetische Markierung mit Nanobeads vorgenommen wird, welche das Material Magnetit oder Maghemit aufweisen. 4. The method according to any one of the preceding claims, wherein the magnetic marking is performed with nanobeads comprising the material magnetite or maghemite.
5. Verfahren nach einem der vorstehenden Ansprüche, bei dem die magnetische Markierung mit Nanobeads vorgenommen wird, welche eine Magnetisierung zwischen 10 (A-m ) /kg und 60 5. Method according to one of the preceding claims, in which the magnetic marking is carried out with nanobeads, which has a magnetization between 10 (Am) / kg and 60
(A-m2) /kg aufweisen. (Am 2 ) / kg.
6. Verfahren nach einem der vorstehenden Ansprüche, bei dem die einzelnen markierten Analyte (1) mittels des magnetischen6. The method according to any one of the preceding claims, wherein the individual labeled analyte (1) by means of the magnetic
Gradientenfeldes über dem magnetoresistiven Bauteil (20) an¬ gereichert werden, so dass sie dort lokal in hoher Konzentra¬ tion vorliegen, welche ausgehend von Probenkonzentrationen von 0,1 bis 104 Analyte pro μΐ zwischen dem lOOfachen bis 10000fachen nach der Anreicherung beträgt. Gradient field over the magnetoresistive component (20) are enriched to ¬ , so that they are locally there in high concentration ¬ tion, which is based on sample concentrations of 0.1 to 10 4 analytes per μΐ between the lOOfachen to 10,000 times after the enrichment.
7. Verfahren nach einem der vorstehenden Ansprüche, bei dem die einzelnen Analyte (1) bei Überfließen des magnetoresisti¬ ven Bauteils (20) in direktem Kontakt miteinander stehen. 7. The method according to any one of the preceding claims, in which the individual analytes (1) with each other at overflow of the magnetoresisti ¬ ven component (20) in direct contact.
8. Verfahren nach einem der vorstehenden Ansprüche, bei dem die Durchflussgeschwindigkeit so eingestellt wird, dass die Analyte (1) mit konstanter Geschwindigkeit über das magneto- resistive Bauteil (20) geführt werden, insbesondere darüber abrollen. 8. The method according to any one of the preceding claims, wherein the flow rate is adjusted so that the analytes (1) at a constant speed over the magnetoresistive member (20) are guided, in particular roll over it.
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