EP1597573A1 - Procede et dispositif de detection selective de particules ferromagnetiques ou supraparamagnetiques - Google Patents

Procede et dispositif de detection selective de particules ferromagnetiques ou supraparamagnetiques

Info

Publication number
EP1597573A1
EP1597573A1 EP04706604A EP04706604A EP1597573A1 EP 1597573 A1 EP1597573 A1 EP 1597573A1 EP 04706604 A EP04706604 A EP 04706604A EP 04706604 A EP04706604 A EP 04706604A EP 1597573 A1 EP1597573 A1 EP 1597573A1
Authority
EP
European Patent Office
Prior art keywords
magnetic field
frequency
particles
modulation
oscillator
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Withdrawn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
EP04706604A
Other languages
German (de)
English (en)
Inventor
Peter Miethe
Hans-Joachim Krause
Yi Zhang
Norbert Wolters
Dmitry Plaksin
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.)
Miehte Peter
Forschungszentrum Juelich GmbH
Original Assignee
Miehte Peter
Forschungszentrum Juelich GmbH
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 Miehte Peter, Forschungszentrum Juelich GmbH filed Critical Miehte Peter
Publication of EP1597573A1 publication Critical patent/EP1597573A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/72—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables
    • G01N27/74—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables of fluids
    • G01N27/745—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables of fluids for detecting magnetic beads used in biochemical assays

Definitions

  • the invention relates to the detection of magnetic particles as markers on analytes.
  • Bioassays based on the magnetic labeling of the analyte are an alternative.
  • the magnetic particles consist of an iron oxide core with defined diameters from a few tens to a few hundred nanometers. They have a biocompatible surface coating with which they attach to the analyte, eg. B. to chemical substances, or to the surface of cells or viruses in a manner known per se.
  • Such markers are advantageously stable, non-toxic and can be manipulated by means of magnetic fields.
  • Iron oxide particles are superparamagnetic.
  • the presence of magnetic particles in a sample volume can be determined by alternating field susceptometry.
  • the concentration of the particles can also be determined quantitatively.
  • the detection of magnetic particles by means of susceptometry is known from US Pat. No. 6,110,660.
  • the magnetic susceptibility of an analyte is measured using a Maxwell bridge in the frequency range around 200 kHz.
  • the measured electrical voltage at an output amplifier of the bridge is proportional to the susceptibility of the solution. With constant particle size, the susceptibility is again proportional to the number of magnetic particles in solution.
  • This method is disadvantageously not selective.
  • the magnetic susceptibility of concentrated nanoparticle solutions is high.
  • immunoassay methods generally require the detection of very low concentrations of biomolecules and, accordingly, very low concentrations of magnetic marker particles.
  • the resulting susceptibility of the solution is then very small and can hardly be distinguished from the susceptibility of a comparison solution without magnetic particles.
  • Increasing the gain at the output of the Maxwell bridge is not a viable solution to this problem because parasitic effects such as susceptibility variations in the sample vessels, the reagents and the laboratory environment lead to scattering in the output voltage, as do thermal effects and electronic drift of the components of the readout circuit.
  • No. 6,046,585 describes the movement of magnetic particle samples in order to achieve low-frequency modulation of the measurement signal using a gradiometric SQUID magnetic field detector. Disadvantageously, any signals from the sample holder and the sample vessels are not suppressed with this method.
  • the object of the invention is to provide a method for the selective detection of superpara- and / or ferromagnetic particles which can detect such particles in a highly sensitive manner with little expenditure on equipment.
  • the object of the invention is further to provide a device for carrying out the method in this regard.
  • a frequency component of magnetic fields generated at a mixed frequency due to the nonlinearity of the magnetization characteristic of the particles is measured.
  • Superpara- and ferromagnetic substances have a non-linear magnetization characteristic, a property which is used according to the invention for the selective detection of these substances.
  • the method uses the dependency of the differential susceptibility (that is, the derivative of the magnetic susceptibility after the magnetic field) of the particles from the magnetic field acting on the particles.
  • a first alternating magnetic field is applied to the particles. This results in modulation of the magnetization characteristic of the particles (modulation magnetic field).
  • the modulation magnetic field preferably has a frequency between 50 and 100 Hertz. By selecting such low-frequency alternating magnetic fields, low currents and voltages can be used.
  • the particles are also subjected to a second alternating magnetic field with a frequency different from the actuation magnetic field.
  • the second alternating magnetic field is used to scan the nonlinearity of the magnetization characteristic of the particles (scanning magnetic field).
  • the scanning magnetic field can advantageously have a frequency between 10 and 100 kilohertz. If an induction coil is used as a magnetic field sensor, this advantageously has the effect that the voltage induced in this coil by the response magnetic field, which is proportional to the frequency of the response magnetic field, is high and therefore easier to measure.
  • the occurring amplitude variation of the response magnetic field is primarily dependent on the type and the concentration of the magnetic particles.
  • superpara- and ferromagnetic materials have a non-linear magnetization characteristic. Due to the non-linear magnetization characteristic, the substances mentioned can be selectively detected.
  • the response magnetic field has frequency mixing components of the two alternating magnetic fields applied to the magnetic particles on a sensor.
  • a component can be detected by suitable phase-sensitive detection (demodulation).
  • the voltage generated in this way corresponds to the amplitude profile of the component and is used as an output signal to determine the concentration of the analyte.
  • the time course of the amplitude (amplitude variation) of the response magnetic field is particularly advantageously measured by phase-sensitive detection at the frequency of the scanning magnetic field.
  • the time course of the amplitude of the response magnetic field has frequency components that are multiples of the frequency of the modulation magnetic field. Even multiples are particularly pronounced, and in particular the component with twice the frequency of the modulation magnetic field.
  • the modulation magnetic field leads to a distorted magnetic induction of the magnetic particles.
  • the additional applied In the event that the instantaneous magnetic induction due to the modulation magnetic field just passes through a zero crossing, the magnetic field results in a large additional magnetic induction. In the event that the modulation induction has just a maximum amount, the scanning magnetic field, on the other hand, only leads to a small, additional induction.
  • the amplitude is controlled by the derivation of the magnetization curve of the magnetic particles, which is lower in the case of large magnetic fields due to the saturation effect than in the case of small magnetic fields.
  • this amplitude varies precisely with twice the frequency of the frequency of the modulation magnetic field. It has its minima at the extremes of the modulation and its maxima at the zero crossings.
  • the amplitude variation of the response magnetic field is expediently demodulated by phase-sensitive detection.
  • the response magnetic field is expediently converted into an electrical voltage by a magnetic field sensor and possibly amplified.
  • a device for the selective detection and / or for the quantification of superparamagnetic and / or ferromagnetic particles by measuring the differential susceptibility of the particles comprises a container with the particles to be detected and / or quantified on the analyte.
  • the container can be made of a non-magnetic material suitable for the method, i.e. e.g. B. made of glass or plastic.
  • the device comprises at least one oscillator.
  • the oscillator is suitable for generating frequencies of alternating magnetic fields, as is necessary in the present case.
  • the device particularly advantageously comprises a base frequency oscillator, from which the frequencies of the scanning and modulation magnetic fields are derived in a phase-locked manner by means of frequency dividers.
  • two oscillators can also be used to generate both alternating magnetic fields independently.
  • a first oscillator then generates the frequency of the scanning magnetic field, a second oscillator the frequency of the modulation magnetic field.
  • the device also has at least one field generator. This serves to apply the alternating magnetic fields to the analyte. There may be two such field generators that are particularly advantageous, which generate the analyte with the first or second alternation.
  • Magnetic field that is, the modulation magnetic field and the scanning magnetic field.
  • the device comprises a magnetic field sensor which serves to record a response magnetic field of the particles.
  • the magnetic field sensor converts and amplifies the amplitude variation of the response magnetic field of the particles and passes them on to a phase-sensitive detector.
  • the device further comprises at least one phase-sensitive detector, which is fed by an oscillator, possibly via a frequency divider, with a reference frequency.
  • the phase sensitive detector determines the amplitude of the response magnetic field at this frequency.
  • the device can comprise at least one second phase-sensitive detector, which is also fed by an oscillator, optionally via a frequency divider, with a reference frequency.
  • the first phase-sensitive detector would expediently be supplied with the sampling frequency as a reference and would accordingly supply the amplitude of the response magnetic field at the sampling frequency.
  • the second phase-sensitive detector would be fed as a reference with a multiple of the frequency of the modulation magnetic field.
  • the second phase-sensitive detector examines the amplitude of the response magnetic field, which is passed via the first phase-sensitive detector to the second phase-sensitive detector, for a frequency component that corresponds to an even number times, in particular twice the frequency of the modulation magnetic field.
  • the second phase sensitive detector generates an output voltage that corresponds to the amplitude of this frequency component.
  • the device may further include phase shifters at the reference inputs of the phase sensitive detectors.
  • Figure 1 illustrates the underlying physical property of superparamagnetic and ferromagnetic materials. Such materials have a non-linear magnetization characteristic (Fig. Lb). In contrast, diamagnetic and paramagnetic materials have a linear magnetization characteristic (Fig. La).
  • An alternating field of frequency f and amplitude H a is applied to the substance.
  • Any existing static magnetic field, e.g. B. the earth's magnetic field, is denoted by H 0 .
  • H (t) H a sin (2 ⁇ ß) + H 0 (1)
  • Figure la shows the magnetic modulation induction 3 of a paramagnetic substance on a modulation magnetic field 1.
  • Superparamagnetic substances have the nonlinear and non-hysteretic magnetization characteristic 5 mentioned.
  • the magnetization M (H) of superparamagnetic substances can generally be determined by the formula
  • the typical magnetic response of a superparamagnetic material is represented by the distorted sine function as magnetic modulation induction 6 in FIG. 1b and can generally be described by inserting formula (1) into formula (3) according to formula (5):
  • a modulation magnetic field 4 is used to modulate the magnetization characteristic 5.
  • the modulation magnetic field 4 is an alternating magnetic field with a specific frequency f.
  • a second alternating field (not shown), a so-called scanning magnetic field, is applied to the analyte.
  • the frequency of the scanning magnetic field is selected differently from the frequency of the modulation magnetic field 4.
  • FIG. 2 explains the principle on which the method is based.
  • FIG. 2b characterizes the case of a superparamagnetic or ferromagnetic analyte and FIG. 2a that of a diamagnetic or paramagnetic analyte.
  • the modulation magnetic field 4 leads, as shown in FIG. 1, to a distorted magnetic modulation Induction 6, the time dependence of which is described by formula (5).
  • the additionally applied second alternating magnetic field leads to a large additional scanning induction at the frequency of the scanning magnetic field as a scanning magnetic field in the event that the magnetic modulation induction 6 generated by the modulation magnetic field 4 passes through its zero crossing ( Figure 2b, No. 9).
  • the amplitude of the response magnetic field 11 is namely controlled by the derivation of the magnetization characteristic.
  • this slope of the magnetization characteristic is considerably smaller in the case of large magnetic fields
  • H than in the origin at H 0.
  • this statement is obtained by considering the derivative of the Langevin -Function,
  • the method has the advantage that only analytes with a non-linear magnetization characteristic 5 show a temporal variation of the amplitude 11, as shown in FIG. 2b.
  • Paramagnetic or diamagnetic substances with a linear magnetization characteristic 2 have a constant amplitude 8 of the magnetic induction at the frequency of the scanning magnetic field (FIG. 2a).
  • This selectivity distinguishes the method according to the invention from the methods according to the prior art, which do not differentiate whether the measured susceptibility is due to a di- or paramagnetic or a super- or ferroparamagnetic material.
  • FIG. 3 shows a first device for the selective detection and / or for the quantification of superpara- and / or ferromagnetic particles on analytes.
  • the components are configured so that a due to the
  • Non-linearity of the magnetization characteristic 5 of the particle-generated frequency component of the magnetic fields 15, 18 is measured at a mixed frequency.
  • a suitable container 12 is required, in which an analyte is located in a measuring volume.
  • the analyte can be introduced into the container 12 by methods known per se, such as selective physisorption reaction, chemisorption reaction, precision, filtration or extraction.
  • the analytes are to be demonstrated send superpara- and / or ferromagnetic particles coupled.
  • An oscillator 16 generates the frequency of the modulation magnetic field 18, which is applied to the analyte by means of a field generator 17.
  • An oscillator 13 generates the frequency of a second alternating magnetic field, the scanning magnetic field 15, which is applied to the analyte by means of a further field generator 14.
  • the response magnetic field 19 emanating from the particles or analytes is converted into an electrical voltage and preamplified by a magnetic field sensor 20.
  • the device comprises a first phase-sensitive detector 21, which receives the frequency of the scanning magnetic field 15 from the oscillator 13 as a reference.
  • This phase-sensitive detector 21 determines the time course of the amplitude (see FIG. 2, No. 11 and 8) of the response magnetic field 19 at the frequency of the scanning magnetic field 15.
  • This amplitude 11 and 8 is determined by a second phase-sensitive detector 23, which receives the frequency of the modulation magnetic field 18 via an m-fold frequency multiplier 22 as a reference by oscillator 16, is examined for a frequency component that corresponds to -multiple the frequency of the modulation magnetic field 18, with an integer, positive number m.
  • the generated output voltage 24 corresponds to the Amplitude of this frequency component.
  • a frequency component is measured at a mixed frequency that only occurs when the particle's magnetization characteristic is non-linear.
  • the output voltage 24 is linearly dependent on the superparamagnetic / ferromagnetic moment in the sample volume of container 12.
  • the concentration of the magnetic markers in the sample is proportional to the output voltage 24.
  • the amplitude of the modulation magnetic field 18 is advantageously chosen such that the analyte is in the magnetic saturation is driven.
  • the amplitude of the modulation magnetic field 18 is selected in the order of magnitude of the characteristic field B c of the superparamagnetic particles used. The curvature of the Langevin function on which the magnetization characteristic (equation 3) is based
  • the frequency of the modulation magnetic field 18 is used to modulate the magnetization Characteristic 5 chosen advantageously low, z. B. between 50 Hz and 100 Hz, because coils with a high number of turns and accordingly low currents and voltages can be used to generate fields of such low frequencies.
  • the scanning magnetic field 15 is advantageously chosen high frequency, for. B. between 10 kHz and 100 kHz.
  • a high frequency of the second magnetic field has the advantage that the induced voltage 24 in the measuring coil 20, which is proportional to the frequency, is high.
  • Any static ambient DC field is advantageously chosen to be as small as possible.
  • FIG. 4 outlines an alternative device.
  • a quartz oscillator 25 is used to generate a frequency which is divided down by means of three frequency dividers 26, 27 and 28.
  • the division ratios of the three dividers 26, 27 and 28 are chosen such that they each
  • the field generators 17 and 14 generate the modulation magnetic field 18 and the scanning magnetic field 15.
  • the response magnetic field 19 is via a differential, that is, gradiometric induction coil recorded as a magnetic field sensor 20.
  • the coil is manufactured in such a way that it consists of two identically manufactured, but in each case wound in opposite directions, connected in series, the analyte in the container 12 being immersed in only one of the two coils.
  • the device includes a first phase-sensitive detector 21, which receives the frequency of the scanning magnetic field 15 from the frequency divider 26 as a reference and which shows the course of the amplitude over time (see FIG. 2, No. 11 and No. 8). of the response magnetic field 19 is determined at the frequency of the scanning magnetic field 15.
  • This amplitude 11 or 8 is examined by a second phase-sensitive detector 23 for a frequency component which corresponds to m times the frequency of the modulation magnetic field 18, with an integer, positive number m.
  • this reference frequency is divided down directly from the frequency of the oscillator 25 by a frequency divider 28 instead of having to use a frequency multiplier, as in the example in FIG. 3. In this way, all three frequencies are advantageously coupled in a phase-locked manner, so that any oscillator drift is irrelevant to the measurement.
  • the output voltage 24 is available as a measurement signal.
  • Figure 5 outlines another device.
  • a crystal oscillator 25 was used as the frequency base, which was divided down by means of three frequency dividers 29, 30 and 28.
  • the division ratios of the three dividers 29, 30 and 28 are chosen such that they each
  • the device from FIG. 5 contains only a phase-sensitive detector 21, which receives the frequency of the oscillator 25 divided down n times as a reference. This phase-sensitive detector 21 determines the time profile of the same magnetic field component as in the two previously presented exemplary embodiments from FIGS. 3 and 4.
  • FIG. 6 shows the measured output voltage with standard deviation as a function of the iron concentration of the samples.
  • FIG. 7 shows how the measurement signals of a sample of a fixed concentration vary as a function of a static ambient constant field Ho. Maximum output voltage is obtained with a minimum DC field. At a DC field amplitude of approximately 1.9 mT, a minimum of the output voltage is observed, the signal rises again for higher DC fields. It can be shown mathematically that the signal has a course like the third derivative of the Langevin function
  • FIG. 8 shows the measured dependency of the output voltage of a sample of a fixed concentration as a function of the amplitude of the modulation magnetic field.
  • the signal to be expected was calculated by integration via the amplitude-dependent signal curve and entered in FIG. 8. The agreement with the measured values is particularly good for small amplitudes.
  • the method / device according to the invention can be used in particular for the following applications:
  • Detection of chemical substances, cells or viruses via the quantitative detection of the molecular interaction, in particular the ligand-receptor interaction, the antigen-antibody interaction and the interaction between oligonucleotides, the superpara- and / or ferromagnetic particles being attached to the substances , Cells or viruses are coupled.

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  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analyzing Materials By The Use Of Magnetic Means (AREA)

Abstract

L'invention concerne un procédé de détection et/ou de quantification sélective de particules ferromagnétiques et/ou supraparamagnétiques sur des analytes. Le procédé est caractérisé en ce qu'une composante fréquentielle de champs magnétiques (15, 18), produite en raison de la non linéarité de la caractéristique de magnétisation des particules, est mesurée à une fréquence mixte. L'invention concerne également un dispositif de détection et/ou de quantification sélective de particules ferromagnétiques et/ou supraparamagnétiques sur des analytes, composé d'un contenant (12) de particules à détecter et/ou quantifier sur des analytes ; d'au moins un oscillateur (13, 16 ; 25) destiné à produire des fréquences de champs magnétiques alternatifs (15, 18) ; d'au moins un générateur de champ (14, 17) destiné à appliquer des champs magnétiques alternatifs (15, 18) à l'analyte ; d'un capteur de champ magnétique (20) destiné à mesurer un champ magnétique de réponse (19) des particules ; et d'un détecteur sensible à la phase (21, 23). Lesdits composants sont configurés de telle manière qu'une composante fréquentielle des champs magnétiques (15, 18), produite en raison de la non linéarité de la caractéristique de magnétisation des particules, est mesurée à une fréquence mixte.
EP04706604A 2003-02-28 2004-01-30 Procede et dispositif de detection selective de particules ferromagnetiques ou supraparamagnetiques Withdrawn EP1597573A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10309132A DE10309132A1 (de) 2003-02-28 2003-02-28 Verfahren und Vorrichtung zum selektiven Nachweis magnetischer Partikel
DE10309132 2003-02-28
PCT/DE2004/000149 WO2004077044A1 (fr) 2003-02-28 2004-01-30 Procede et dispositif de detection selective de particules ferromagnetiques ou supraparamagnetiques

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EP1597573A1 true EP1597573A1 (fr) 2005-11-23

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US (1) US8071027B2 (fr)
EP (1) EP1597573A1 (fr)
JP (1) JP2006519366A (fr)
DE (1) DE10309132A1 (fr)
WO (1) WO2004077044A1 (fr)

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US20070155024A1 (en) 2007-07-05
WO2004077044A1 (fr) 2004-09-10
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JP2006519366A (ja) 2006-08-24
DE10309132A1 (de) 2004-11-18
US8071027B2 (en) 2011-12-06

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