[go: up one dir, main page]

US20040235197A1 - Process for detecting binding reactions with use of the measurement of the relaxation of the double refraction of magnetic particles - Google Patents

Process for detecting binding reactions with use of the measurement of the relaxation of the double refraction of magnetic particles Download PDF

Info

Publication number
US20040235197A1
US20040235197A1 US10/796,003 US79600304A US2004235197A1 US 20040235197 A1 US20040235197 A1 US 20040235197A1 US 79600304 A US79600304 A US 79600304A US 2004235197 A1 US2004235197 A1 US 2004235197A1
Authority
US
United States
Prior art keywords
relaxation
substance
sample
ferromagnetic
double refraction
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.)
Abandoned
Application number
US10/796,003
Inventor
Roman Kotitz
Julia Lange
Julian Browaeys
Regine Perzynski
Jean-Claude Bacri
Virginie Ponsinet
Thomas Rheinlander
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE19938384A external-priority patent/DE19938384A1/en
Application filed by Individual filed Critical Individual
Priority to US10/796,003 priority Critical patent/US20040235197A1/en
Publication of US20040235197A1 publication Critical patent/US20040235197A1/en
Abandoned legal-status Critical Current

Links

Images

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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/21—Polarisation-affecting properties
    • G01N21/23—Bi-refringence
    • 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
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/806—Electrical property or magnetic property
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/24—Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Definitions

  • the invention relates to a new in-vitro process for detection of analytes or binding reactions, in which ferromagnetic or ferrimagnetic substances are used as labeling in immunoassays or other binding assays, which is characterized in that the relaxation of the double refraction is intended as a measurement variable, as well as the use of suitable ferromagnetic or ferrimagnetic substances in this process.
  • German Patent DE 195 03 664 C2 describes a process for magnetorelaxometric quantitative detection of analytes in liquid and solid phases.
  • this is an assay process, in which first structure-specific substances with freely movable ferrimagnetic or ferromagnetic colloidal particles of suitable magnetic relaxation time and with suitable magnetic moment are labeled, and then these labeled structure-specific substances are used in a liquid or immobilized sample that is to be measured.
  • the sample that is to be measured is then magnetized with a magnetic field that is applied from the outside and, after the external field is turned off, the relaxation of the magnetization of the colloidal particles is measured with the use of suitable magnetic field sensors, whereby the relaxation time that is changed by specific binding and/or the relaxation amplitude that is changed by the extent of the binding is used for analysis. In this way, it is possible, e.g., to determine quantitatively the concentration of an antibody vis a vis collagen.
  • the drawback of the invention consists in that, i.a., the experimental set-up for implementing the process is very expensive.
  • SQUIDs Superconducting Quantum Interference Devices
  • To measure the magnetic field SQUIDs (Superconducting Quantum Interference Devices) are used that are located in a special cryostat and must be cooled in an expensive manner with liquid helium.
  • magnetic noise fields must be suppressed for highly sensitive measurements, and thus at this time, the process can be carried out only in a very expensive magnetically shielded space. Further problems of magnetic measuring techniques are produced by a technically produced dead time, by which data on the decisive initial part of the relaxation signal are not available, as well as a background signal, which must be compensated.
  • ferrofluids show double refraction, if when a magnetizing field is applied, the ferrimagnetic or ferromagnetic colloidal particles of the ferrofluid line up overall in the direction of the field. After the field is turned off, a relaxation of the double refraction then results by thermal reorientation of the magnetic particles.
  • This relaxation signal is also known as a “magnetic transient birefringence.” The time constant of this relaxation process depends on the temperature, the viscosity of the carrier liquid and the hydrodynamic volume of the magnetic particles.
  • the object of this invention is to develop a new in-vitro process for detecting analytes or binding reactions with use of magnetic particles, which overcomes the drawbacks of the known process according to DE 195 03 664 C2, is especially significantly simpler to perform, and makes it possible to detect the analytes or binding reactions with a comparable sensitivity.
  • the new process is based on a special measuring technique, which makes it possible to produce a double refraction according to the orientation of the ferromagnetic or ferrimagnetic substances by application of an external magnetic field in the sample and to determine the relaxation of the double refraction after the external magnetic field is turned off.
  • the drawbacks of the known measuring technique that are described above are overcome with the new process.
  • the process is implemented with a measurement arrangement that first allows an orientation of the ferromagnetic or ferrimagnetic substances of the sample that is to be examined with use of a suitable magnetic field and then makes possible the measurement of the relaxation of the double refraction of these substances.
  • This measurement arrangement generally contains a device for producing polarized light, a device for receiving the sample, a device for magnetizing the sample with magnetic pulses or a magnetic field of variable frequency, as well as a device for analysis of the polarization direction of polarized light.
  • An embodiment of a device that is suitable for implementing the process comprises, e.g., an optical bank, on which a laser, a polarizer, an optical cell with the sample, an analyzer and a detector are arranged.
  • the sample is introduced into a magnetizing coil, which is actuated by a power supply and a pulse generator, and the measurement signal is fed to a computer for evaluation.
  • a device is depicted diagrammatically in FIG. 1.
  • (1) shows an He-Ne-laser, (2) a polarizer, (3) an optical cell with a sample, (4) the magnetizing coils, (5) a ⁇ /4-plate (optional), (6) an analyzer and (7) a photodetector.
  • Such a device is described in, e.g., J. Physique Lett., Vol. 46, 1985, L-1199-L-1205.
  • the relaxation of the double refraction can be measured in an especially sensitive manner using highly sensitive photosensors, such as, e.g., pin-diodes or avalanche diodes. To improve the signal-noise ratio, several measurements are averaged.
  • a measurement of the double refraction in the frequency range can also be performed.
  • a number of measurements are made, in which the sample is exposed to alternating magnetic fields, and the double refraction is detected with distance and phase relative to the magnetizing field.
  • This measurement variable can be transformed into a complex form, whereby data on the relaxation times of the double refraction can be obtained from real and imaginary parts of this complex double refraction.
  • ferrofluids do not show any double refraction.
  • a requirement for the production of the double refraction is the turning of the whole particle through an external magnetic field, in other words, a reaction to the applied magnetic field according to the Brownian mechanism.
  • Particles that react to an external magnetic field according to the Néelian mechanism do not contribute to the double refraction.
  • the process with shorter relaxation time is predominant. Only particles whose Brownian relaxation time is shorter than their Néelian relaxation time therefore contribute to the double refraction.
  • ferromagnetic or ferrimagnetic substances are used in immunoassays or other binding assays, their Brownian relaxation under the measuring conditions in the unbonded state proceeds faster than the Néelian relaxation, and the proportion of unbonded magnetic markers in addition to unbonded magnetic markers that are present simultaneously in the measuring sample can be determined by the change in the predominant relaxation mechanism or by the scaling-up of the particle volume.
  • the structure-specific substances that bind the analytes are first labeled with ferrimagnetic or ferromagnetic colloidal particles. These magnetically labeled structure-specific substances are added to the liquid or immobilized sample that is to be measured, and the sample that is to be measured is magnetized with a magnetic field that is applied from the outside. After the external field is turned off, the relaxation of the double refraction or the double refraction in the frequency range is determined.
  • the measurement of the double refraction that is changed by the binding can also be used for analysis in the frequency range.
  • analytes can be identified in that the structure-specific substances that bind the analytes first
  • (i) are labeled with ferrimagnetic or ferromagnetic substances, whereby these substances are selected so that the Brownian relaxation of at least a portion of these substances has a shorter relaxation time under the measuring conditions than the Néelian relaxation and then
  • the sample that is to be measured is magnetized with a magnetic field of suitable strength that is applied from the outside, and
  • the double refraction of the sample can also be determined in the frequency range.
  • Structure-specific substances are defined as all substances that bind specifically to certain structures. Structure-specific substances are defined as especially antibodies, antibody fragments, biotin, or substances that bind biotin such as avidin or streptavidin, extravidin or neutravidin, agonists that bind specifically to receptors, such as cytokines, lymphokines, endothelins or their antagonists, specific peptides and proteins, receptors, enzymes, enzyme substrates, nucleotides, ribonucleic acids, deoxyribonucleic acids, carbohydrates, lipoproteins, etc.
  • substances are preferred whose binding constant lies in the range of 10 5 -10 15 (mol/l) ⁇ 1 . Especially preferred are substances whose binding constant is in the range of 10 7 -10 15 (mol/l) ⁇ 1 .
  • the structure-specific substances or analytes that are to be identified can be labeled with the ferrimagnetic or ferromagnetic particles with the aid of processes that are familiar in immunochemistry, peptide chemistry, and protein chemistry. Especially advantageous are covalent bonds between the structure-specific substances or the analytes that are to be identified with the substances that form the stabilizing shell of ferrimagnetic or ferromagnetic particles. Examples of especially suitable methods are activation and coupling with the aid of carbodiimides [Jakoby and Wilchek, eds.; Methods Enzymol.
  • all ferromagnetic or ferrimagnetic materials can be used that can be dispersed in a medium that is suitable for detection, whereby the Néelian relaxation time of at least a part of the magnetic labelings under the measurement conditions is longer than the Brownian relaxation time of these magnetic labelings.
  • all ferromagnetic or ferrimagnetic colloidal particles with Brownian relaxation times in aqueous media in the range of 10 ⁇ 8 -10 ⁇ 1 second and correspondingly longer Néelian relaxation times.
  • the viscosity of the dispersing medium used must be matched to the relaxation times of the ferromagnetic and ferrimagnetic particles and the measurement time since the suspension medium basically determines the time constant of Brownian relaxation. In this case, the temperature dependency of the viscosity of the dispersing medium is taken into consideration.
  • Magnetic labelings with narrowly distributed particle sizes and/or magnetic moments or Brownian and Néelian relaxation times is generally advantageous. Separation of magnetic labelings into fractions with a narrow distribution of particle sizes can be achieved by, e.g., chromatographic processes or by using special filtration processes (e.g., glass capillary systems or tangential filtration), by using molecular sieves, or by means of centrifuging. Magnetic labelings with moments that are as uniform as possible can be produced by, e.g., classification in a magnetic gradient field.
  • the ferromagnetic and ferrimagnetic substances can be stabilized with a shell that is made of oligomeric or polymeric carbohydrates, proteins, peptides, nucleotides, surfactants, other monomers, oligomers, or polymers and/or lipids.
  • the particle sizes of the ferromagnetic and ferrimagnetic substances are advantageously between 1 nm and 100 nm.
  • Preferred are colloidal particles with particle sizes of between 1 nm and 400 nm.
  • Especially preferred are particle sizes of between 1 nm and 100 nm.
  • ferromagnetic or ferrimagnetic substances can also be produced with a stabilizing shell that is made of the structure-specific substance or the analyte that is to be identified, by the particles being put after production directly into a solution of the structure-specific substance, optionally in the presence of other adjuvants, such as, e.g., proteins, carbohydrates, as well as natural, synthetic, or partially synthetic surface-active substances, etc., or by being produced directly in the presence of structure-specific substances.
  • a stabilizing shell that is made of the structure-specific substance or the analyte that is to be identified, by the particles being put after production directly into a solution of the structure-specific substance, optionally in the presence of other adjuvants, such as, e.g., proteins, carbohydrates, as well as natural, synthetic, or partially synthetic surface-active substances, etc., or by being produced directly in the presence of structure-specific substances.
  • Suitable magnetic markers and suspensions that contain these particles are described in, for example, WO 92/12735, WO 92/22586, EP 0 186 616 and U.S. Pat. No. 4,101,435.
  • magnetic particles can also be used that are usually used as contrast media for nuclear resonance tomography, such as, e.g., Resovist, Lumirem, Feridex, Combidex, Abdoscan and Clariscan.
  • the compounds can also consist of combinations of several ferromagnetic or ferrimagnetic particles with relaxation times that can be discriminated, since measurement results that can be discriminated individually can be achieved through the use of different magnetic labelings with respectively a very narrow distribution of relaxation times and/or magnetic moments for various structure-specific substances or analytes inside a sample. As a result, direct simultaneous determination of several analytes is made possible.
  • suspension media all liquids in which the compounds can move freely are suitable. Especially suitable are water, aqueous solutions of surface-active adjuvants, such as, e.g., surfactants or oligomeric or polymeric carbohydrates and proteins, as well as mixtures of water with alcohols, such as, e.g., glycerol and polyethylene glycol.
  • the suspension media can additionally contain adjuvants that change the osmotic pressure, such as, e.g., common salt.
  • buffer substances that determine pH such as, e.g., phosphates, can be contained.
  • suspension media with suitable optical properties such as lower absorption and double refraction.
  • the light source can be selected so that as little light as possible is absorbed from the suspension medium, and especially also biological suspension media can be used.
  • the compounds that consist of ferromagnetic or ferrimagnetic colloidal particles with structure-specific substances or analytes that are to be identified can also be present in dried form, optionally in combination with other adjuvants which, e.g., facilitate drying or increase the stability of the dried product (e.g., as lyophilizates) and are converted into the suspension medium only shortly before the measurement.
  • adjuvants e.g., facilitate drying or increase the stability of the dried product (e.g., as lyophilizates) and are converted into the suspension medium only shortly before the measurement.
  • the process according to the invention can be used in, e.g., fertility, histocompatibility, allergology, infectiology, hygiene, genetics, virology, bacteriology, toxicology, pathology, environmental analysis, food chemistry and medical diagnosis.
  • the amount of Fe substance contained in the sample volume of 1 ml of this sample is 10 nmol. It is observed that actually only the volume of about 2 ⁇ 2 ⁇ 10 mm (40 ⁇ l) that is in the laster beam contributes to the signal, and a detection amount of 0.4 nmol of Fe is produced. This is on the order of the detection sensitivity of the process that is described in DE 195 03 664 C2. The new measuring position was not specially optimized to a high detection sensitivity. The particles that are used contain only a very small fraction of particles that are lined up according to Brown and thus contribute to the signal.
  • a sample 2 that consists of 100 ⁇ l of dextran-magnetite, coupled with streptavidin (diluted in 400 ⁇ l of BSA-PBS buffer), was saturated with 5 ⁇ l of free biotin (1:10 diluted, initial concentration of 1 mg/ml). Also, here, 40 ⁇ l of biotin-BSA diluted in BSA-PBS buffer (absolute amount 400 ng of biotin-BSA) was added one time, and the relaxation signal was measured at various times after mixing.
  • Sample 2 also showed a clear relaxation signal.
  • biotin-BSA in the BSA-PBS buffer did not result, however, in a change of the relaxation time (cf. FIG. 6).

Landscapes

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

Abstract

The invention relates to a new process for detecting analytes or binding reactions using measurement of the double refraction, as well as the use of the compounds in analytical chemistry that are necessary for this purpose.

Description

  • The invention relates to a new in-vitro process for detection of analytes or binding reactions, in which ferromagnetic or ferrimagnetic substances are used as labeling in immunoassays or other binding assays, which is characterized in that the relaxation of the double refraction is intended as a measurement variable, as well as the use of suitable ferromagnetic or ferrimagnetic substances in this process. [0001]
  • German Patent DE 195 03 664 C2 describes a process for magnetorelaxometric quantitative detection of analytes in liquid and solid phases. In this case, this is an assay process, in which first structure-specific substances with freely movable ferrimagnetic or ferromagnetic colloidal particles of suitable magnetic relaxation time and with suitable magnetic moment are labeled, and then these labeled structure-specific substances are used in a liquid or immobilized sample that is to be measured. The sample that is to be measured is then magnetized with a magnetic field that is applied from the outside and, after the external field is turned off, the relaxation of the magnetization of the colloidal particles is measured with the use of suitable magnetic field sensors, whereby the relaxation time that is changed by specific binding and/or the relaxation amplitude that is changed by the extent of the binding is used for analysis. In this way, it is possible, e.g., to determine quantitatively the concentration of an antibody vis a vis collagen. [0002]
  • The drawback of the invention consists in that, i.a., the experimental set-up for implementing the process is very expensive. To measure the magnetic field, SQUIDs (Superconducting Quantum Interference Devices) are used that are located in a special cryostat and must be cooled in an expensive manner with liquid helium. In addition, magnetic noise fields must be suppressed for highly sensitive measurements, and thus at this time, the process can be carried out only in a very expensive magnetically shielded space. Further problems of magnetic measuring techniques are produced by a technically produced dead time, by which data on the decisive initial part of the relaxation signal are not available, as well as a background signal, which must be compensated. [0003]
  • It is also known that ferrofluids show double refraction, if when a magnetizing field is applied, the ferrimagnetic or ferromagnetic colloidal particles of the ferrofluid line up overall in the direction of the field. After the field is turned off, a relaxation of the double refraction then results by thermal reorientation of the magnetic particles. This relaxation signal is also known as a “magnetic transient birefringence.” The time constant of this relaxation process depends on the temperature, the viscosity of the carrier liquid and the hydrodynamic volume of the magnetic particles. By measuring the relaxation of the double refraction, the hydrodynamic volume of the magnetic particles can then be determined (see Bacri et al., “Magnetic Transient Birefringence of Ferrofluids: Particle Size Determination,” J. Physique 48 (1987), 1385-1391).[0004]
  • The object of this invention is to develop a new in-vitro process for detecting analytes or binding reactions with use of magnetic particles, which overcomes the drawbacks of the known process according to DE 195 03 664 C2, is especially significantly simpler to perform, and makes it possible to detect the analytes or binding reactions with a comparable sensitivity. [0005]
  • This object is achieved by this invention, which is defined in the claims. [0006]
  • It has been found that the detection of analytes or binding reactions is possible in liquid phases if ferromagnetic or ferrimagnetic substances are used as labeling in immunoassays or other binding assays, and the relaxation of the double refraction is determined as a measurement variable. The new process no longer detects the magnetic field of the sample that dies down by the reorientation of the magnetic moments of the ferromagnetic or ferrimagnetic substances, but rather the relaxation of the double refraction as a measure of the reorientation of the ferromagnetic or ferrimagnetic substances in the sample. It has been found, surprisingly enough, that the sensitivity of the measurement of the relaxation of the double refraction is comparable to the sensitivity of the direct measurement of the relaxation of the magnetization of the sample. [0007]
  • The new process is based on a special measuring technique, which makes it possible to produce a double refraction according to the orientation of the ferromagnetic or ferrimagnetic substances by application of an external magnetic field in the sample and to determine the relaxation of the double refraction after the external magnetic field is turned off. The drawbacks of the known measuring technique that are described above are overcome with the new process. [0008]
  • The process is implemented with a measurement arrangement that first allows an orientation of the ferromagnetic or ferrimagnetic substances of the sample that is to be examined with use of a suitable magnetic field and then makes possible the measurement of the relaxation of the double refraction of these substances. [0009]
  • This measurement arrangement generally contains a device for producing polarized light, a device for receiving the sample, a device for magnetizing the sample with magnetic pulses or a magnetic field of variable frequency, as well as a device for analysis of the polarization direction of polarized light. [0010]
  • An embodiment of a device that is suitable for implementing the process comprises, e.g., an optical bank, on which a laser, a polarizer, an optical cell with the sample, an analyzer and a detector are arranged. The sample is introduced into a magnetizing coil, which is actuated by a power supply and a pulse generator, and the measurement signal is fed to a computer for evaluation. Such a device is depicted diagrammatically in FIG. 1. In this case, (1) shows an He-Ne-laser, (2) a polarizer, (3) an optical cell with a sample, (4) the magnetizing coils, (5) a λ/4-plate (optional), (6) an analyzer and (7) a photodetector. Such a device is described in, e.g., J. Physique Lett., Vol. 46, 1985, L-1199-L-1205. [0011]
  • After the sample is magnetized (strength of the pulses, e.g., 10 mT; period, e.g., 2 ms; pause, e.g. 20 ms) and after the field is turned off, the relaxation of the double refraction can be measured in an especially sensitive manner using highly sensitive photosensors, such as, e.g., pin-diodes or avalanche diodes. To improve the signal-noise ratio, several measurements are averaged. [0012]
  • As an alternative to measuring the relaxation of the double refraction in the time range, a measurement of the double refraction in the frequency range can also be performed. In this case, a number of measurements are made, in which the sample is exposed to alternating magnetic fields, and the double refraction is detected with distance and phase relative to the magnetizing field. This measurement variable can be transformed into a complex form, whereby data on the relaxation times of the double refraction can be obtained from real and imaginary parts of this complex double refraction. [0013]
  • As was already described in German Patent DE 195 03 664 C2, after an external magnetizing field is turned off, the magnetization of freely moveable ferromagnetic or ferrimagnetic colloidal particles relaxes within the measuring time by two different mechanisms: [0014]
  • (i) Turning of the whole colloidal particle inside the surrounding liquid, whereby the time constant depends on the hydrodynamic diameter of the particles, the viscosity of the carrier liquid, and temperature, which mainly reflects parameters of the environs of the particles; this mechanism is also referred to below as Brownian relaxation [0015]
  • and [0016]
  • ii) Turning of the internal magnetizing vector inside the magnetic core of the colloidal particles, whereby the time constant depends in a very sensitive manner on material and shape (the anisotropy constants of the particle material used), volume and the temperature of the magnetic core of the particles that are used. These are basically intrinsic parameters of the particles; this mechanism is also referred to below as Néelian relaxation. [0017]
  • These two mechanisms also determine the change in magnetization of a system of freely moveable ferromagnetic or ferrimagnetic colloidal particles when an external magnetic field is activated. [0018]
  • In the absence of an external magnetic field, ferrofluids do not show any double refraction. A requirement for the production of the double refraction is the turning of the whole particle through an external magnetic field, in other words, a reaction to the applied magnetic field according to the Brownian mechanism. Particles that react to an external magnetic field according to the Néelian mechanism do not contribute to the double refraction. The process with shorter relaxation time is predominant. Only particles whose Brownian relaxation time is shorter than their Néelian relaxation time therefore contribute to the double refraction. [0019]
  • If ferromagnetic or ferrimagnetic substances are used in immunoassays or other binding assays, their Brownian relaxation under the measuring conditions in the unbonded state proceeds faster than the Néelian relaxation, and the proportion of unbonded magnetic markers in addition to unbonded magnetic markers that are present simultaneously in the measuring sample can be determined by the change in the predominant relaxation mechanism or by the scaling-up of the particle volume. [0020]
  • As in the process that is described in DE 195 03 664 C2, the structure-specific substances that bind the analytes are first labeled with ferrimagnetic or ferromagnetic colloidal particles. These magnetically labeled structure-specific substances are added to the liquid or immobilized sample that is to be measured, and the sample that is to be measured is magnetized with a magnetic field that is applied from the outside. After the external field is turned off, the relaxation of the double refraction or the double refraction in the frequency range is determined. [0021]
  • The process is preferably implemented so that [0022]
  • (i) the structure-specific substances that bind the analytes are labeled first with ferrimagnetic or ferromagnetic substances and then [0023]
  • (ii) these labeled structute-specific substances are used in a sample that is to be measured, [0024]
  • (iii) the sample that is to be measured is magnetized with a magnetic field that is applied from the outside, and [0025]
  • (iv) after the external field is turned off, the relaxation of the double refraction of the magnetic marker is measured. [0026]
  • The process can also be implemented so that [0027]
  • (i) analytes first are labeled with ferrimagnetic or ferromagnetic substances, and then [0028]
  • (ii) these magnetically labeled analytes are used in a sample that is to be measured, to which substances were added that specifically bind the analytes, and [0029]
  • (iii) the sample that is to be measured is magnetized with a magnetic field that is applied from the outside, and [0030]
  • (iv) after the external field is turned off, the relaxation of the double refraction of the magnetic marker is measured. [0031]
  • The evaluation of the measuring results is carried out here, as also in the competitive assay processes that are described below, in a way that is known to one skilled in the art, i.e., analogously to the processes as they are used in immunoassays or radioassays. [0032]
  • In both previously mentioned cases, the measurement of the double refraction that is changed by the binding can also be used for analysis in the frequency range. [0033]
  • The discrimination between bonded and unbonded markers that are possible to date only in exceptional cases is made possible by the use of their different relaxation mechanisms or the influence of the relaxation time of the magnetic marker that is caused by the binding. [0034]
  • In a liquid phase, analytes can be identified in that the structure-specific substances that bind the analytes first [0035]
  • (i) are labeled with ferrimagnetic or ferromagnetic substances, whereby these substances are selected so that the Brownian relaxation of at least a portion of these substances has a shorter relaxation time under the measuring conditions than the Néelian relaxation and then [0036]
  • (ii) these magnetically labeled substances are used in a sample that is to be measured, and [0037]
  • (iii) the sample that is to be measured is magnetized with a magnetic field of suitable strength that is applied from the outside, and [0038]
  • (iv) after the external field is turned off, the relaxation of the double refraction is measured, whereby the different relaxation behavior of the magnetic markers that are bonded to the analytes compared to the unbonded magnetic markers is used for analysis. [0039]
  • As a measurement variable, the double refraction of the sample can also be determined in the frequency range. [0040]
  • Also in this case, it is possible to combine the analytes that are to be identified, instead of structure-specific substances, with the magnetic labelings. [0041]
  • Structure-specific substances are defined as all substances that bind specifically to certain structures. Structure-specific substances are defined as especially antibodies, antibody fragments, biotin, or substances that bind biotin such as avidin or streptavidin, extravidin or neutravidin, agonists that bind specifically to receptors, such as cytokines, lymphokines, endothelins or their antagonists, specific peptides and proteins, receptors, enzymes, enzyme substrates, nucleotides, ribonucleic acids, deoxyribonucleic acids, carbohydrates, lipoproteins, etc. As structure-specific substances, substances are preferred whose binding constant lies in the range of 10[0042] 5-1015 (mol/l)−1. Especially preferred are substances whose binding constant is in the range of 107-1015 (mol/l)−1.
  • The structure-specific substances or analytes that are to be identified can be labeled with the ferrimagnetic or ferromagnetic particles with the aid of processes that are familiar in immunochemistry, peptide chemistry, and protein chemistry. Especially advantageous are covalent bonds between the structure-specific substances or the analytes that are to be identified with the substances that form the stabilizing shell of ferrimagnetic or ferromagnetic particles. Examples of especially suitable methods are activation and coupling with the aid of carbodiimides [Jakoby and Wilchek, eds.; Methods Enzymol. (1974) [0043] 34], the formation of Schiff bases after periodates are exposed to compounds that contain carbohydrates (Wichek and Bayer, eds., Methods Enzym 184:177), which are then optionally reduced for further stabilization, coupling with the aid of glutaric dialdehyde [Heitzmann and Richards, Proc. Natl. Acad. Sci. USA 71 (1974) 3537], cross-linking of bromoacetylated particles with thiolylated substances [Angerer et al.; Cell 9 (1976) 81], as well as reductive alkylation (Bayer et al.: J. Histochem. Cytochem. 24 (1976) 933].
  • As substances for magnetic labeling, all ferromagnetic or ferrimagnetic materials can be used that can be dispersed in a medium that is suitable for detection, whereby the Néelian relaxation time of at least a part of the magnetic labelings under the measurement conditions is longer than the Brownian relaxation time of these magnetic labelings. Especially suitable are all ferromagnetic or ferrimagnetic colloidal particles with Brownian relaxation times in aqueous media in the range of 10[0044] −8-10−1 second and correspondingly longer Néelian relaxation times. To carry out the measurements, the viscosity of the dispersing medium used must be matched to the relaxation times of the ferromagnetic and ferrimagnetic particles and the measurement time since the suspension medium basically determines the time constant of Brownian relaxation. In this case, the temperature dependency of the viscosity of the dispersing medium is taken into consideration.
  • Preferred are especially ferromagnetic or ferrimagnetic colloidal particles that are made of iron, iron oxides, barium ferrites, strontium ferrites, cobalt, nickel, nickel ferrites, cobalt ferrites, and chromium dioxide, whose Néelian relaxation time is longer than the Brownian relaxation time. [0045]
  • The use of magnetic labelings with narrowly distributed particle sizes and/or magnetic moments or Brownian and Néelian relaxation times is generally advantageous. Separation of magnetic labelings into fractions with a narrow distribution of particle sizes can be achieved by, e.g., chromatographic processes or by using special filtration processes (e.g., glass capillary systems or tangential filtration), by using molecular sieves, or by means of centrifuging. Magnetic labelings with moments that are as uniform as possible can be produced by, e.g., classification in a magnetic gradient field. [0046]
  • The ferromagnetic and ferrimagnetic substances can be stabilized with a shell that is made of oligomeric or polymeric carbohydrates, proteins, peptides, nucleotides, surfactants, other monomers, oligomers, or polymers and/or lipids. [0047]
  • The particle sizes of the ferromagnetic and ferrimagnetic substances are advantageously between 1 nm and 100 nm. Preferred are colloidal particles with particle sizes of between 1 nm and 400 nm. Especially preferred are particle sizes of between 1 nm and 100 nm. [0048]
  • As magnetic markers, ferromagnetic or ferrimagnetic substances can also be produced with a stabilizing shell that is made of the structure-specific substance or the analyte that is to be identified, by the particles being put after production directly into a solution of the structure-specific substance, optionally in the presence of other adjuvants, such as, e.g., proteins, carbohydrates, as well as natural, synthetic, or partially synthetic surface-active substances, etc., or by being produced directly in the presence of structure-specific substances. [0049]
  • Suitable magnetic markers and suspensions that contain these particles are described in, for example, WO 92/12735, WO 92/22586, EP 0 186 616 and U.S. Pat. No. 4,101,435. In principle, magnetic particles can also be used that are usually used as contrast media for nuclear resonance tomography, such as, e.g., Resovist, Lumirem, Feridex, Combidex, Abdoscan and Clariscan. [0050]
  • Compounds, which consist of colloidal suspensions of freely movable ferrimagnetic or ferromagnetic particles and structure-specific substances or analytes that are to be identified, were already described in German Patent DE 195 03 664 C2. [0051]
  • The compounds can also consist of combinations of several ferromagnetic or ferrimagnetic particles with relaxation times that can be discriminated, since measurement results that can be discriminated individually can be achieved through the use of different magnetic labelings with respectively a very narrow distribution of relaxation times and/or magnetic moments for various structure-specific substances or analytes inside a sample. As a result, direct simultaneous determination of several analytes is made possible. [0052]
  • As suspension media, all liquids in which the compounds can move freely are suitable. Especially suitable are water, aqueous solutions of surface-active adjuvants, such as, e.g., surfactants or oligomeric or polymeric carbohydrates and proteins, as well as mixtures of water with alcohols, such as, e.g., glycerol and polyethylene glycol. The suspension media can additionally contain adjuvants that change the osmotic pressure, such as, e.g., common salt. In addition, buffer substances that determine pH, such as, e.g., phosphates, can be contained. Especially preferred are suspension media with suitable optical properties, such as lower absorption and double refraction. As an alternative, the light source can be selected so that as little light as possible is absorbed from the suspension medium, and especially also biological suspension media can be used. [0053]
  • The compounds that consist of ferromagnetic or ferrimagnetic colloidal particles with structure-specific substances or analytes that are to be identified can also be present in dried form, optionally in combination with other adjuvants which, e.g., facilitate drying or increase the stability of the dried product (e.g., as lyophilizates) and are converted into the suspension medium only shortly before the measurement. [0054]
  • Due to the binding identification based on physical mechanisms, non-specific measurement signals (matrix phenomena) can be largely ruled out. The specificity of the process thus depends only on the “true” specificity of the structure-specific substance (cross reactivity of antibodies, non-specific binding of ligands). Based on the high sensitivity of the process according to the invention, it is easy to remain under the other commonly used detection limits of binding assays. [0055]
  • The process according to the invention can be used in, e.g., fertility, histocompatibility, allergology, infectiology, hygiene, genetics, virology, bacteriology, toxicology, pathology, environmental analysis, food chemistry and medical diagnosis. [0056]
  • The following examples are used for a more detailed explanation of the subject of the invention, without intending that they be limited to this subject. [0057]
    • EXAMPLES
  • 1. Measurement of a Series of Dilutions of Dextran-Magnetite: [0058]
  • Procedure: The starting sample (iron oxide that is coated with dextran, produced according to the method of M. Hasegawa and S. Hakukoku, U.S. Pat. No. 4,101,435 (1978), manufacturer: Meito Sangyo) was present at a concentration of 1 mol of Fe/l. From this, in each case, samples diluted with distilled water by the factor 10 were produced (concentration range of 10[0059] −1 to 10−6 mol of Fe/l). A comparison sample with distilled water was also examined. For measurement, a volume of 1 ml of all samples was loaded into the optical cell.
  • All measurements produced here were performed with the same procedure. The samples were magnetized for a period of 2 ms repeatedly with a magnetic pulse of strength 100 Oe (10 mT). The pause between the pulses was 20 ms. To improve the signal-noise ratio, 256 individual measurements were averaged. [0060]
  • Result: For the comparison sample with distilled water and the sample of concentration of 10[0061] −6 mol of Fe, no relaxation signal could be detected (cf. FIG. 2 or FIG. 3).
  • The relaxation of a sample of the concentration of 10[0062] −5 mol/l of Fe was detectable and is shown in FIG. 4.
  • The amount of Fe substance contained in the sample volume of [0063] 1 ml of this sample is 10 nmol. It is observed that actually only the volume of about 2×2×10 mm (40 μl) that is in the laster beam contributes to the signal, and a detection amount of 0.4 nmol of Fe is produced. This is on the order of the detection sensitivity of the process that is described in DE 195 03 664 C2. The new measuring position was not specially optimized to a high detection sensitivity. The particles that are used contain only a very small fraction of particles that are lined up according to Brown and thus contribute to the signal.
  • 2. Measurement of the Influence of the Addition of Biotin-BSA to Dextran-Magnetite that is Coupled with Streptavidin in the Relaxation of the Double Refraction: [0064]
  • Procedure: The sample (dextran-magnetite of Example 1, coupled with streptavidin) was present in an initial concentration of 2.66 mmol of Fe/l. For the measurements, a [0065] sample 1 that consists of 100 μl of the sample that is diluted with 400 μl of BSA-PBS (PBS: phosphate buffered saline) buffer was produced. The relaxation signal of this sample was measured. Then, 40 μl of biotin-BSA that was diluted in BSA-PBS buffer (absolute amount of 400 ng of biotin-BSA) was added one time to this sample. Then, the relaxation signal was measured at various times after mixing.
  • As a control, a [0066] sample 2 that consists of 100 μl of dextran-magnetite, coupled with streptavidin (diluted in 400 μl of BSA-PBS buffer), was saturated with 5 μl of free biotin (1:10 diluted, initial concentration of 1 mg/ml). Also, here, 40 μl of biotin-BSA diluted in BSA-PBS buffer (absolute amount 400 ng of biotin-BSA) was added one time, and the relaxation signal was measured at various times after mixing.
  • Result: The relaxation signal of [0067] sample 1 was clearly observable. After biotin-BSA was added, a significant increase in the relaxation time (cf. FIG. 5) was shown. This extension of the relaxation time can be explained by the increase of the hydrodynamic particle diameter of the sample by biotin-BSA-induced aggregation or cross-linking.
  • [0068] Sample 2 also showed a clear relaxation signal. The addition of biotin-BSA in the BSA-PBS buffer to this sample did not result, however, in a change of the relaxation time (cf. FIG. 6).
  • The unchanged hydrodynamic particle diameter that is to be concluded from the above shows that by the saturation of the binding sites of streptavidin with free biotin, it no longer results in an aggregation or cross-linking of the sample that is induced by biotin-BSA. Thus, the specificity of the binding reaction was demonstrated in [0069] sample 1.

Claims (13)

1-18. (Cancelled)
19. A composition that comprises combinations of a ferromagnetic or ferrimagnetic substance with a structure-specific substance, wherein the structure-specific substance is such that its Brownian relaxation proceeds faster than its Néelian relaxation under measuring conditions for detection of an analyte or binding reaction by its double refraction behavior or relaxation behavior of double refraction in the composition.
20. A composition that comprises combinations of a ferromagnetic or ferrimagnetic substance with a substance that is to be identified, wherein the substance that is to be identified is such that its Brownian relaxation proceeds faster than its Néelian relaxation under measuring conditions for detection of a binding reaction by its double refraction behavior or relaxation behavior of double refraction in the composition.
21. A composition according to claim 19, wherein the ferromagnetic or ferrimagnetic substance's particle size is 1 nm to 100 μm.
22. A composition according to claim 19, wherein the ferromagnetic or ferrimagnetic substance is stabilized with a shell that comprises an oligomeric or polymeric carbohydrate, protein, peptide, nucleotide, surfactant, polymer and/or lipid.
23. A composition according to claim 19, wherein the structure-specific substance is an antibody, antibody fragment, biotin, a substance that binds biotin, agonist that binds specifically to receptors or their antagonists, peptide, protein, receptor, enzyme, enzyme substrate, nucleotide, ribonucleic acid, deoxyribonucleic acid, carbohydrate, or lipoprotein.
24-25. (Cancelled)
26. An apparatus comprising a light polarizer, a device that receives a sample, a device that magnetizes the sample with magnetic pulses or a magnetic field of variable frequency, and an analysis of analyzer that analyzes the polarization direction of polarized light, wherein said apparatus is capable of implementing a process for detecting an analyte or a binding reaction in a binding assay conducted in a sample labeled with a ferromagnetic or ferrimagnetic substance comprising determining double refraction behavior or relaxation behavior of double refraction in the sample, and correlating said behavior with the presence of the analyte or binding reaction.
27. An apparatus according to claim 26, that contains an optical bank, a laser, a polarizer, an optical cell containing the sample, an analyzer and a photodetector.
28. An apparatus according to claim 26 further comprising a λ/4-plate between the sample and the analyzer.
29. A composition according to claim 20, wherein the ferromagnetic or ferrimagnetic substance's particle size is 1 nm to 100 μm.
30. A composition according to claim 20, wherein the ferromagnetic or ferrimagnetic substance is stabilized with a shell that comprises an oligomeric or polymeric carbohydrate, protein, peptide, nucleotide, surfactant, polymer and/or lipid.
31. A composition according to claim 20, wherein the substance that is to be identified is an antibody, antibody fragment, biotin, a substance that binds biotin, agonist that binds specifically to receptors or their antagonists, peptide, protein, receptor, enzyme, enzyme substrate, nucleotide, ribonucleic acid, deoxyribonucleic acid, carbohydrate, or lipoprotein
US10/796,003 1999-08-06 2004-03-10 Process for detecting binding reactions with use of the measurement of the relaxation of the double refraction of magnetic particles Abandoned US20040235197A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US10/796,003 US20040235197A1 (en) 1999-08-06 2004-03-10 Process for detecting binding reactions with use of the measurement of the relaxation of the double refraction of magnetic particles

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE19938384.7 1999-08-06
DE19938384A DE19938384A1 (en) 1999-08-06 1999-08-06 Detecting analytes or binding reactions, useful e.g. for diagnosis, based on measuring relaxation of birefringence of magnetic marker compounds
US14868699P 1999-08-16 1999-08-16
US09/631,630 US6979574B1 (en) 1999-08-06 2000-08-04 Process for detecting binding reactions with use of the measurement of the relaxation of the double refraction of magnetic particles
US10/796,003 US20040235197A1 (en) 1999-08-06 2004-03-10 Process for detecting binding reactions with use of the measurement of the relaxation of the double refraction of magnetic particles

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US09/631,630 Division US6979574B1 (en) 1999-08-06 2000-08-04 Process for detecting binding reactions with use of the measurement of the relaxation of the double refraction of magnetic particles

Publications (1)

Publication Number Publication Date
US20040235197A1 true US20040235197A1 (en) 2004-11-25

Family

ID=33458010

Family Applications (2)

Application Number Title Priority Date Filing Date
US09/631,630 Expired - Fee Related US6979574B1 (en) 1999-08-06 2000-08-04 Process for detecting binding reactions with use of the measurement of the relaxation of the double refraction of magnetic particles
US10/796,003 Abandoned US20040235197A1 (en) 1999-08-06 2004-03-10 Process for detecting binding reactions with use of the measurement of the relaxation of the double refraction of magnetic particles

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US09/631,630 Expired - Fee Related US6979574B1 (en) 1999-08-06 2000-08-04 Process for detecting binding reactions with use of the measurement of the relaxation of the double refraction of magnetic particles

Country Status (1)

Country Link
US (2) US6979574B1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006079998A1 (en) 2005-01-31 2006-08-03 Koninklijke Philips Electronics N.V. Rapid and sensitive biosensing
US20090085557A1 (en) * 2006-04-19 2009-04-02 Anatol Krozer Detection device and method

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AT503845B1 (en) * 2007-04-11 2008-03-15 Arc Austrian Res Centers Gmbh Determining the relaxation behavior e.g. relaxation time of micro- or nano-particles, on which target molecules e.g. viruses are bound, comprises aligning the particles in dispersion and/or suspension by electrical and/or magnetic field
US8076161B2 (en) * 2007-05-31 2011-12-13 Canon Kabushiki Kaisha Target substance detection kit and target substance detection method
CN103025859A (en) 2010-05-25 2013-04-03 阿尔利克斯公司 Methods and apparatuses for detection of positional freedom of particles in biological and chemical analyses and applications in immunodiagnostics
US9642923B2 (en) 2014-02-24 2017-05-09 Verily Life Sciences Llc Engineered particles with polarization contrast and alignment control for enhanced imaging

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4261989A (en) * 1979-02-19 1981-04-14 Kaken Chemical Co. Ltd. Geldanamycin derivatives and antitumor drug
US5650430A (en) * 1990-06-06 1997-07-22 Sankyo Company, Limited Radicicol derivatives, their preparation and their anti-tumor activity
US5932566A (en) * 1994-06-16 1999-08-03 Pfizer Inc. Ansamycin derivatives as antioncogene and anticancer agents
US5968921A (en) * 1997-10-24 1999-10-19 Orgegon Health Sciences University Compositions and methods for promoting nerve regeneration
US6239168B1 (en) * 1996-10-25 2001-05-29 Kyowa Hakko Kogyo Co., Ltd. Radicicol derivatives

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19503664C2 (en) * 1995-01-27 1998-04-02 Schering Ag Magnetorelaxometric detection of analytes
DE19503644A1 (en) 1995-02-06 1996-08-14 Wolfgang Dr Ing Lorenz Emission test chamber

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4261989A (en) * 1979-02-19 1981-04-14 Kaken Chemical Co. Ltd. Geldanamycin derivatives and antitumor drug
US5650430A (en) * 1990-06-06 1997-07-22 Sankyo Company, Limited Radicicol derivatives, their preparation and their anti-tumor activity
US5932566A (en) * 1994-06-16 1999-08-03 Pfizer Inc. Ansamycin derivatives as antioncogene and anticancer agents
US6239168B1 (en) * 1996-10-25 2001-05-29 Kyowa Hakko Kogyo Co., Ltd. Radicicol derivatives
US5968921A (en) * 1997-10-24 1999-10-19 Orgegon Health Sciences University Compositions and methods for promoting nerve regeneration

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006079998A1 (en) 2005-01-31 2006-08-03 Koninklijke Philips Electronics N.V. Rapid and sensitive biosensing
US20080191688A1 (en) * 2005-01-31 2008-08-14 Koninklijke Philips Electronics, N.V. Rapid and Sensitive Biosensing
US8828740B2 (en) 2005-01-31 2014-09-09 Koninklijke Philips N.V. Rapid and sensitive biosensing
US20090085557A1 (en) * 2006-04-19 2009-04-02 Anatol Krozer Detection device and method
US8624584B2 (en) * 2006-04-19 2014-01-07 Acreo Swedish Ict Ab Method and arrangement relating to analyses of a substance

Also Published As

Publication number Publication date
US6979574B1 (en) 2005-12-27

Similar Documents

Publication Publication Date Title
US6027946A (en) Process and compounds for the magnetorelaxometric detection of analytes and use thereof
US8217647B2 (en) Measuring agglutination parameters
US8034634B2 (en) Ultra-sensitive magnetoreduction measurement system and ultra-sensitive, wash-free assay using the same
EP2069769B1 (en) Method of detecting interactions on a microarray using nuclear magnetic resonance
US10488408B2 (en) Detection of target molecules in a sample by using a magnetic field
JP2003528289A (en) A method for detecting binding reactions using measurements of the relaxation of the birefringence of magnetic particles
RU97114745A (en) METHOD AND COMPOUNDS FOR MAGNETORELAXOMETRIC DETECTION OF ANALYTES AND THEIR APPLICATION
JP2002509242A (en) Force discrimination test
AU2009333937A1 (en) Quantitative analyte assay device and method
JP3628704B2 (en) Methods and compounds for detection utilizing remanence and their use
Larsson et al. Magnetic transducers in biosensors and bioassays
US6979574B1 (en) Process for detecting binding reactions with use of the measurement of the relaxation of the double refraction of magnetic particles
EP1936350A1 (en) A method for quantitatively measuring agglutination parameters
EP1966613A1 (en) Sensing device and method for determination of the amount of target molecule in an analyte
MXPA97005543A (en) Processes and compounds for magnetorrelaxometric detection of analytics and its
CN116840094A (en) Magnetic labeling biological molecule detection method and measurement system
HK1008563A (en) Process and compounds for the magnetorelaxometric detection of analytes and use thereof
Hawkins et al. 9 Magnetic Nanoparticles
DE19938384A1 (en) Detecting analytes or binding reactions, useful e.g. for diagnosis, based on measuring relaxation of birefringence of magnetic marker compounds

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION