WO2010086772A1 - Système et procédé de détection - Google Patents

Système et procédé de détection Download PDF

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Publication number
WO2010086772A1
WO2010086772A1 PCT/IB2010/050289 IB2010050289W WO2010086772A1 WO 2010086772 A1 WO2010086772 A1 WO 2010086772A1 IB 2010050289 W IB2010050289 W IB 2010050289W WO 2010086772 A1 WO2010086772 A1 WO 2010086772A1
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WO
WIPO (PCT)
Prior art keywords
analyte
magnetic particles
reaction chamber
region
sensor region
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PCT/IB2010/050289
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English (en)
Inventor
Wendy U. Dittmer
Wilhelmina M. Hardeman
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Koninklijke Philips Electronics N.V.
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Application filed by Koninklijke Philips Electronics N.V. filed Critical Koninklijke Philips Electronics N.V.
Publication of WO2010086772A1 publication Critical patent/WO2010086772A1/fr

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Classifications

    • 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
    • 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
    • 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
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection

Definitions

  • the invention relates to systems, apparatus and methods for detecting and/or quantifying molecules in a sample using magnetic particles, including (disposable) cartridges for use with such systems apparatus and methods.
  • Diagnostic devices using lateral flow chromatography for performing sandwich or competition assays are well known. Typically, the detection is performed using colloidal gold or latex particles. In such devices, all or part of the reagents necessary for performing the assays may be present in dry form prior to use.
  • US4693912 describes for example methods and materials for the lyophilization of reagent-coated latex particles. More recently immunoassays have been developed wherein analytes or antibodies are labeled with magnetic particles. The magnetic particles are used for manipulation but can also function as a detectable label. Also for this type of assays reagents can be provided in dry form prior to performing the assay. In the most simple configuration, magnetic particles are mixed with a sample prior to the introduction of the sample into the diagnostic device.
  • US20060257958 describes a lateral flow assay wherein the flow of magnetic particles, functionalized with various receptors such as for example antibodies, nucleic acids, etc., is assisted by a magnetic field.
  • magnetic particles are deposited and lyophilized in a channel of a device, which is used for transport of the sample to the reaction and detection chamber of the device.
  • the magnetic particles are redispersed by the introduction of the sample and are transferred to the reaction and detection chamber by the flow of the sample or by the application of a magnetic field.
  • WO2007072380 and WO2007129275 disclose the introduction of magnetic particles, optionally in dried from, into the reaction chamber of a diagnostic device.
  • Drying and redispersion of dried magnetic particles results in an increased clustering of magnetic particles.
  • the deposition of magnetic particles in a dry form has the disadvantage that such particles have a tendency to irreversibly aggregate or remain attached to a substrate even after the addition of a redispersion liquid.
  • magnetic particles are not mobile and are in contact with neighboring particles for a long period of time during the storage period prior to use. It is during these long periods that non-specific, irreversible interactions take place between the particles themselves e.g. via van der Waals or other electrostatic interactions. Similar interactions can take place between a magnetic particle and a substrate.
  • As magnetic particles are larger than the usual molecular species, necessary for the assay to be performed, that are dried down in an immunoassay, the number and chance of such interactions that promote aggregation are greater.
  • the invention relates to a device and system that is capable of using the device for performing biological binding assays.
  • the device comprising a reaction chamber (2) with a first surface (3) and a second surface (4).
  • the first surface (3) comprises a sensor region (31), with immobilized thereto at least one of an analyte-specific probe, an analyte and an analyte-analogue.
  • the second surface (4) comprises a reagent region (41), which comprises prior to the use of the system, magnetic particles in dry form.
  • the sensor region (31) and the reagent region (41) are located on opposite sides of the reaction chamber.
  • the magnetic particles may be non-covalently immobilized to the reagent region (41) prior to performing the biological assay with the sensor device or system.
  • the immobilization must be such that during performance of the assay, the magnetic particles can be at least partly resuspended in a sample liquid present in the reaction chamber.
  • the device and system are suitable for use in a sandwich immunoassay, wherein the sensor region comprises a first analyte-specific probe and wherein the magnetic particles are bound to a second analyte-specific probe.
  • the device and system are suitable for use in a competitive immunoassay, wherein the sensor region comprises an analyte or analyte analogue wherein the magnetic particles are bound to an analyte or analyte-specif ⁇ c probe.
  • the device and system are suitable for use in another type competitive immunoassay, wherein the sensor region comprises an analyte-specif ⁇ c probe and wherein the magnetic particles are bound to an analyte or analyte analogue.
  • the device of the invention may take the form of a cartridge that can disconnected from and/or (re)connected to the system in order to allow the reuse of the device or the disposal of the device (cartridge).
  • the first surface and/or second surface or at least the reagent region and /or the sensor region may be curved or flat depending on for example design or other parameters to be optimized. They may be cylindrical if they are to form a part of a cylindrical tubular reaction chamber for example. They are however preferably flat.
  • the first surface and second surface or at least the sensor region and the reagent region are parallel to each other. This may have the effect that distance to travel by magnetic particles from the reagent region to the sensor region is substantially the same over the regions, therewith improving the assay reproducibility, and/or sensitivity and/or accuracy.
  • the first surface and second surface may have the same area and shape, but these may also differ.
  • the reagent region may have a larger area than the sensor region or vice versa.
  • the area of the regent region may have a shape different from the area of the sensor region.
  • the sensor region may be square while the reagent region may be rectangular.
  • the reagent region may overlap the sensor region symmetrically or asymmetrically.
  • the largest of the overlapping areas of the reagent region and the sensor region may be 5, 10 15 20 or even 50 % larger than the smallest.
  • the system further comprises means for magnetic actuation (5) of the magnetic particles.
  • the magnetic particles in dry form are present within a mixture comprising a buffer, a sugar and a carrier protein, which upon filling of the reaction chamber with water results in a concentration of about 1 to 100 mM of buffer, 1 to 25 % (w/v) of sugar and 1 to 10 % (w/v) of carrier protein, typically in a concentration of about 25 to 75 rnM of buffer, 2.5 to 7.5 % (w/v) of sugar and 2.5 to7.5 % (w/v) of carrier protein.
  • the buffer is a phosphate buffer
  • the sugar is sucrose
  • the carrier protein is BSA (Bovine Serum Albumin).
  • the first and second surface are planar surfaces separated from each other by about 500 ⁇ m.
  • first and second surface are planar surfaces, parallel to each other.
  • the first surface (3) and the sensor region (31) have the same size.
  • the reagent region has a surface of between 0.5 and 2.0 mm 2 , e.g. 1 mm 2 .
  • the volume of the reaction chamber is between 0.1 and 5 ⁇ l, more particularly between 0.5 and 2 ⁇ l, e.g. 1 ⁇ l.
  • the magnetic particles have a diameter of between 250 and
  • the device and system are suitable for a variety of assays wherein the analyte-specific probe can be an oligonucleotide, an antibody or fragment thereof, a lectin, a pharmaceutical compound, a peptide or a protein.
  • the system further comprises a detection means (6) for detection at least one magnetic particle on the sensor region.
  • the detection means detects an optical property of the at least one magnetic particle. This is obtained for example by Frustrated Total Internal Reflection (FTIR) method.
  • FTIR Frustrated Total Internal Reflection
  • Yet another aspect of the invention relates to a method for preparing a reaction cartridge comprising a reaction chamber (2) comprising the steps of, providing a surface (4), free of bound analyte, analyte-analogue or analyte- specific probe, applying to a reagent region (41) on the surface (4) a solution comprising magnetic particles, drying the solution, assembling the surface (4) with dried solution into a reaction chamber (2), wherein after assembly the reagent region (41) on the surface (4) is positioned opposite the sensor region (31) of the sensor region (3) of the reaction chamber.
  • FTIR Frustrated Total Internal Reflection
  • the solution further comprises a buffer, a sugar and a carrier protein, their concentration being adapted to the volume of the reaction chamber, to provide upon redispersion a buffer with a concentration of about 1 to 100 mM buffer, 1 to 10 % (w/v) carrier protein, 1 to 25 % (w/v) sugar, more particularly a concentration of about 25 to 75 mM of buffer, 2.5 to 7.5 % (w/v) of sugar and 2.5 to 7.5 % (w/v) of carrier protein.
  • a buffer with a concentration of about 1 to 100 mM buffer, 1 to 10 % (w/v) carrier protein, 1 to 25 % (w/v) sugar, more particularly a concentration of about 25 to 75 mM of buffer, 2.5 to 7.5 % (w/v) of sugar and 2.5 to 7.5 % (w/v) of carrier protein.
  • the drying is performed by lyophilization.
  • the magnetic particles have an average diameter of between 250 and 750 nm, more particularly between 400 and 600 nm, e.g. 500 nm.
  • the magnetic particles are applied at the surface at a density of between 10 to 50 ⁇ g per mm 2 .
  • Another aspect of the present invention relates to a method for quantifying and/or detecting an analyte, comprising the steps of:- introducing a liquid sample suspected to contain an analyte, into the reaction chamber of a device according to the invention, thereby resuspending the disposed magnetic particles in the liquid sample in said reaction chamber, applying a magnetic field to manipulate the magnetic particles, and - detecting said magnetic particles bound to the sensor region (31) of said reaction chamber.
  • the manipulation of the magnetic particles, using the means for magnetic actuation may be understood in its broadest from and can include steps such as loosening the magnetic particles from the first surface for their suspension in the sample fluid, shaking the particles back and for such as to effectuate mixing, displacement towards detection region for detection and subsequent displacing those not bound to the detection region away from the detection region.
  • the detection is based on an optical property of the magnetic particles, for example by using frustrated total internal reflection technique (FTIR).
  • FTIR frustrated total internal reflection technique
  • This detection method is applicable to a variety of samples, in particular to blood plasma, saliva or other bodily fluids
  • the sample may be water based or organic solvent based
  • the present invention describes materials and methods for performing a one- step assay, typically an immunoassay in a fluid sample wherein the magnetic particle labels are present in the reaction chamber, remote from the sensor region, in a dry form prior to the application of a sample. This reduces the transportation time and distance considerably and results in a decreased loss of reagents. Methods are described for the detection of analytes (e.g. proteins, drugs and drug metabolites) in a binding assay using analyte-binding molecules (e.g. antibodies) conjugated to magnetic particles.
  • analytes e.g. proteins, drugs and drug metabolites
  • compositions for applying magnetic particles in dry form to a surface of a reaction chamber Particular embodiments of the invention describe materials and methods for performing immunoassays in e.g. plasma, serum and blood, for example in the detection of disease bio markers in the picomolar range such as cardiac Troponin cTnl, which is a diagnostic marker for myocardial infarct.
  • the invention is equally applicable to detection of other markers of disease or disposition.
  • Compositions and methods of the present invention are in particular applicable in handheld biosensor system for use in rapid medical diagnosis outside of laboratory environments such as the physician's office, hospital bedside, ambulance and patient's home. Systems and methods of the present invention are compact, robust and have as few user- aided steps as possible.
  • An assay generally requires only the addition of a sample to a disposable cartridge whereby all reagents necessary for the test are already present in the cartridge in a dry form.
  • Systems and methods as described herein overcome the disadvantages of wet reagents where there is a risk of leaking and drying out, in which case it is difficult to control the concentrations of the reagents in the final assay. Dry reagents are not mobile after deposition. As a consequence reagents will remain at the desired location until to start of an assay.
  • FIG. 1 shows a system of the present invention, illustrated for a sandwich assay.
  • Fig. 2 shows the detection of magnetic particles in a sandwich assay, in accordance with an embodiment of the present invention, for cardiac Troponin I (cTnl), using dried magnetic particles with Troponin antibody in various amounts in the range of 3 to 15 ⁇ g and various amounts of troponin analyte in the range of 0- 1000 picomol.
  • Fig. 3 shows a dose response curve of the detection of various amounts of analyte according to an embodiment of the present invention.
  • the dotted line shows the background signal in the absence of analyte.
  • the grey area represents 2x the value of the background.
  • Fig. 4 shows in accordance with an embodiment of the present invention, the detection of an analyte in buffer, 50 % (v/v) plasma in buffer or plasma.
  • the assay was performed with antibody labeled magnetic beads in dry form on the reagent region (dry) or by adding a suspension antibody labeled magnetic beads in liquid (wet) to the sample.
  • analyte refers to a compound in a sample of which the detection of presence and or/ concentration is desired.
  • analyte-analogue refers to a compound which has a chemical structure which is different, but resembling the structure of the analyte. Both analyte and analyte-analogue are recognized by the same analyte specific probe
  • analyte-specific probe refers to a compound which can bind with the analyte or the analyte analogue.
  • reaction chamber refers to a region within a device or a cartridge, where different reagents taking part in a reaction are contacted with each other.
  • sensor region refers to the part of the reaction chamber, wherein probes, such as analyte-specif ⁇ c probes are bound or immobilized. Generally, it is also the area where the most important sensitive detection takes place.
  • reaction region refers to the part of the reaction chamber, where prior to assay magnetic particles are deposited.
  • buffer refers to compounds that have a pH stabilizing effect.
  • the device may be made of any kind of material as long as it is compatible with magnetic actuation and or the necessary detection to be performed on the sample. The latter may require appropriate optical windows if for example optical detection such as FTIR is used.
  • Preferred materials are plastics especially in view of disposability of such a device and/or cost during production. Plastics may also have improved compatibility with materials used during biological assay.
  • the parts of the device being in contact with the solvent should be chosen such that they can withstand the solvents and/or reagents at least within the timescale of the assay.
  • the device may be in the form of a cartridge that is removable from the system according to the invention. In that case appropriate lock features may be added to the device and system.
  • buffer components, sugars, carrier proteins and other ingredients are mixed with the magnetic particles in solution and applied on the reagent region as one spot or zone or as a plurality of different spots or zones.
  • one or more of the above compounds are in separate solutions and applied next the each other on the reagent region or on top of each other.
  • the reagents can be deposited via several drying techniques including lyophilization, vacuum drying and ambient pressure drying.
  • the magnetic particles can also be applied as millimeter-sized lyophilized spheres (also known as accuspheres or lyospheres). Lyophilization prevents the formation of crystals and allows the reagents to be dried to an amorphous glassy state that is readily redispersed upon the addition of a fluid.
  • An amount of 1 to 40 ⁇ g of magnetic particles can be applied in an area as small as 1 mm 2 and can be redispersed in a volume as small as 1 ⁇ l.
  • a high concentration of particles (40 ⁇ g) is particularly suitable for applications in which a high concentration of analyte is to be measured (InM) whereas concentrations of approximately 5 ⁇ g and less (2 or 1 ⁇ g) are typical for the detection of an analyte present at picomolar concentrations in a sample.
  • the concentration of buffer component, sugar and carrier protein thus depends on their concentration in the solution for applying the magnetic particles, the volume of this solution and the volume of the reaction chamber. Taking in account these factors, magnetic particles in dry form are generally present on the reagent region within a mixture comprising a buffer, a sugar and a carrier protein, which upon filling of the reaction chamber with water results in a concentration of about 50 mM of buffer (e.g. between 25 and 75 mM), 5 % (w/v) of sugar (e.g. between 2.5 and 7.5 % (w/v)) and 5 % (w/v) of carrier protein (e.g. between 2.5 and 7.5 % (w/v)).
  • buffer e.g. between 25 and 75 mM
  • 5 % (w/v) of sugar e.g. between 2.5 and 7.5 % (w/v)
  • carrier protein e.g. between 2.5 and 7.5 % (w/v)
  • Reaction chambers which are used in the present systems and methods are typically rectangularly, or box-like shaped with flat top and bottom parts, separated from each other by a wall.
  • the flat top and bottom parts comprising the sensor and reagent regions respectively are parallel to each other.
  • such reaction chambers have a volume between 0.1 and 10 ⁇ l, and the bottom and top part are separated from each other by a distance ranging from 100 to 500 ⁇ m.
  • Other configurations of reaction chambers may have grooves, ridges or protruding posts.
  • reaction chambers may have a tube-like shape with a bottom half and top half corresponding respectively to the top and bottom parts of a box- like reaction chamber.
  • the sensor region comprises one analyte-specific probe.
  • Alternative embodiments are also envisaged whereby a variety of analyte- specific probes are arrayed on the sensor region to allow simultaneous detection of different compounds in a sample (sensor multiplexing).
  • different sensor regions (31) are present on the first surface (3), comprising different analyte specific probes, analytes or analyte analogues.
  • appropriate magnetic particles are present at the corresponding reagent regions (41) on the second surface.
  • aspecific binding of magnetic particles to inappropriate sensor regions is minimized because magnetic particles are localized opposite of the sensor regions where the assay takes place.
  • a system has multiple reaction chambers.
  • the sample is spread over different chambers to run different assays in parallel.
  • a magnetic field is applied to retain magnetic particles to the reagent region upon addition of the sample, to avoid that magnetic particles are transported with the liquid flow of the sample upon entry into the reaction chamber.
  • the system of the present invention either further comprises or is used in combination with a detection means, capable of detecting the binding of magnetic particles to the sensor region. Detection of the bound magnetic particles on the sensor region can be done by various means, either based on the properties of the magnetic particles themselves or using a label.
  • the label can be attached to the magnetic particles, or can be bound to or incorporated into the analyte.
  • the detection means present in or used in combination with the systems of the present invention are detection means capable of detecting the relevant signal such as, but not limited, to a magnetic signal, magnetoresistance, a Hall effect, an optic signal (reflection, absorption, scattering, fluorescence, chemiluminescence, RAMAN, etc.), an acoustical signal (quartz crystal microbalance (QCM), surface acoustic waves (SAW), Bulk Acoustic Wave (BAW) etc.).
  • a magnetic signal magnetoresistance, a Hall effect
  • an optic signal reflection, absorption, scattering, fluorescence, chemiluminescence, RAMAN, etc.
  • an acoustical signal quartz crystal microbalance (QCM), surface acoustic waves (SAW), Bulk Acoustic Wave (BAW) etc.
  • QCM quartz crystal microbalance
  • SAW surface acoustic waves
  • BAW Bulk Acoustic Wave
  • Such vesicles may be filled with a liquid, a gas, a gaseous precursor, and/or a solid or solute material.
  • Typical labels useful in the context of the present invention are those labels which are classically used in in vitro assays such as, but not limited to, chromophoric groups, radioactive labels, electroluminescent, chemiluminescent, phosphorescent, fluorescent or reflecting labels.
  • the sensor of the detection unit is integrated into the reaction chamber (e.g. magnetoresistive sensor is integrated), which can be provided as a disposable cartridge.
  • the sensor is provided as separate part from the reaction chamber (e.g. optical unit).
  • the reaction chamber optionally comprises a detection window, which allows the detection of the signal of the magnetic particles and/or labels bound to the sensor region.
  • the location of the detection window is of course determined by the location of the sensor region and the detection means. Most particularly, the detection window is opposite to the sensor region.
  • the sensor region of the reaction chamber is provided on the detection window. Where the detection is based on magnetic field or optical methods, the material of the reaction chamber may render the provision of a specific detection window superfluous.
  • the detection of magnetic particles is performed by Frustrated Total Internal Reflection (FTIR), wherein the presence of magnetic particles on a transparent surface is detected.
  • FTIR Frustrated Total Internal Reflection
  • this transparent surface coincides with the surface used for binding the analyte probe or the analyte(analogue).
  • a sensor device using such FTIR is described in WO/2008/072156. All FTIR measurement techniques described therein can be applied to a system according to the present invention for the measurement of the capture of target molecules in the sensor region.
  • the input light beam (Ll) is transmitted into the device of the present invention and totally internally reflected at the sensor region of the device.
  • the amount of light in the output light beam (L2) and optionally also of fluorescence light emitted by target components at the sensor region is then detected by a light detector (31).
  • Evanescent light generated during the total internal reflection is affected (absorbed, scattered) by target components and/or label particles (1) at the sensor region and will therefore be missing in the output light beam (L2). This can be used to determine the amount of target components at the binding surface (12) from the amount of light in the output light beam (L2, L2a, L2b).
  • Magnetic particles used in the present invention can be completely inorganic or can be a mixture of an inorganic and an organic material (e.g. a polymer). Accordingly, labels can be attached via the inorganic or via the organic component at the outside or can be incorporated into the particle.
  • an organic material e.g. a polymer
  • Magnetic particles are widely used in biological analysis, e. g. in high- throughput clinical immunoassay instruments, sample purification, cell extraction, etc.
  • diagnostic companies Roche, Bayer, Johnson & Johnson, Abbott, BioMerieux, etc.
  • fabricate and sell reagents with magnetic particles e.g. for immunoassays, nucleic-acid extraction, and sample purification.
  • Magnetic particles are commercially available in various sizes, ranging from nanometers to micrometers.
  • the particles may carry functional groups such as hydroxyl, carboxyl, aldehyde or amino groups.
  • These may in general be provided, for example, by treating uncoated monodisperse, superparamagnetic particles, to provide a surface coating of a polymer carrying one of such functional groups, e. g. polyurethane together with a polyglycol to provide hydroxyl groups, or a cellulose derivative to provide hydroxyl groups, a polymer or copolymer of acrylic acid or methacrylic acid to provide carboxyl groups or an aminoalkylated polymer to provide amino groups.
  • a polymer carrying one of such functional groups e. g. polyurethane together with a polyglycol to provide hydroxyl groups, or a cellulose derivative to provide hydroxyl groups, a polymer or copolymer of acrylic acid or methacrylic acid to provide carboxyl groups or an aminoalkylated polymer to provide amino groups.
  • US Patent 4654267 describes the introduction of many such surface coatings.
  • Other coated particles may be prepared by modification of the particles according to the US Patents 4336173, 4459378
  • the surface carries-OH groups connected to the polymeric backbone through (CH 2 CH 2 O) 8-10 linkages.
  • Other preferred carry -COOH groups obtained through polymerization of methacrylic acid.
  • the NH 2 groups initially present in the particles may be reacted with a diepoxide as described in US Patent 4654267 followed by reaction with methacrylic acid to provide a terminal vinyl grouping.
  • Solution copolymerization with methacrylic acid yields a polymeric coating carrying terminal carboxyl groups.
  • amino groups can be introduced by reacting a diamine with the above product of the reaction with a diepoxide, while reaction with a hydroxylamine such as aminoglycerol introduces hydroxy groups.
  • the coupling of a bio active molecule to a particle can be irreversible but can also be reversible by the use of a linker molecule for the crosslinking between particle and bioactive molecule.
  • linkers include peptides with a certain proteolytic recognition site, oligonucleotide sequences with a recognition site for a certain restriction enzyme, or chemical reversible crosslinking groups as those comprising a reducible disulfide group.
  • a variety of reversible crosslinking groups can be obtained from Pierce Biotechnology Inc. (Rockford, IL, USA).
  • Magnetic particles suitable for use in the methods and devices of the present invention are known to the skilled person.
  • Magnetic particles of different size (10 nm to 5 ⁇ m, typically between 50 nm and 1 ⁇ m, or between 250 nm and 750 nm) shape (spheres, spheroids, rods), composition, and magnetic properties (magnetic, paramagnetic, superparamagnetic, ferromagnetic, i.e. any form of magnetism which has a magnetic dipole in a magnetic field, either permanently or temporarily) have been described.
  • different types of magnetic particles e.g. with different magnetic and/or optical properties can be used simultaneously within one reaction chamber (magnetic particle multiplexing).
  • Attachment of the analytes or probes to the surface of magnetic particles can be performed by methods described in the art, as described above in this application.
  • the system uses one or more magnetic fields, each of which is generated by one or more magnetic field generating means.
  • magnetic field generating means are envisaged in the context of the present invention, such as permanent magnets, electromagnets, coils and/or wires.
  • the magnetic field(s) generated by the one or more magnetic field generating means can be constant, pulsating, or can vary in strength.
  • their exact orientation may be fixed or may vary, provided that the field gradient is essentially parallel to the detection surface or has at least a component parallel to the sensor region.
  • the magnetic field generating means is an electromagnet. This makes it possible to avoid mechanical moving of parts in the device. Alternatively permanent magnets may be arranged to move to and from the reaction chamber.
  • magnetic fields can be combined with other forces for moving magnetic particles.
  • forces envisaged in this context are other (non parallel) magnetic fields, electrical fields, acoustic forces, hydrodynamic forces, gravitational forces... etc.
  • the system of the present invention is a single-chamber (bio)sensor, with low reagent use and small required sample volume.
  • a (bio)sensor in accordance with the present invention comprises can be operated with a minimum of equipment, washing steps and buffers.
  • the first surface of the reaction chamber is typically completely occupied by the sensor region and this sensor region coincides with the region, which is used for detection. This allows performing an assay within one single chamber, which functions both a reaction chamber and detection chamber.
  • an optionally disposable cartridge comprising a reaction chamber having a first surface (3) and a second surface (4).
  • the first surface (3) comprises a sensor region (31), with immobilized thereto at least one of an analyte-specific probe, an analyte and an analyte-analogue.
  • the second surface (4) comprises a reagent region (41).
  • This reagent region (41) comprises, prior to use of the system, magnetic particles in dry form, non-covalently immobilized to the reagent region (31).
  • the sensor region (31) and the reagent region (41) are located on the opposite sides of the reaction chamber.
  • the cartridge is made of glass or a synthetic material, such as plexiglass [poly(methy)methacrylate] or clear PVC (polyvinyl chloride) or PC (polycarbonate) or COP (e.g. Zeonex) or PS (polystyrene).
  • plexiglass poly(methy)methacrylate
  • clear PVC polyvinyl chloride
  • PC polycarbonate
  • COP e.g. Zeonex
  • PS polystyrene
  • the system of the invention will usually comprise one or more inlet means for introducing sample or reagents into the reaction chamber, and optionally an outlet means for removing reagents, reaction waste, and optionally, magnetic particles, from the reaction chamber. These can optionally be coupled to sources comprising each of the reagents. Additionally or alternatively, one or more inlet and outlet means are provided so as to ensure the direct delivery of the sample and/or magnetic particles and/or other reagents or buffers in the reaction chamber.
  • the different inlet and/or outlet means can be connected to connection means such as valves and tubing, which can be driven by pumps.
  • a liquid sample suspected to contain an analyte is introduced into the reaction chamber of a system as described above.
  • the liquid sample used in the devices and methods of the present invention can be as diverse as environmental water samples, microbiological samples or samples of vegetable or animal (including human) origin. More particularly the sample is of human origin, such as urine, saliva, sweet, blood or plasma.
  • buffer components, sugar and carrier proteins dissolve and magnetic particles are redispersed.
  • This process can be enhanced by applying a magnetic mixing force when the sample enters the chamber or even prior to the entry of the same.
  • the use of mixing forces also improves the speed at which particles separate from the dried mass.
  • Magnetic mixing also enhances the rate at which a homogeneous solution with respect to buffer composition is obtained after sample fluid addition. This ensures that the assay is performed under buffered conditions that are relatively constant in time and optimal for binding.
  • Magnetic actuation can be accomplished for example by using an electromagnet consisting of two (ID683682) or three subunits (ID 678545) in one plane that allows the precise control of the x, y position of particles and a magnet above the sensor that allows the simultaneous control of the z-position.
  • magnetic particles bound to the sensor region of the reaction chamber are detected.
  • magnetic particles can be manipulated several times towards and away from the sensor region.
  • the system and methods described in the present invention can be used as rapid, robust, and easy to use point-of-care biosensors for small sample volumes.
  • the reaction chamber can be a disposable item to be used with a compact reader, containing the one or more magnetic field generating means and one or more detection means.
  • Methods as described in the present invention offers three key advantages over the current state of the art: a sensitivity in the picomolar range, an assay time of less than 5 minutes and a reaction which is performed using sample volumes less than 30 ⁇ l.
  • the assays are performed in undiluted plasma, serum or even whole blood.
  • Example 3 Influence of dried or suspended magnetic particles on assay sensitivity.
  • An experiment as described in example 1 is repeated whereby in one experimental setting the magnetic particles with the antibody are mixed with a buffer or with plasma, where after the sample is introduced in the reaction chamber (indicated as "wet” in Fig. 4).
  • a same amount of magnetic particles is dried on the reagent region opposite from the sensor region ((indicated as "dry” in Fig. 4), where after the buffer or sample is introduced in the reaction chamber.
  • Fig. 4 shows that the sensitivity of an assay is increased when the magnetic particles are provided in dry form in the reaction chamber prior to the introduction of the sample. This increased sensitivity is even more pronounced for a sample comprising plasma.

Abstract

La présente invention concerne un système de biodétecteur permettant de réaliser des immunodétections biologiques et comprenant une enceinte réactionnelle comportant deux surfaces se faisant face. A l'intérieur, la première surface comporte une région servant à la détection sur laquelle sont immobilisés une sonde spécifique d'un analyte, un analyte ou un analogue d'un analyte. La seconde surface comprend une région réactive, faisant face à la région servant à la détection, la région réactive comprenant, préalablement à l'utilisation dudit système, des particules magnétiques sous forme sèche, disposées à la surface de ladite région réactive (31).
PCT/IB2010/050289 2009-01-29 2010-01-22 Système et procédé de détection WO2010086772A1 (fr)

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WO2013001461A1 (fr) 2011-06-30 2013-01-03 Koninklijke Philips Electronics N.V. Préparation de chambres réactionnelles avec des protéines sèches
WO2013102850A1 (fr) * 2012-01-04 2013-07-11 Insituto De Engenharia De Sistemas E Computadores Para Os Microsistemas E As Nanotecnologias Dispositif monolithique combinant des capteurs cmos et magnétorésistifs
CN103344753A (zh) * 2013-07-24 2013-10-09 公安部第三研究所 基于磁免疫分析技术实现毒品含量快速检测的装置
EP2664914A1 (fr) * 2012-05-16 2013-11-20 Koninklijke Philips N.V. Outil de forage
US8895320B2 (en) 2009-11-16 2014-11-25 Silicon Biodevices, Inc. Filtration device for assays
CN107407675A (zh) * 2015-03-26 2017-11-28 皇家飞利浦有限公司 生物传感器盒的制造

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US8895320B2 (en) 2009-11-16 2014-11-25 Silicon Biodevices, Inc. Filtration device for assays
US9244068B2 (en) 2009-11-16 2016-01-26 Silicon Biodevices, Inc. Filtration device for assays
WO2013001461A1 (fr) 2011-06-30 2013-01-03 Koninklijke Philips Electronics N.V. Préparation de chambres réactionnelles avec des protéines sèches
WO2013102850A1 (fr) * 2012-01-04 2013-07-11 Insituto De Engenharia De Sistemas E Computadores Para Os Microsistemas E As Nanotecnologias Dispositif monolithique combinant des capteurs cmos et magnétorésistifs
US9567626B2 (en) 2012-01-04 2017-02-14 Magnomics, S.A. Monolithic device combining CMOS with magnetoresistive sensors
EP2664914A1 (fr) * 2012-05-16 2013-11-20 Koninklijke Philips N.V. Outil de forage
WO2013171600A1 (fr) * 2012-05-16 2013-11-21 Koninklijke Philips N.V. Traitement assisté par champ magnétique d'un milieu
CN104285143A (zh) * 2012-05-16 2015-01-14 皇家飞利浦有限公司 对介质的磁辅助处理
CN103344753A (zh) * 2013-07-24 2013-10-09 公安部第三研究所 基于磁免疫分析技术实现毒品含量快速检测的装置
CN107407675A (zh) * 2015-03-26 2017-11-28 皇家飞利浦有限公司 生物传感器盒的制造
CN107407675B (zh) * 2015-03-26 2020-03-17 皇家飞利浦有限公司 生物传感器盒的制造
US10690663B2 (en) 2015-03-26 2020-06-23 Koninklijke Philips N.V. Manufacturing of a biosensor cartridge

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