WO2010086772A1

WO2010086772A1 - System and method for assay

Info

Publication number: WO2010086772A1
Application number: PCT/IB2010/050289
Authority: WO
Inventors: Wendy U. Dittmer; Wilhelmina M. Hardeman
Original assignee: Koninklijke Philips Electronics N.V.
Priority date: 2009-01-29
Filing date: 2010-01-22
Publication date: 2010-08-05

Abstract

The invention relates to a biosensor system for performing biological binding assays, comprising a reaction chamber with two opposing surfaces. Herein, the first surface comprises a sensor region, and immobilized thereto an analyte-specific probe, an analyte or an analyte-analogue. The second surface comprises a reagent region, opposite of the sensor region, the reagent region comprising, prior to the use of said system, magnetic particles in dry form, disposed on said reagent region (31).

Description

SYSTEM AND METHOD FOR ASSAY

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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. Herein 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 however 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. In dry form 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.

This clustering is disadvantageous when quantitative detection using magnetic particles is intended. Quantitative detection requires that particles be as close to monodisperse as possible (< 5 in a cluster). Clusters that are formed upon redispersion, will behave also as clusters upon detection and may even increase in size upon actuation.

SUMMARY OF THE INVENTION It is an object of the invention to reduce the above described disadvantageous property.

The invention is defined by the independent claims. The dependent claims define advantageous embodiments.

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). Herein, 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. In the system 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. In any case 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-specifϊc probe.

The device and system are suitable for use in another type competitive immunoassay, wherein the sensor region comprises an analyte-specifϊc 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.

Preferably, 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. Thus, the reagent region may have a larger area than the sensor region or vice versa. Alternatively or additionally, the area of the regent region may have a shape different from the area of the sensor region. Thus, 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. In embodiments, the system further comprises means for magnetic actuation (5) of the magnetic particles. These may include one or more permanent and or electromagnets disposed under and/or above and/or aside the sensor region and/or the reagent region such that they are able to exert magnetic forces on the magnetic particles during the use of the system. According to one embodiment of the invention 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.

In particular embodiments the buffer is a phosphate buffer, the sugar is sucrose and the carrier protein is BSA (Bovine Serum Albumin). In embodiments the first and second surface are planar surfaces separated from each other by about 500 μm.

In embodiments the first and second surface are planar surfaces, parallel to each other.

In embodiments the first surface (3) and the sensor region (31) have the same size.

According to certain embodiments, the reagent region has a surface of between 0.5 and 2.0 mm², e.g. 1 mm².

According to certain embodiments the volume of the reaction chamber is between 0.1 and 5 μl, more particularly between 0.5 and 2μl, e.g. 1 μl. In embodiments the magnetic particles have a diameter of between 250 and

750 nm, more particularly between 400 and 600 nm .

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. In an embodiment of the invention, the system further comprises a detection means (6) for detection at least one magnetic particle on the sensor region.

In another embodiment of the invention, 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. 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. In embodiments of this method 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.

In this method, the drying is performed by lyophilization. In particular embodiments, the magnetic particles have an average diameter of between 250 and 750 nm, more particularly between 400 and 600 nm, e.g. 500 nm. In particular embodiments, the magnetic particles are applied at the surface at a density of between 10 to 50 μg per mm².

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.

According to one embodiment of this method, the detection is based on an optical property of the magnetic particles, for example by using frustrated total internal reflection technique (FTIR).

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.

The present invention describes 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.

BRIEF DESCRIPTION OF THE FIGURES

The following Figures show embodiments of the present invention in schematic form. Fig. 1 shows a system of the present invention, illustrated for a sandwich assay.

1 : system; 2: reaction chamber; 3: first surface; 31 sensor region; 4: second surface; 41 : reagent region; means for magnetic actuation; detection means; M: magnetic particle; squares and vertical lines: analyte specific probes

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 μg 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.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention will hereafter be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. The terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The terms or definitions used herein are provided solely to aid in the understanding of the invention.

The term "analyte" as used herein refers to a compound in a sample of which the detection of presence and or/ concentration is desired.

The term "analyte-analogue" as used herein 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

The term "analyte-specific probe", as used herein, refers to a compound which can bind with the analyte or the analyte analogue.

The term "reaction chamber" as used herein refers to a region within a device or a cartridge, where different reagents taking part in a reaction are contacted with each other. The term "sensor region", as used herein, refers to the part of the reaction chamber, wherein probes, such as analyte-specifϊc probes are bound or immobilized. Generally, it is also the area where the most important sensitive detection takes place.

The term "reagent region", as used herein, refers to the part of the reaction chamber, where prior to assay magnetic particles are deposited.

The term "buffer" as used herein refers to compounds that have a pH stabilizing effect.

The invention comprises a device comprising a reaction chamber (2) with a first surface (3) and a second surface (4). Herein, 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). The sensor region and the reagent region are located on opposite sides of the reaction chamber This reagent region (41) comprises, prior to use of the system, magnetic particles in dry form, non- covalently immobilized to the reagent region (41). The device may be and generally is supplemented by a sample inlet and channel for supply to the reaction chamber, which are not shown for convenience. These may for example be situated such that the sample is supplied from the side between the first and second surfaces. There may also be an exit channel located on a different or same side of the reaction chamber for removing sample after or washing fluids before or during assay from the reaction chamber. Such channels may have microfluidic dimensions to facilitate small volume sample handling. 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. On the other hand, if organic solvents or other reagents are to be used during assay, then 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. In order to provide the magnetic particles in dry form, 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. Alternatively 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.

Deposition of magnetic particles and subsequent drying may require particular conditions. Immobilized magnetic particles that are in close proximity with another for a long time have a tendency to irreversibly aggregate as well as to adhere irreversibly to the substrate on which they were dried. Accordingly in particular embodiment magnetic particles are applied and dried separately from the other reagents, as one can specifically choose the drying reagents that are optimal to co -deposit with the magnetic particles such that the particles easily redisperse upon fluid addition. Conjugate-particles containing antibodies (for example anti-Troponin antibodies) redisperse rapidly, are monodisperse and detach completely from both hydrophilic and hydrophobic reagent regions (e.g. polystyrene), when they are dried in a buffered solution, comprising a sugar and a carrier protein. The solution used for applying magnetic particles to the reagent region comprise buffer components, a sugar and a so-called carrier protein, to increase stability and the overall shelf- life of the protein, such as BSA pH buffering components are typically present in a molar concentration of between 1 and 100 mM at a pH between 5 and 10, and include but are not limited to Tris or phosphate buffers (or other biological buffers such as (MES, Bis-Tris, ADA, Aces, PIPES, MOPSO, Bis-Tris Propane, BES, TES, HEPES, DIPSO, MOBS, TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS, Tricine, Gly-Gly Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO). Sugars are typically present in a concentration between 1 and 25 % (w/v), between 1 and 10 % (w/v), or between 2.5 and 7.5 % (w/v). Suitable sugars include but are not limited to sucrose and trehalose. Carrier proteins are typically present in a concentration between 1 and 10 % (w/v) or between 2.5 and 7.5 % (w/v). Suitable carrier proteins are for example albumin, casein or gelatin. A particular buffered composition comprises 5X PBS (Phosphate Buffer

Saline, i.e. to 685 mM NaCl, 2.7 mM KCl, 50 mM phosphate, pH 7.4), 5 % (w/v) BSA, and 5 % (w/w) sucrose. These buffered compositions allow the redispersion of the dried magnetic particles later on in a variety of fluids, such as PBS, serum, plasma and blood. In a particular embodiment, magnetic particles are applied at the reagent region at a concentration of between 1 to 40 μg per μl. The solution used to apply the magnetic particles typically has a volume that is equal to, or is 5, 10, 20, 50 or 100 times smaller than the volume of the reaction chamber. It will be appreciated by the skilled person, that the buffer, sugar and carrier protein concentration in solution used to apply the magnetic particles is adjusted depending on the volume of the reaction chamber of the device.

An amount of 1 to 40 μg of magnetic particles can be applied in an area as small as 1 mm² and can be redispersed in a volume as small as 1 μl. A high concentration of particles (40 μg) is particularly suitable for applications in which a high concentration of analyte is to be measured (InM) whereas concentrations of approximately 5 μg and less (2 or 1 μg) 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)).

With the localization of the particles on a reagent region, opposite from the sensor region, the labels only have to traverse a small and defined distance to the sensor region, which improves the reproducibility of an assay. Moreover, in particular embodiment, one can arrange the particles to be deposited on the reagent region and actuate the particles such that there is minimal lateral motion of particles over the detection area. This reduces the chance that particles that are already bound become displaced by a laterally moving particle. A configuration of the device wherein the first and the second surface are parallel to each other has the advantage that the particles upon manipulation are transported over the same distance towards and backwards from the sensor region regardless from their position on the reagent region. This is particularly advantageous for multiplexing assays.

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. Preferably the flat top and bottom parts comprising the sensor and reagent regions respectively are parallel to each other. Generally, such reaction chambers have a volume between 0.1 and 10 μl, and the bottom and top part are separated from each other by a distance ranging from 100 to 500 μm. Other configurations of reaction chambers may have grooves, ridges or protruding posts. Alternatively 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 method of the invention can also be performed in a flow-through system with devices or cartridges made of porous aluminum oxide, porous silicon, or a porous column containing microparticles. When such material is used, methods of the invention can also be performed by applying magnetic particles at a region of the cartridge opposite to the region where probe, or analyte(analogue) is applied. The sensor region is typically a specially derivatized surface to which molecules, more particularly probes can be immobilized by eg binding. Examples of suitable surfaces include glass, metal, plastic, an organic crystal or an inorganic crystal (e. g. silicon), an amorphous organic or an amorphous inorganic material (e. g. silicon nitride, silicon oxide, silicon oxinitride, aluminum oxide). Suitable surface materials and linking chemistries are known to the person skilled in the art, and are described for instance in "Diagnostic

Biosensor Polymers" , by A. M. Usmani and N. Akmal, American Chemical Society, 1994 Symposium Book Series 556, Washington DC, USA, 1994, in "Protein Architecture, Interfacing Molecular Assemblies and Immobilization Biotechnology" , edited by Y. Lvov and H.Mhwald (Marcel Dekker, New York, 2000), in "The Immunoassay Handbook" by David Wild (Nature Publishing Group, London, 2001, ISBN 1-56159-270-6) or "Handbook of Biosensors and Electronic Noses. Medicine, Food and the Environment" by Kress-Rogers (ISBN 0-8493-8905-4). Supports for coupling proteins to coated and uncoated plastic and glass supports are disclosed in Angenendt et al. (2002) Anal Biochem. 309, 253-260. Dufva (2005) in Biomol Eng 22, 173-184, review the methodology to attach oligonucleotides and factors influencing this process.

Systems and methods of the present invention are applicable in the detection of any molecule, more particularly biomolecules such as DNA, RNA, proteins, carbohydrates, lipids and organic anabolites or metabolites. The nature of the sample comprising the analyte to be detected is not critical and can be for instance any sample of a living or dead organism (body fluid such as, but not limited to blood or urine, hair, stool, etc.), an environmental sample (water, soil, plant material), food or feed products or products used in the manufacturing thereof, a sample of a chemical reaction process etc...The detection can be performed on a sample which is the result of a pre-processing step such as a semi-purification, purification, semi-purification and/or amplification of the analyte. The methods of the present invention provide for a detection of an analyte based on the reaction/binding of the analyte with an analyte-specific probe. Typical specific interactions include DNA/DNA or DNA/RNA binding, protein/protein, protein/DNA and protein/carbohydrate interactions antibody/ antigen interactions, receptor/ligand binding. Also synthetic molecules can be used to detect an analyte (e.g. enzyme inhibitors, pharmaceutical compounds, lead compounds isolated from library screenings). Accordingly, examples of analyte-specific probes include but are not limited to oligonucleotides, antibodies, enzyme substrates, receptor ligands, lectins etc... For example, the analyte- specific probe is an analyte-specific oligonucleotide, i.e. an oligonucleotide comprising a sequence which is complementary to a sequence specific for the analyte.

According to a particular embodiment of the present invention, the system is used 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. According to another particular embodiment of the present invention, the system is used 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-specific probe. In an alternative setting of a competitive immunoassay, the sensor region comprises an analyte-specific probe and the magnetic particles are bound to an analyte or analyte analogue.

According to yet another particular embodiment, 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). According to this embodiment, different sensor regions (31) are present on the first surface (3), comprising different analyte specific probes, analytes or analyte analogues. At the corresponding reagent regions (41) on the second surface, appropriate magnetic particles are present. In this type of multiplexing methods, 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.

Another modification is chamber multiplexing. Herein a system has multiple reaction chambers. In one embodiment, the sample is spread over different chambers to run different assays in parallel. In such configurations, optionally 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. Accordingly, 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.). These may be generated by liposomes, micelles, bubbles, microbubbles, microspheres, lipid-, or polymer coated bubbles, microbubbles and/or microspheres, microballoons, aerogels, clathrate bound vesicles, and the like. 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.

According to a particular embodiment, 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. Alternatively, the sensor is provided as separate part from the reaction chamber (e.g. optical unit). In this embodiment, 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. Alternatively, 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. In particular embodiments, 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. When optical detection methods are detected 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. Thereto, and with reference to Fig 1 of WO/2008/072156, 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.

Magnetic particles are widely used in biological analysis, e. g. in high- throughput clinical immunoassay instruments, sample purification, cell extraction, etc. Several 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. For attachment or binding of the particles of the invention to the bioactive molecules, 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. 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 and 4654267. In the case of a preferred type of particle, the surface carries-OH groups connected to the polymeric backbone through (CH₂CH₂O) 8-10 linkages. Other preferred carry -COOH groups obtained through polymerization of methacrylic acid. For example, the NH₂ 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. Similarly, 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. Examples of such 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).

The 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 5μm, typically between 50 nm and 1 μm, 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. It is envisaged that 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.

According to the present invention, the system uses one or more magnetic fields, each of which is generated by one or more magnetic field generating means. Different types of magnetic field generating means are envisaged in the context of the present invention, such as permanent magnets, electromagnets, coils and/or wires. According to the present invention, the magnetic field(s) generated by the one or more magnetic field generating means can be constant, pulsating, or can vary in strength. Moreover, where more than one magnetic field is generated, 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.

According to a particular embodiment, 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.

In addition to magnetic fields can be combined with other forces for moving magnetic particles. Examples of other forces envisaged in this context are other (non parallel) magnetic fields, electrical fields, acoustic forces, hydrodynamic forces, gravitational forces... etc.

Typically 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. For this purpose 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.

Another aspect of the present invention provides an optionally disposable cartridge comprising a reaction chamber having a first surface (3) and a second surface (4). Herein, 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). Herein, the sensor region (31) and the reagent region (41) are located on the opposite sides of the reaction chamber.

For example, 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).

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.

Another aspect of the present invention relates to a method for quantifying and/or detecting an analyte. Herein, 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. Upon entry of the sample in the reaction chamber, 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.

Hereafter the magnetic articles are manipulated to bind with the analyte and to interact with the sensor region. 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.

After removal of unbound magnetic particles, the magnetic particles bound to the sensor region of the reaction chamber are detected. In the methods of the present invention, 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.

Also, the systems and methods of the present invention can be used in automated high-throughput testing for centralized laboratories. In this case, the reaction chamber is e.g. a cuvette, fitting into an automated instrument. When applied in e.g. immunoassays, a minimum number of fluid manipulation steps are needed, incubation occurs at high speed and washing steps are reduced to a minimum with a minimal fluid waste.

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 μl. In particular embodiments, the assays are performed in undiluted plasma, serum or even whole blood.

Other arrangements for embodying the invention will be obvious for those skilled in the art. It is to be understood that although preferred embodiments, specific constructions and configurations, as well as materials, have been discussed herein for devices and methods according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention.

The invention is illustrated by the Examples provided below which are to be considered for illustrative purposes only and the invention is not limited to the specific embodiments described therein.

Example 1 :

Detection of cTnl using 500 nm magnetic particles coated with goat polyclonal anti-cTnl (Hytest) in a dry form.

The reaction chamber described in the examples has a box-like shape with an internal volume of approximately 1 μl. This reaction chamber has a transparent polystyrene region for detecting magnetic particles by FTIR. A monoclonal antibody against cardiac Troponin I (M 16/560 Hytest) was immobilized on the polystyrene sensor region. A buffer of 1 μl with 3-15 μg magnetic particles (500 nm diameter) coated with goat polyclonal anti- cTnl (Hytest) was applied on the reagent region positioned opposite of the sensor region. This buffer contained 5 x concentrated PBS, 5 % (w/v) BSA and 5 % (w/v) sucrose. The liquid was applied at one surface of an open cartridge and allowed to dry initially in ambient air and later under conditions which lower the ambient humidity (e.g. vacuum chamber) Reagent and sensor region are separated in the reaction chamber from each other by about 500 nm.

A sample (plasma) containing the analyte cTnl in a concentration between 100 and 1000 pg was added to the above prepared reaction chamber. Upon addition of the buffer with the analyte the particles were mixed for 1 minute using an electromagnet consisting of 2 subunits in a plane beneath the cartridge surface and 1 subunit above the cartridge. Subsequently the particles were attracted with a magnetic force to the sensor region where the labeled antibody with the analyte bound to the antibody on the sensor region. Unbound particles were removed by reversing the magnetic force. Bound particles were detected with a frustrated total internal reflection technique.

Fig. 2 shows the detection of magnetic particles at the sensor region using different amounts of analyte and magnetic particles. Herein the detection is performed by Frustrated Total Internal Reflection. Light is reflected at the bottom site of the binding area, which creates an evanescent field in the binding area. The reflected light is measured at the detector. If no beads are present, all light is reflected (100 %), the decrease in signal as indicated in Fig. 2 is an indication of bound particles.

Example 2:

Detection of cTnl using 500 nm magnetic particles coated with goat polyclonal anti-cTnl (Hytest) in a dry form. The experiment as described in Example 1 is repeated, whereby Troponin was added in varying concentrations to plasma, buffered with PBS. 10 μg of the above mentioned magnetic particles were used in this experiment. The results (performed in triplicate or duplicate) are shown in Table 2 and in Fig. 3. The dotted line in Fig. 3 shows the background signal obtained in the absence of analyte and the width of the grey area is 2x the error in the blank measurement.

Table 2. Detection of Troponin in plasma

CV: variation as a percentage of the mean These results show that the analyte can be reproducibly detected in a buffered plasma sample at a concentration as low as about 10 pM.

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). In the other setting, 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.

Claims

CLAIMS:

1. A device (1) for performing biological assays, comprising a reaction chamber (2) with a first surface (3) and a second surface (4),wherein 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, and wherein the second surface (4) comprises a reagent region (41), in which prior to performance of the biological assay, magnetic particles in dry form are disposed and wherein the sensor region (31) and the reagent region (41) are located on opposite sides of the reaction chamber (2).

2. The device according to claim 1, wherein said 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 10OmM of buffer, 1 to 25% (w/v) of sugar and 1 to 10 % (w/v) of carrier protein.

3. The device according to claim 2, wherein the buffer is phosphate buffer, the sugar is sucrose and the carrier protein is Bovine Serum Albumin.

4. The device according to any of claims 1 to 3, wherein said magnetic particles have a diameter of between 250 and 750 nm.

5. The device according to any of claims 1 to 4, wherein the sensor region and the reagent region are parallel to each other.

6. The device according to any claims 1 to 5, wherein said first and second surface are planar surfaces that are parallel to each other at least at the sensor region and the reagent region.

7. The device according to any of claims 1 to 6, wherein the sensor region and the reagent region have the same areal size.

A system comprising the device according to any one of the previous claims.

9. The system according to claim 8, further comprising a detection means (6) for detection at least one magnetic particle on the sensor region.

10. The system according to claim 9 wherein said detection means are optical detection means, the device comprises windows for the optical detection radiation and the detection means are for detecting an optical property of said at least one magnetic particle.

11. The system according to claims 8 9 or 10, comprising means for magnetic actuation (5) of said magnetic particles.

12. A method for preparing a device according to claim 1 the method comprising the steps of: - providing a surface (4), free of bound analyte, analyte-analogue or analyte- specifϊc probe,- applying to a reagent region (41) on said surface (4) a solution comprising magnetic particles, drying said solution, assembling said surface (4) with dried solution into the reaction chamber (2), wherein after assembly said reagent region (41) on said surface (4) is positioned opposite the sensor region (31) of the sensor region (3) of said reaction chamber.

13. The method according to claim 12 wherein said solution further comprises a buffer, a sugar and a carrier protein, their concentration being adapted to the volume of said reaction chamber, to provide upon redispersion during use of the device 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.

14. The method according to claims 12 or 13, wherein said magnetic particles are applied at said surface at a density of between 10 to 50 micrograms per square millimeter.

15. 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 claim 1 , 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.