WO2009039239A2

WO2009039239A2 - Lateral flow assay using centrifugal force

Info

Publication number: WO2009039239A2
Application number: PCT/US2008/076788
Authority: WO
Inventors: Charles Carpenter; Giosi Farace; Robert Farrell; Keith Nassif
Original assignee: Idexx Laboratories, Inc.
Priority date: 2007-09-18
Filing date: 2008-09-18
Publication date: 2009-03-26
Also published as: WO2009039239A3

Abstract

An apparatus, device, and method for laterally moving or assisting in the lateral movement of at least one of a liquid sample and a reagent along a porous carrier matrix is disclosed. The method comprises contacting the matrix with the at least one of the sample and the reagent and exposing the at least one of a liquid sample and a reagent to a centrifugal force, thereby moving or assisting in the lateral movement of the at least one of a liquid sample and a reagent along the porous carrier matrix.

Description

Lateral Flow Assay Using Centrifugal Force CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 60/994,220, filed September 18, 2007, which is incorporated herein by reference in its entirety.

BACKGROUND OF TBDE INVENTION

Field of the Invention

[0002] In general, the invention relates to the detection of analytes in samples. More specifically, the invention relates to the detection of analytes using rotational forces applied to lateral flow devices.

Description of Related Art

[0003] Various analytical procedures and devices are commonly employed in specific binding assays to determine the presence and/or amount of substances of interest or clinical significance which may be present in biological or non-biological fluids. Such substances are commonly termed "analytes" and can include antibodies, antigens, drugs, hormones, etc.

[0004] The ability to use materials which specifically bind to an analyte of interest has created a burgeoning diagnostic device market based on the use of binding assays. Binding assays incorporate specific binding members, typified by antibody and antigen immunoreactants, wherein one member of the specific binding pair is labeled with a signal-producing compound (e.g., an antibody labeled with an enzyme, a fluorescent compound, a chemiluminescent compound, a radioactive isotope, a direct visual label, etc.). For example, in a binding assay the test sample suspected of containing analyte can be mixed with a labeled anti-analyte antibody (an example of a conjugate reagent), and incubated for a period of time sufficient for the immunoreaction to occur. The reaction mixture is subsequently analyzed to detect either that label which is associated with an analyte/conjugate complex (bound conjugate) or that label which is not complexed with analyte (free conjugate). As a result, the amount of label in one of these species can be correlated to the amount of analyte in the test sample. [0005] The solid phase assay format is a commonly used binding assay technique. There are a number of assay devices and procedures wherein the presence of an analyte is indicated by the analyte's binding to a conjugate and/or an immobilized complementary binding member. The immobilized binding member is bound, or becomes bound during the assay, to a solid phase such as a dipstick, test strip, flow- through pad, paper, fiber matrix or other suitable solid phase material. The binding reaction between the analyte and the assay reagents results in a distribution of the conjugate between that which is immobilized upon the solid phase and that which remains free. The presence or amount of analyte in a test sample is typically indicated by the extent to which the conjugate becomes immobilized upon the solid phase material.

[0006] The use of reagent-impregnated test strips in specific binding assays is also well-known. In such procedures, a test sample is applied to one portion of the test strip and is allowed to migrate or wick through the strip material. Thus, the analyte to be detected or measured passes through or along the material, possibly with the aid of an eluting solvent which can be the test sample itself or a separately added solution. The analyte migrates into a capture or detection zone on the test strip, wherein a complementary binding member to the analyte is immobilized. The extent to which the analyte becomes bound in the detection zone can be determined with the aid of the conjugate which can also be incorporated in the test strip or which can be applied separately.

[0007] Other detection technologies employ magnetic particles or microbeads, for example, such as superparamagnetic iron oxide impregnated polymer beads. These beads are associated with, for example, a specific binding partner for the analyte. The beads bind with the target analytes in the sample being tested and are then typically isolated or separated out of solution magnetically. Once isolation has occurred, other testing may be conducted, including observing particular images or labels, whether directly optically or by means of a camera.

[0008] Despite their cost-effectiveness and simplicity of use, typical lateral flow assays may be time consuming as a result of the time associated with the lateral flow of sample and reagents along a porous carrier matrix. Accordingly, the inventors have identified a need in the art for way to decrease the time required to perform an assay using such devices.

SUMMARY OF THE INVENTION

[0009] In one aspect, the invention is directed to a method for affecting the lateral movement of at least one of a liquid sample and a reagent along a porous carrier matrix. The method includes contacting the matrix with a sample and/or reagent and exposing the sample and/or reagent to a centrifugal force. The porous carrier matrix may be associated with a lateral flow immunoassay device.

[0010] In another aspect, the invention is directed to a method for determining the presence or amount of an analyte in a liquid sample. The method includes contacting the liquid sample with an application zone of a porous carrier matrix. The matrix includes a detection zone laterally spaced from the application zone. Centrifugal force is used to move or assist in the movement of the liquid sample from the application zone to the detection zone. The presence or amount of the analyte in the liquid sample is determined in the detection zone.

[0011] Optionally, the detection zone may be populated with an analyte binding partner prior to contacting the liquid sample with the sample application zone. In this aspect, an analyte specific binding reagent is contacted with the application zone and centrifugal force is used to move or assist in the movement of the reagent from the application zone to the detection zone. The reagent may also include the analyte binding partner bound to magnetic particles and the detection zone may be associated with a magnet.

[0012] In another aspect, the invention is directed to an apparatus for detecting analytes in liquid samples. The apparatus includes a rotary system arranged to provide centrifugal force to a liquid sample on a porous carrier matrix of a lateral flow device and a detection instrument that detects a signal generated by a label associated with the analyte or a reagent in a detection zone of the matrix.

[0013] In a further aspect, the invention is directed to a method for determining the presence or amount of an analyte in a liquid sample. The method includes forming a mixture of a sample and a reagent of a complex comprising a magnetic nanoparticle bound to a metal colloid wherein at least one of the nanoparticle or the colloid includes an analyte-specific binding partner, and the colloid includes a first label attached to the colloid. The mixture is applied to a device having a porous carrier matrix with a detection zone including a magnet. The matrix has an average pore size that allows for the substantially unimpeded lateral flow of the magnetic particles. The matrix is contacted with a conjugate reagent including a second label bound to a second analyte-specific binding partner or an analog of the analyte. The mixture and/or the conjugate reagent are exposed to centrifugal force to affect the movement of the mixture and/or the conjugate reagent from one or more zones for the application of the mixture and/or reagent to the detection zone. A signal is simultaneously or sequentially detected from the first label and the second label in the detection zone to determine the presence or amount of the analyte in the sample.

[0014] In an even further aspect, the invention is directed to an apparatus for detecting analytes in one or more liquid samples. The apparatus includes a rotor for mounting and rotating one or more lateral flow devices and a detection instrument operable to detect a signal generated by a label associated with the analyte or a reagent in a detection zone on the device.

[0015] The invention also includes a method for lyophilizing antibody functionalized paramagnetic particles. The method includes freezing the paramagnetic particles at approximately -40 degrees Celsius; and drying the paramagnetic particles at approximately 40 to 45 degrees Celsius, wherein the drying time is at least about 72 hours thereby producing stable cakes of lyophilized paramagnetic particles.

BRIEF DESCRIPTION OF THE FIGURES

[0016] Figure 1 is a block diagram showing a lateral flow device according to an example embodiment of the invention.

[0017] Figure 2 is a block diagram showing an apparatus for providing centrifugal force to a sample or reagent according to an example embodiment of the invention.

[0018] Figure 3 is an illustration of an apparatus for providing centrifugal force to a sample or reagent according to an example embodiment of the invention. [0019] Figure 4 is another illustration of an apparatus for providing centrifugal force to a sample or reagent according to an example embodiment of the invention.

[0020] Figure 5 is a graph showing test results from an apparatus for providing centrifugal force to a reagent according to an example embodiment of the invention.

[0021] Figure 6 is a graph showing results of an assay for Canine Pancreatic Lipase cPL using an apparatus for providing centrifugal force according to an example embodiment of the invention.

[0022] Figure 7 is another illustration of an apparatus for detecting analytes in one or more liquid samples.

DETAILED DESCRIPTION

[0023] The invention provides apparatus and methods for conducting assays using the principle of lateral flow through a porous carrier matrix for the qualitative or quantitative analysis of selected analytes in samples. In several aspects, the invention is directed to an apparatus and a method for use in a lateral flow assay wherein the lateral flow is affected by using the principle of centrifugal force. For instance, centrifugal force can initiate, maintain, accelerate, prohibit, inhibit or stop lateral flow. The invention can be used for a wide variety of assays, both ligand-based and non-ligand-based. Applicable ligand-based methods include, but are not limited to, competitive immunoassays, non-competitive or so-called sandwich technique immunoassays, and blocking assays. The use of the invention is not limited to any particular analyte. The embodiments described herein are solely for illustrative purposes and are not intended to limit the scope of the invention to any particular set of binding partners or assay format.

[0024] Before describing the present invention in more detail, a number of terms will be defined. As used herein, the singular forms "a," "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0025] By "analyte" is meant a molecule or substance to be detected. For example, an analyte, as used herein, may be a ligand, which is mono- or polyepitopic, antigenic or haptenic; it may be a single compound or plurality of compounds that share at least one common epitopic site; it may also be a receptor or an antibody. [0026] A "sample" refers to an aliquot of any matter containing, or suspected of containing, an analyte of interest. For example, samples include biological samples, such as samples taken from animals (e.g., saliva, whole blood, serum, and plasma, urine, tears and the like), cell cultures, plants, etc.; environmental samples (e.g., water); and industrial samples. Samples may be required to be prepared prior to use in the methods of the invention. For example, samples may require diluting, filtering, centrifuging or stabilizing prior to use with the invention. For the purposes herein, "sample" refers to the either the raw sample or a sample that has been prepared.

[0027] A "reagent" refers to a substance that participates in a chemical reaction or physical interaction. A reagent can comprise an active component, that is, a component that directly participates in a chemical reaction (e.g. covalent binding) or physical interaction (e.g. non-covalent binding), such as a labeled conjugate, and other materials or compounds directly or indirectly involved in the chemical reaction or physical interaction. It can include a component inert to the chemical reaction or physical interaction, such as catalysts, stabilizers, buffers, and the like.

[0028] "Binding specificity" or "specific binding" refers to the substantial recognition of a first molecule for a second molecule, for example a polypeptide and a polyclonal or monoclonal antibody, an antibody fragment (e.g. a Fv, single chain Fv, Fab', or F(ab')2 fragment) specific for the polypeptide, enzyme—substrate interactions, and polynucleotide hybridization interactions.

[0029] "Non-specific binding" refers to non-covalent binding between molecules that is relatively independent of specific surface structures. Non-specific binding may result from several factors including electrostatic and hydrophobic interactions between molecules.

[0030] "Member of a specific binding pair" or "specific binding partner" refers one of two different molecules, having an area on the surface or in a cavity which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule. The members of the specific binding pair are referred to as ligand and receptor (antiligand). These will usually be members of an immunological pair such as antigen-antibody, although other specific binding pairs such as biotin-avidin, hormones-hormone receptors, IgG-protein A, polynucleotide pairs such as DNA-DNA, DNA-RNA, and the like are not immunological pairs but are included in the invention and the definition of specific binding pair member.

[0031] "Analyte-specific binding partner" refers to a specific binding partner that is specific for the analyte.

[0032] "Substantial binding" or "substantially bind" refer to an amount of specific binding or recognizing between molecules in an assay mixture under particular assay conditions. In its broadest aspect, substantial binding relates to the difference between a first molecule's incapability of binding or recognizing a second molecule, and the first molecules capability of binding or recognizing a third molecule, such that the difference is sufficient to allow a meaningful assay to be conducted distinguishing specific binding under a particular set of assay conditions, which includes the relative concentrations of the molecules, and the time and temperature of an incubation. In another aspect, one molecule is substantially incapable of binding or recognizing another molecule in a cross-reactivity sense where the first molecule exhibits a reactivity for a second molecule that is less than 25%, preferably less than 10%, more preferably less than 5% of the reactivity exhibited toward a third molecule under a particular set of assay conditions, which includes the relative concentration and incubation of the molecules. Specific binding can be tested using a number of widely known methods, e.g., an immunohistochemical assay, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), or a western blot assay.

[0033] "Ligand" refers any organic compound for which a receptor naturally exists or can be prepared.

[0034] "Analyte analog" or an "analog of the analyte" refers to a modified form of the analyte which can compete with the analyte for a receptor, the modification providing means to join the analyte to another molecule. The analyte analog will usually differ from the analyte by more than replacement of a hydrogen with a bond that links the analyte analog to a hub or label, but need not. The analyte analog can bind to the receptor in a manner similar to the analyte.

[0035] "Receptor" refers to any compound or composition capable of recognizing a particular spatial and polar organization of a molecule, e.g., epitopic or determinant site. Illustrative receptors include naturally occurring receptors, e.g., thyroxine binding globulin, antibodies, enzymes, Fab fragments, lectins, nucleic acids, protein A, complement component CIq, and the like.

[0036] "Antibody" refers to an immunoglobulin that specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of another molecule. The antibody can be monoclonal or polyclonal and can be prepared by techniques that are well known in the art such as immunization of a host and collection of sera (polyclonal) or by preparing continuous hybrid cell lines and collecting the secreted protein (monoclonal), or by cloning and expressing nucleotide sequences or mutagenized versions thereof coding at least for the amino acid sequences required for specific binding of natural antibodies. Antibodies may include a complete immunoglobulin or fragment thereof, which immunoglobulins include the various classes and isotypes, such as IgA, IgD, IgE, IgGl, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof may include Fab, Fv and F(ab')2, Fab', and the like. In addition, aggregates, polymers, and conjugates of immunoglobulins or their fragments can be used where appropriate so long as binding affinity for a particular molecule is maintained.

[0037] In one aspect, the invention involves the rotation of a porous carrier matrix to introduce a centrifugal force in the direction of lateral flow along the porous carrier matrix. The centrifugal force may act on any sample or reagent placed on or in the porous carrier matrix. Centrifugal force can move or assist in the movement of the sample or reagent by initiating, maintaining liquid movement, or increasing the speed with which liquids move, along a porous carrier matrix. Accordingly, the invention shortens the time within which specific binding-pair assays, and other assays associated with a lateral flow device, may be performed.

[0038] In one aspect, one or more porous carrier matrices may be rotated so as to apply centrifugal force to samples and/or reagents within the matrices. The porous carrier matrix may include an application zone and a detection zone. Samples may be applied to the porous carrier matrix at the application zone and moved towards the detection zone using centrifugal force. More specifically, for example, the rotation of a porous carrier matrix induces a centrifugal force on the sample and/or reagents. [0039] In a particular aspect of the invention, the porous carrier matrix may be arranged so that the direction of the centrifugal force is approximately aligned with the direction of lateral flow on the porous carrier matrix. For example, the detection zone and application zone of the porous carrier matrix may be arranged so that lateral flow from the application zone to the detection zone is approximately perpendicular to the axis of rotation of a rotary device upon which the matrix is mounted. As such, exposing the porous carrier matrix to centrifugal force affects the lateral flow of the sample and/or reagent outward from the application zone to the detection zone. Accordingly, the centrifugal force affects the lateral flow of the sample and/or reagents by moving or assisting in the movement of sample and/or reagents along the matrix.

[0040] In another aspect, orientation of the matrix is reversed, so that the centrifugal force opposes the lateral flow of the sample and/or reagents induced by capillary force. In this aspect, centrifugal force can affect the lateral flow of sample and reagents by prohibiting, inhibiting, or stopping flow the flow of liquids along the carrier matrix.

[0041] In another aspect, a porous carrier matrix may include, or be used in combination with, a magnet associated with the detection zone. Magnetic particles having bound thereto analyte-specifϊc binding reagents and/or labels may be placed in the application zone and accelerated along the matrix towards the detection zone, at least in part as a result of centrifugal force. More specifically, the invention provides for a porous carrier matrix that allows for the unimpeded flow of magnetic particles suspended in a carrier liquid, such as the sample or other liquid reagent. The porous carrier matrix allows for the lateral flow of the liquid containing the magnetic particles to a point in the matrix associated with a magnet that stops the flow of the particles but not the liquid. As the liquid carrying the magnetic particles passes through the magnetic field, the particles are attracted to the field and form a detection zone in discreet location on the matrix. The analyte may be captured in the detection zone and detected with a label.

[0042] In most cases, porous carrier matrices will be referred to by way of example with reference to an exemplary embodiment having one or more porous carrier matrices having one or more detection zones associated with a magnet. While the invention is generally described by way of example, the exemplary embodiment is not intended to limit the scope of the invention. Many variations and/or configurations will be apparent to one skilled in the art. As one example, reagent- impregnated test trips are included in the scope of the invention in addition to or in place of test strip with a magnet associated with detection zones. For instance, a detection zone on a porous carrier matrix may be defined by immobilized reagents such as a specific-binding partner for the analyte or an analyte-analog.

[0043] I. Porous Carrier Matrix

[0044] The invention may employ a porous carrier matrix capable of providing lateral flow to a liquid test sample and/or liquid reagents. Generally, the porous carrier matrix can be selected from any available material having appropriate thickness, pore size, lateral flow rate, and color. Lateral flow refers to liquid flow in which all of the dissolved or dispersed components of the liquid are carried at substantially equal rates and with relatively unimpaired flow laterally through the matrix, as opposed to the preferential retention of one or more components of the liquid, such as a chromatographic separation of the sample. Examples of suitable porous carrier matrices include glass fiber mats, non-woven synthetic mats, sintered particulate structures, cast or extruded matrix materials, or other materials characterized by the presence of adhesion within the material. These materials may be a formed (molded or cast) from open pore structures such as nylon or nitrocellulose. The porous carrier matrix may also be a particulate material such as glass particles or polymer particles.

[0045] The porous carrier matrix may be made from a material which has a low affinity for the analyte and test reagents. This is to minimize or avoid pretreatment of the test matrix to prevent nonspecific binding of analyte and/or reagents. However, materials that require pretreatment may provide advantages over materials that do no require pretreatment. Therefore, materials need not be avoided simply because they require pretreatment. Hydrophilic matrices generally decrease the amount of nonspecific binding to the matrix.

[0046] In one aspect, the porous carrier matrix has an open pore structure with an average pore diameter of 1 to 250 micrometers and, in further aspects, about 3 to 100 micrometers, or about 10 to about 50 micrometers. The matrixes are from a few mils (0.001 in) to several mils in thickness, typically in the range of from 5 or 10 mils and up to 200 mils. The matrix should be translucent to allow for the visualization or photometric determination of the light and or color throughout the thickness of the matrix. The matrix may be backed with a generally water impervious layer, or may be totally free standing.

[0047] An example of a suitable porous carrier matrix in which lateral flow occurs is the high density or ultra high molecular weight polyethylene sheet material manufactured by Porex Technologies Corp. of Fairburn, Georgia, USA. This material is made from fusing spherical particles of ultra-high molecular weight polyethylene (UHWM-PE) by sintering. This creates a porous structure with an average pore size of eight microns. The polyethylene surface is treated with an oxygen plasma and then coated with alternating layers of polyethylene imine (PEI) and poly acrylic acid (PAA) to create surfactant-free hydrophilic surface having wicking rate of 70 sec/4 cm.

[0048] While matrices made of polyethylene have been found to be highly satisfactory, lateral flow materials formed of other olefin or other thermoplastic materials, e.g., polyvinyl chloride, polyvinyl acetate, copolymers of vinyl acetate and vinyl chloride, polyamide, polycarbonate, polystyrene, etc., can be used. Examples of suitable materials include Magna Nylon Supported Membrane from GE Osmonics (Minnetonka, Minnesota), Novylon Nylon Membrane from CUNO Inc. (Meriden, Connecticut) and Durapore Membrane from Millipore (Billerica, Massachusetts).

[0049] Generally the shape of the matrix should be suitable for the analyte or analytes being detected. The matrix may include two or more discreet channels for measuring multiple analytes in a sample. The matrix may be divided physically, such as by providing fluid impermeable barriers between the channels, or by treating discreet areas of the matrix with reagents that render the matrix impermeable to liquid flow. For example, a fluoro methacrylate polymer can be used to create discrete channels in the matrix by rendering portions of the matrix impermeable to liquid.

[0050] The matrix materials may be slit, cut, die-cut or punched into a variety of shapes prior to incorporation into a device. Examples of alternative shapes of the matrix include circular, square/rectangular-shaped, flattened ellipse shaped or triangularly shaped. If desired, biological reagents may be applied to the materials before or after forming the desired shape. Biological reagents may be attached to the materials by any available method, for example, either by passively, diffusively, non- diffusively, by absorption, or covalently, depending upon the application and the assay.

[0051] II. Magnetic Nanoparticles

[0052] In one aspect, the invention may employ a magnetic nanoparticle that is separable from solution with a conventional magnet. Such particles are usually provided as magnetic fluids or ferrofluids, as they are often called, and mainly consist of nano sized iron oxide particles (Fe₃O₄ or γ-Fe₂O₃) suspended in carrier liquid. Although generally characterized as magnetic, the particles of the invention are preferably paramagnetic or superparamagnetic particles. Paramagnetic and superparamagnetic particles are particles are only magnetic in the presence of an external magnetic force. Therefore, paramagnetic and superparamagnetic particles may be easily magnetized with an external magnetic field and redispersed immediately once the magnet is removed. Generally the particles have a small size distribution and uniform surface properties.

[0053] The magnetic particles can be prepared by a variety of established methods, generally by the precipitation of iron oxide in the presence of polymers, coating of iron oxide with polymers according to the core-shell principle, or high pressure homogenization. The precipitation methods generally provide particles having a diameter of 50-100 nanometers, the core shell method provide particles with diameters from about 200 to 500 nanometers, and the homogenization technique generally provides particles between those ranges.

[0054] For example, particles may be prepared from superparamagnetic iron oxide by precipitation of ferric and ferrous salts in the presence of sodium hydroxide and subsequent washing with water. The size of the iron oxide cores can be determined by photon correlation spectroscopy (PCS). The iron oxide cores can be coated with polysaccharides such as dextran, starch, chitosan, and fϊcell, or with synthetic polymer polyethylene imine and polyvinylpyrrolidine at temperatures depending on the solubility of the polymers in water. Generally, the molecular weight of the polymer corresponds to the size of the magnetic particle. The temperature and strength of the applied base for the iron oxide precipitation can influence the size of the particles and their percentage of iron oxide within the particle. The polymer used can also influence the amount of iron oxide in the particle. See Griittner, C, et al, Preparation and Characterization of Magnetic Nanospheres for In Vivo Application, in Scientific and Clinical Applications of Magnetic Carriers, Haefeli, et al., Eds., Plenum Press, New York, 1997, pp. 53-67.

[0055] In another example, biodegradable magnetic nanoparticles which includes a mixture of non-toxic biodegradable magnetic metal oxide nanophases (i.e. Fe₃O₄Or γ -Fe₂O₃) and active t-PA, is packed in biodegradable poly(lactic acid) (PLA) nanospheres having sizes ranging from 100 nm to several microns. PLA nanospheres are encapsulated with poly(ethylene glycol) (PEG) to provide protection against nonspecific binding with sample components.

[0056] The magnetic particles can be functionalized to provide a surface for coupling the particles to a molecule or biomolecule. Depending upon the polymer, the surfaces can be activated with a variety of functional groups readily known to those skilled in the art. These groups include, for example, amino, carboxy, alcohol, and aldehyde groups. In one aspect of the present invention, the particle is attached to a metal colloid. A variety of attachment chemistries can be used, including covalent attachment or attachment through specific binding partners. Linking molecules may also be employed.

[0057] Currently available formats of particles can be broadly classified into unmodified or naked particles, chemically derivatized particles with general specificity ligands (streptavidin, Protein A, etc) and chemically derivatized particles with specific recognition groups such as monoclonal and polyclonal antibodies. Suitable particles with diameters ranging from 50 to 1000 nanometers, and functionalized with a variety surfaces, are available from a number of sources including Micromod Partikeltechnologie GmbH, Rostock-Warnemuende, Germany; AdemtechSA, Pare scientifique Unitec 1, 4, Allee du Doyen George Brus, 33600 Pessac, France; and EMD Biosciences Inc., Estapor® Microspheres, Division Life Science Products, 1658 Apache Dr. Naperville, Illinois (USA). In one aspect, the magnetic particles are Carboxyl-Adembeads; Ademtech; catalog # 02121.

[0058] Polymer coated magnetic particles as used in the invention can be functionalized with specific binding partners, such as an antibody, using well known EDC (l-Emyl-3-[3-dmiethylaminopropyl]carbodiiniide Hydrochloride) chemistry, or other chemistry known to those of skill in the art for attaching a binding partner to a solid support.

[0059] m. Labels

[0060] A "label" is any molecule that is bound (via covalent or non-covalent means, alone or encapsulated) to another molecule or solid support and that is chosen for specific characteristics that allow detection of the labeled molecule. Generally, labels include, but are not limited to, the following types: particulate metal and metal- derivatives, radioisotopes, catalytic or enzyme-based reactants, chromogenic substrates and chromophores, fluorescent and chemiluminescent molecules, and phosphors. The utilization of a label produces a signal that may be detected by means such as detection of electromagnetic radiation or direct visualization, and that can optionally be measured.

[0061] In various aspects of the invention, one, two, or more labels may be employed. For example, a first label is associated with the magnetic particle used in the invention. A second label is part of a conjugate reagent that includes the label and an analyte-specifϊc binding partner or an analyte analog. In one aspect, the magnetic nanoparticle is attached to a metal colloid. In typical immunoassays, colloidal gold provides a label for visual or qualitative detection. In the primary aspect of the present invention, however, the colloid serves as a carrier for a different label. To avoid confusion, the label carried by the metal colloid will be referred to as a label attached to colloid to distinguish this label from the colloid itself. Recently, for example, colloidal gold particles have been coated with fluorescent metal chelates. U.S. Patent No. 7,053,249, which is incorporated by reference herein its entirely, describes Europium chelates coated onto colloidal gold particles. Also, the colloid may be coupled to other conventional and unconventional fluorescent, chemiluminescent and enzyme labels, including for example, fluoresceins, rhodamines, Texas Red, luminol, anthracenes, luciferaces, peroxidases, dehydogenases, and others. While the possibilities for the label attached to the colloid are extensive, the label must produce a signal that is distinguishable from signal produce by the label of the conjugate reagent. In one aspect of the invention, the label should be capable of producing a signal in an aqueous environment that is a quantifiable, preferably by conventional instrumentation.

[0062] Metal colloids are primarily gold and silver, but other metals, especially rare earth metals, are appropriate. The size of the colloid is not significant as long as the colloid and the magnetic particle, when attached, are capable of remaining in suspension in carrier liquids.

[0063] In certain aspects of the invention, the label attached to the colloid includes a chelate of a tripositive lanthanide ion, such as Eu³⁺, Tb³⁺ and Sm³⁺. These chelates may include chelating ligands, enhancing ions, and synergistic agents to increase the intensity of the signal from the label. In certain aspects, the label attached to the colloid operates on the principle of time-resolved fluorimetry to eliminate the effects of background signal and increase the sensitivity of the assay.

[0064] The fluorescent properties of tripositive lanthanide ions, especially the chelates OfEu³⁺, Yb³⁺ and Sm³⁺, are particularly well suited for time-resolved fluorimetry. In these chelates, a strong ion emission originates from an intrachelate energy transfer, where an organic ligand absorbs the excitation radiation in the ultraviolet (UV) range and transfers the excited energy to the emitting ion. The ligand field around the ion also prevents the quenching caused by coordinated water molecules which in aqueous solution tend to create an efficient deactivation route. The ion-specific emission appears at narrow banded lines at long wavelengths (Tb³⁺ 544 nm, Eu³⁺ 613 nm, Sm³⁺ 643 nm) with a long Stokes' shift (230-300 nm). The most important feature in this context is the long fluorescence lifetime, ranging from 1 μs to over 2 ms, which makes it possible to apply time-resolved detection for the effective eUmination of the background and to increase the sensitivity.

[0065] β-Diketones have gained the widest use as the ligands to increase lanthanide fluorescence. As bidentate chelating agents, they form relatively stable chelates; the six-membered ring involved in the chelate structure directly absorbs the excitation light and efficiently transfers the energy to the chelated ion. For some chelates, however, fluorescence is essentially limited to organic solvents, making them unattractive or impractical for biological applications. In one aspect of the invention, the label attached to the colloid is a β-diketone that forms a fluorescent chelate with lanthanide (III) rare earth metal ions in an aqueous solvent as described in U.S. Patent No. 7,053,249, which is incorporated herein by reference in its entirety. This label can be detected by irradiating the label with energy equal to or greater than 360 nm and detecting the fluorescence from label. In addition, several other β- diketones are known effective chelating agents as described in Xu, Y, et al., Co- fluorescence Effect in Time-resolved Fluoroimmunoassay: A Review, Analyst 1992 (117:1061-1069):

Thenoyltrifluoroacetone (TTA)

Benzoyltrifiuoroacetone (BTA)

2-Furoyltrifluoroacetone (FTA) p-Fluorobenzoyltrifluoroacetone (FBTA) β-Naphthoyltrifluoroacetone (β-NTA)

1,1,1 ,2,2-Pentafluoro-5-phenylpentane-3 ,5-dione (PFPP)

Dibenzoylmethane (DBM)

Di-p-fluorobenzoylmethane (DFBM)

Pivaloyltrifluoroacetone (PTA)

1,1,1 -Trifluoro-6-methylheptane-2,4-dione (TFMH)

Dipivaloylmethane (DPM)

1,1, 1 ,5,5,5-Hexafluoroacetylacetone (HFAcA)

1,1,1 ,2,2-Pentafluorohexane-3,5-dione(PFH)

1,1,1 ,2.2-Pentafluoro-6,6-dimethyUieptane-3,5-dione (PFDMH)

1,1,12,2,2,3 ,3-Heptafluoro-7,7-dimethyloctane-4,6-dione(HFDMO)

1,1,1 ,2,2-Pentafluorotetradecane-2,4-dione(PFTD)

1,1,1 -Trifluorotridecane-2,4-dione(TFTD) 1,1,1 -Trifluoroacetylacetone(TFAc A) Acetylacetone(AcA)

[0066] In many instances, the strong fluorescence intensity of lanthanide β- diketone chelates requires a non-aqueous chelate environment. For instance, the three β-diketone molecules in a tris-chelate occupy only six of the nine available coordination sites of the ion, which still remains sensitive to quenching by water. Synergist ligands have been introduced to work as an 'insulating sheet,' in which a synergistic ligand (or a synergistic agent) is involved in the chelate structure to replace water molecules from the ligand field. The agent can act as a shield, protecting the chelate from external interactions and thus efficiently reducing nonradioactive energy degradation. Examples of synergistic ligands applicable for co- fluorescence enhancement are 1,10-phenanthroline (Phen) and its derivatives, e. g. , 4,7-(or 5,6)-dimethyl-l,10-phenanthroiine, 4,7-diphenyl- 1,10-phenanthroline, 2.9- drmethyl-4,7-diphenyl-l,10-phenanthroline, pyridine derivatives such as 2,2'-dipyridyl (DP), 2,2'-dipyridylamine, 2,4.6-trimethylpyridine, 2,2':6', 2"-tetrapyridine and 1.3- diphenylguanidine. Tri-n-octyl-phosphene oxide (TOPO) has been found to be one of the most effective synergistic agents. Usually, the fluorescence on the lanthanide chelate increases with increasing concentration of the synergistic ligand until a maximum and almost stable fluorescence is reached.

[0067] In another aspect, the fluorescent label may be used in the presence of enhancing ions. The enhancing ions generally applied are Gd³⁺, Tb³⁺, Lu³⁺, La³⁺ and Yb³⁺. Enhancing ions are useful because, under some conditions involving time resolved fluorimetry, a weak co-fluorescence enhancement effect is obtained, for example with Yb³⁺ and Dy³⁺. This is because, in the co-fluorescence enhancement system, the enhancing ion used must not have excited 4f or 4d levels situated below the excited triplet level of the β-diketone used. Hence the energy absorbed by these chelates cannot be dissipated through these non-existing energy levels. Instead, the energy is transferred to the fluorescent ions through an intermolecular energy transfer. Enhancing ions are needed to provide the high molar excess of the triplet sensitizer to ensure a linear response for the acceptor detection. The high concentration is also needed to create the aggregates for necessary for energy transfer. [0068] Non-ionic detergents have been found to protect the chelates against nonradioactive processes and solubilize the chelates and stabilize the solutions by preventing sedimentation. Examples of suitable detergents include TRITON® X-IOO and TWEEN® 20. Water soluble organic solvents may also be used to enhance fluorescence.

[0069] In another aspect, a conjugate reagent includes a label for detecting the analyte. This label is attached to an analyte-specific binding partner or an analyte analog. This label is independent and distinct from the label attached to the colloid and its signal must be distinguishable from the label attached to the colloid. In various aspects of the invention, the two labels are detected sequentially or simultaneously. The attachment of the labels to the analyte-specific binding partners may be accomplished directly, through a linker, or through a pair of specific binding partners (e.g. biotin/avidin) as is well known in the art. If the label on the conjugate reagent and the label attached to the colloid are both fluorescent labels, then the labels should distinguishable, for example by excitation at different wavelengths or by different emission spectra.

[0070] TV. Particulate Reagent

[0071] A reagent for use in one aspect of the invention includes, for example the colloid bound to the magnetic particle. More specifically, for example, a particulate reagent may include a complex of the magnetic particle and the metal colloid. In this aspect, the metal colloid is functionalized with a label attached to the colloid.

[0072] The colloid and the particle may be attached using well known linking chemistries. These chemistries include, for example, direct attachment using zero linkers such as EDC, and long chain linkers, or indirectly through a pair of binding partners such as biotin and avidin/streptavidin/neutravidin. To assist in the linking, the magnetic particle may be functionalized with amino, aldehyde, hydroxyl, or carboxyl groups.

[0073] Colloidal gold has been used extensively as a label in immunoassays. Therefore methods for functionalizing the colloids and attaching them to various molecules are well characterized. For example, U.S. Patent No. 7,053,249 describes a gold colloid coated with a Europium chelate and a bound to an antibody. [0074] In another aspect, the colloid includes an analyte-specific binding partner. When label and binding partners are added to the colloid together they are added sequentially, label first. In general, the specific concentrations of the label and binding partners are kept the same but concentrations may vary depending on the binding partners. When all three components (i.e., the label, the specific binding partner and the analyte-specific binding partner) are included on the colloid, the components are added in the following order: label, analyte-specific binding partner, and specific binding partner.

[0075] Li general, the particulate reagent includes the label attached to the colloid. A second label associated with a conjugate reagent is used to detect the presence or amount of analyte in the sample. In one embodiment, however, the second label is attached (either through a linking group or through a pair of binding partners) to the colloid or the particle. Thus, this reagent will provide two signals regardless of the presence of the analyte in the sample. Accordingly, this reagent can be used as a control to determine the quality of other reagents in the assay, such as a substrate reagent.

[0076] In one aspect, the particulate reagent is provided in a lyophilized form that allows for their convenient use in a commercial assay. The particulate reagents may be lyophilized by standard lyophilization protocols. In general, the particulate reagent is mixed with trehalose (or any other cyroprotectant) and dispensed into individual vials. They are then lyophilized using a standard lyophilization protocol and stoppered under either vacuum or dry nitrogen. To reduce meltback post lyphophilization, the lyophilized material is dried at elevated temperatures for an extended period of time following the completion of the lyophilization cycle; for example, at 45°C for at least 60 hours. This procedure produces stable cakes of the lyophilized paramagnetic particles.

[0077] In one aspect of the invention, lyophilization of paramagnetic particles may involve an extended terminal drying step. In particular, paramagnetic particles functionalized with antibodies are be mixed with approximately 30% sucrose in a about a 1:1 ratio to yield a final concentration of approximately 15% sucrose. In one example, aliquots of the solution having a volume of between about 200 to 500 μL can then placed in 2ml glass vials. Then, a butyl stopper can be placed in the vial and the vials placed into a lyophilizer. The vials are then frozen at approximately -40° C and lyophilized according to standard protocols for proteins. At the end of the standard lyophilization cycle, the particles are heated to approximately 40-45° C and dried at approximately this temperature for a period of approximately 48-72 hours or more.

[0078] In one aspect, a reagent for detecting an analyte in a sample may include a metal colloid with a label attached to the colloid. The reagent may also include one or more of an analyte-specific binding partner, a protein or another (non-analyte) specific binding partner such as an antibody, antigen, streptavidin or biotin. The (non-analyte) specific binding partner and the protein may be used to couple the colloid to the magnetic particle, either via a complementary binding partner on the particle or through an appropriate linker molecule.

[0079] V. Lateral Flow Device

[0080] Figure 1 shows a general example of a lateral flow device 100, such as a lateral flow immunoassay device. The device includes a porous carrier matrix 102. The shape of porous carrier matrix 102 is irrelevant, so long as it provides a suitable format for conducting an assay and allows for the substantially unimpeded lateral flow of liquid samples and/or reagents along the porous carrier matrix. The porous carrier matrix 102 may include a sample application zone 104 and a detection zone 106. In an exemplary embodiment, a magnet 108 may be associated with the detection zone so that the magnetic field defines the detection zone 106 of porous carrier matrix 102. Device 100 may serve to position porous carrier matrix 102 and magnet 108 appropriately, so that detection zone 106, will bind with an analyte flowing through porous carrier matrix 102. It should be understood that while a rectangular magnet is shown, the magnet, and thus the detection zone, may take various shapes or forms. For example, some embodiments may employ a circular magnet, which creates a circular detection zone.

[0081] In an example configuration, the magnet 108 is underneath the porous carrier matrix 102 so that magnetic particles in a solution flowing past the detection zone 106 may be captured in the magnetic field of the magnet 108. The magnetic particles may be bound to analyte-specific binding particles in the detection zone 106. Therefore, while the solution continues to traverse the matrix, the magnetic particles are held at the detection zone 108 by the magnetic field of magnet 108. The magnetic particles, having bound thereto an analyte-specific binding partner, may function to detect an analyte by binding to and separating the analyte from a test sample flowing through the detection zone.

[0082] In one aspect, the porous carrier matrix 102 includes a sample application zone 104, which is laterally spaced from the detection zone 106. The sample application zone 104 may include a separate pad, cup, well or other member that facilitates the application of the sample solution and/or other reagents at a discreet location on the matrix. The sample application zone 104 may also include a conjugate reagent non-diffusively bound the matrix, a separate pad or other member so that the reagent is solubilized by the sample solution upon addition of the solution. Device 100 may be configured in any manner so as to provide access to sample application zone 104. Further, while only one application zone is depicted, multiple application zones may exist for samples and/or reagents.

[0083] In one aspect, the porous carrier matrix and the magnet are held within a rigid body of a device for convenient handing by the operator. The magnet and the matrix may be held in place within the rigid body so that the location of the magnet is fixed in relation to the matrix. The material for the rigid body is unimportant as long as it is compatible with the sample and reagents, and does not affect the magnetic field. Preferably, the body will be readily and inexpensively manufactured and packaged. In other aspects of the invention as described more fully below, the magnet is associated with a platform for mounting the device on a rotor for providing centrifugal force. Therefore, when the device is mounted on the platform, a detection zone is created as a result of the vicinity of the magnet to the porous carrier matrix.

[0084] The shape of the detection zone is generally defined by the shape of the magnetic field, which in turn may be defined by the shape of the magnet, as well as other characteristics of the magnet and/or the apparatus in which the porous carrier matrix is implemented. In one example, the matrix is a strip with and the magnet is a circular disk located under the strip. In operation, the detection zone may take a circular shape of the surface of the matrix. Other shapes should be compatible with the shape of the matrix and the number of analytes under consideration. It has been found that when the magnetic particles are deposited upstream of the detection area, and are allowed to flow to the magnet, a majority of the particles are retained at a location where the laterally flowing solvent front first contacts the magnetic field.

[0085] The magnet may be held directly against the matrix or spaced apart from the matrix to any extent so long as the magnetic field in the detection zone is strong enough to prevent particles from passing through the detection zone. In one aspect, the magnet prevents substantially all of the particles from passing through the detection zone so that the sensitivity of the assay is not substantially different than if all of the particles had been detained in the detection zone. In various aspects of the invention, the magnet prevents at least a majority of the particles, and preferably at least about 70%, about 80%, about 90%, or about 95% of the particles should be retained in the detection zone, and more preferably, about 99% or more of the particles are retained in the detection zone.

[0086] Suitable magnets are numerous. One example is a rare-earth neodymium- iron-boron rod magnet that is 0.25 inches x 0.25 inches. This magnet has a strength of 40 MGOe (Mega Gauss Oersted). Other geometries including discs and cubes in other sizes are also appropriate. While different strength magnets are available, the distance of the magnet from the matrix affects the efficiency of the particle capture. For example, it has been found that when a 40MGOe magnet is placed 0.25 inches away from the matrix, only 0.8% of the particles are seen in the detection zone compared to when the magnet was touching the underside of the matrix.

[0087] Generally, the size of the device will provide for the ease of handling by an operator as well as provide sufficient signal from the labels such that the signals can be visually, photometrically, or spectrophotometrically detected. In on aspect, the porous carrier is a strip of about 6 mm by about 100 mm, and the device includes a rigid body of suitable size for holding the strip and the magnet in fixed relationship.

[0088] In devices for measuring more than one analyte, one magnet may serve to provide a magnetic field for several discreet detection zones, such as one magnet underlying several discreet portions of the matrix, or each zone may have its own magnet. When more than one magnet is used, the field from one magnet should not attract particles that are intended to be attracted to the field of the other magnet(s). [0089] VI. Apparatus

[0090] Figure 2 shows an example of an apparatus 200 arranged to provide centrifugal force to one or more porous carrier matrices or more specifically, to one or more lateral flow immunoassay devices. The apparatus 200 includes a rotor 204 with the porous carrier matrices 202 mounted thereon. In operation, the rotor 204 creates centrifugal force that initiates, maintains or accelerates liquids 206 laterally along the matrices, in the direction of lateral flow vectors LF. Also shown is a vector in the direction of rotation (R) and a vector in the direction of the momentary direction of rotation (R¹). While four porous carrier matrices 202 are shown, other embodiments may exist. For example, the apparatus may include any number of porous carrier matrices, or only a single porous carrier matrix. Further, while the rotor 204 is shown as centered among the sample application ends of matrices 202, other configurations resulting in the application of centrifugal force along a carrier matrix or matrices are contemplated within the scope of the invention.

[0091] After contacting one or more sample liquids or reagents 206 to one or more of porous carrier matrices 202, the matrices may be rotated, exposing the sample liquids or reagents to centrifugal force. While rotation of apparatus 200 is shown as counter-clockwise, rotation in either direction may provide centrifugal force. Viewed from a rotating frame of reference, the centrifugal force may be directed along each carrier matrix and perpendicular to the momentary direction of rotation R'. Accordingly, centrifugal force is directed in substantially the same direction as LF, which initiates, moves or accelerates lateral flow in the direction of LF.

[0092] Lateral flow along a porous carrier matrix 202 may occur for various reasons. As one example, capillary action is the result of the adhesive intermolecular forces between the liquid and the material of the porous carrier matrix being stronger than the cohesive intermolecular forces within the liquid, resulting in lateral flow. The speed of lateral flow may be increased by rotating the porous carrier matrix and thus introducing a centrifugal force. Alternatively, lateral flow may result solely from rotation of the porous carrier matrix, which exposes the liquid to centrifugal force.

[0093] More specifically, the velocity with which lateral flow of samples 206 move along the carrier matrices 202 (e.g. the magnitude of LF), may be the cumulative effect of centrifugal force and the forces resulting from capillary action. Thus, the speed with which a sample or reagent moves through the matrix may be increased due to centrifugal force. As a result the time required to move a sample from the application zone to the detection zone may be reduced. In alternative embodiment, centrifugal force may work alone or in combination with other forces to move a sample laterally, in the direction of LF.

[0094] In addition, the orientation of the matrix may be reversed so that the centrifugal force oppose the lateral flow of the sample and reagents. Therefore, centrifugal force can be used to delay the time that the sample and/or reagents encounter the detection zone, or may reverse the flow of sample and reagents. In one aspect, the device includes a zone for application of reagents on the matrix at the end of the matrix opposite of the detection zone such that the reagents flow through the detection zone toward the sample application zone. Decelerating or stopping (halting temporarily, or stopping for the remainder of the assay time) of the flow may be used, for example, to allow chemical reactions to proceed to a greater degree of completion. To stop the lateral flow, the centrifugal force should approximate the capillary force.

[0095] "Centrifugal force" is used in accordance with the well understood meaning of that term. Specifically, in a fixed frame of reference, no actual force is exerted on the liquid or reagent. Rather, in theory, the inward rotational force, or centripetal force, forces the surface upon which the liquid rests or flows through (e.g. the porous carrier matrix) toward the axis of rotation. Thus, rotating the matrices results in the appearance that a force is causing the liquids to move outward from the axis of rotation along the matrices 202. This apparent force may be referred to as "centrifugal force," which is in the direction of lateral flow LF.

[0096] Various configurations of an apparatus with magnets arranged to create magnetic fields in the detection zones of porous carrier matrices are contemplated within the scope of the invention. In one exemplary embodiment, a magnet is associated with each porous carrier matrix. The magnet may be held in place by an adhesive, a clamp or any other device that keeps the magnet in place in the detection zone. For example, the porous carrier matrix and the magnet are held within a rigid body so that the location of the magnet is fixed in relation to the matrix. In addition, the magnets may be associated with the rotary apparatus instead of the porous carrier matrix. Therefore, the matrix can be disposed of while the magnet stays fixed to the apparatus to provide a magnetic field for the next matrix that is mounted on the apparatus.

[0097] The rotor shown in FIG. 3 can be constructed to include positions for mounting one or more flow immunoassay devices to a rotor. Figure 3 shows an example where four lateral flow immunoassay devices 302 are attached to a rotor 304. Alternatively, a single lateral flow immunoassay device may include multiple porous carrier matrices. In either case, the devices may be positioned so that all matrices are rotated around a common axis. This configuration may allow for substantially similar lateral movement of samples and/or reagents along each the matrices from an application zone 305 to a detection zone 306.

[0098] In Fig. 3, mounting platforms 307 provide a position for mounting devices 302 on the rotor 304. In one embodiment of the invention, each platform 307 includes a magnet positioned to provide a magnetic field that creates a magnetic zone for immobilizing magnetic particles. Therefore, in this embodiment, it is not necessary that each device 302 include a magnet. Instead, after use, the device 302 can be replaced with a new device while the magnet stays fixed on the mounting platform 307.

[0099] In another aspect, each mounting platform 307 may be configured to rotate its respective device 302, which is preferably a porous carrier matrix, independently from the rotation applied by rotor 302. In this aspect, each mounting platform 307 may rotate its respective device 302 by at least 180 degrees around the z-axis. Each platform 307 may, after rotating its respective device 302, hold its respective device 302 substantially fixed, so that rotation of rotor 304 may cause the devices 302 to be rotated, and apply centrifugal force to the devices. However, when platforms 307 have rotated devices 302 by 180 degrees, devices 302 will be oriented in the opposite direction, relative to the direction of the applied centrifugal force. As such, the centrifugal force will act in the opposite direction upon any of the mobile particles, reagents, liquids, etc. in each device 302.

[00100] In a further aspect, rotation of devices 302 by 180 degrees or more may be achieved using various mechanical and/or manual techniques. As one example, systems and techniques for reversing the direction of applied centrifugal force are described in U.S. Patent No. 4,814,282 (Holen et al.), which is incorporated herein by reference, may be employed in the apparatus shown in Fig. 3. Other examples are also possible. In general, the apparatus by which the orientation of devices 302 is reversed may take on any form, without departing from the scope of the invention.

[00101] Figure 4 shows an example of an apparatus 400 for detecting analytes in one or more liquid samples. The apparatus 400 is operable to expose a liquid sample or reagent in a lateral flow device to centrifugal force, thereby decreasing the time required to perform an assay. In addition, the apparatus 400 may be operable to rotate multiple lateral flow devices in order to perform multiple assays approximately simultaneously. The apparatus 400 includes a housing unit 402 for housing a rotor 404 with mounting platforms 406 for mounting one or more lateral flow device 407. Preferably, the rotor should be capable of rotating the platforms at about 50 to 5000 RPM. The rate of rotation may be constant, or varied during a given run. For example, the rotor may spin at 150 rpm initially be gradually slowed to 100 rpm.

[00102] The housing may also include a pipetting system 408 and a detection instrument 410. In one aspect, pipetting system 408 operates to contact a liquid sample or liquid samples to the application zones of lateral flow devices 407 that are mounted on the platforms 406. The pipetting system 408 transfers liquid samples and/or reagents from a number of reservoirs 412 that store the samples and/or reagents to the devices 407 on the platforms 406 of the rotor 404.

[00103] In one aspect, the rotor 404 may be configured to rotate lateral flow devices 407 mounted on platforms 406. Generally, the axis of rotation is approximately parallel to the Z-axis shown in FIG. 4. Further, while the platforms 406 are shown as perpendicular to the Z-axis, the platforms may be configured so that the lateral flow devices mounted thereon are angled upward, downward, tilted, sideways, offset from center, etc., so long as rotation exposes a liquid sample or reagent to centrifugal force, resulting in flow of the liquid and/or reagent from the application zone to the detection zone.

[00104] The detection instrument 410 may operate to detect one or more types of signals generated by a sample and/or reagent in the detection zone of a device mounted on platform 406. For example, the detection instrument 410 may include a spectrometer, a fluorometer, or both. Therefore, the detection instrument may function to detect chemoluminescence, fluorescence, or other energy emitting signals that are capable of being generated by suitable labels. As a result, signals emitted from reagents and/or samples retained in the detection zone may be detected by the detection instrument 410.

[00105] In one aspect, the rotor 404 may be associated with a mechanism for moving the rotor along the X-axis and/or the Y-axis as shown in FIG. 4. With such a configuration, a lateral flow device mounted on platform 406 may be located so that the pipetting system 408 can introduce a liquid sample or reagent to an application zone of a porous carrier matrix of the device. In particular, the mechanism may be used to locate the application zone under pipetting system 408. After assays are complete, the mechanism can then move the rotor in order to locate the detection zone of a lateral flow device under the detection system 410, which can detect a signal generated by a label associated with an analyte in the detection zone. In particular, a porous carrier matrix of the device 407 may be positioned so that the detection zone is located under the detection instrument 410.

[00106] The order of the application or sample and reagent to and detection of signal from multiple devices on the rotor 404 is not critical. For example, the pipetting system 408 may contact a sample with the porous carrier matrix of a first lateral flow device. Rotor 404 may then rotate so that a second lateral flow device is positioned under pipetting system 408. A sample or reagent may then be contacted with the porous carrier matrix of the second lateral flow device. This process may then be repeated for each lateral flow device. A similar process may occur to position the lateral flow devices under the detection instrument 410. In addition, the detection of a signal from one device may occur sequentially or simultaneously with the application of sample and reagents to another device.

[00107] VTI. Methods

[00108] In one aspect, the invention provides a method for laterally moving or assisting in the movement of a liquid sample and/or reagent along a porous carrier matrix. The method includes (i) contacting the matrix with the sample and/or the reagent, and (ii) exposing the liquid sample and/or the reagent to centrifugal force by rotating the matrix, thereby moving or assisting in the movement of the sample and/or the reagent in the direction of the centrifugal force. By way of example, the porous carrier matrix may be included in a lateral flow immunoassay device, although other configurations are possible. The description will refer to an example where the porous carrier matrix is included in a lateral flow immunoassay device, but this should not be construed as limiting the scope of the invention.

[00109] Contacting the matrix with the sample and/or the reagent may occur at an application zone on a lateral flow immunoassay device. Once the liquid is contacted with the matrix at the application zone, at which point lateral flow of the liquid along the matrix may occur, at least in part, due to capillary or wicking action. Following the application of the liquid to the application zone, the device (including the carrier matrix) is rotated, creating a centrifugal force upon the liquids in the carrier matrix. The centrifugal force can move or assist in the movement of the liquid laterally along the carrier matrix. Moving or assisting in the movement of the liquid includes the initiation and acceleration of the liquids along the matrix.

[00110] Prior to adding the liquid sample, a particulate reagent may be flowed through the porous carrier matrix as a result of capillary or centrifugal force, or both. The particulate reagent includes a carrier liquid for magnetic nanoparticles. Further, the magnetic nanoparticles may be attached or bonded to an analyte-specifϊc binding partner and/or a label. The particulate reagent may enter the device via an application zone, which may be the same or different from the application zone for the sample liquid.

[00111] After the magnetic nanoparticles have contacted the matrix, the carrier matrix may be rotated, introducing a centrifugal force to the particulate reagent. The centrifugal force may accelerate the carrier liquid, and thus accelerate the magnetic nanoparticles, analyte specific binding partner, and/or label as well. In addition, a magnet associated with the detection zone captures the magnetic nanoparticles and analyte specific binding partner in the detection zone (and at the same time separating them from the carrier liquid). Therefore, as the liquid carrying the magnetic particles passes through the magnetic field, the particles are attracted to the field and form a detection zone in discreet location on the matrix. [00112] After the magnetic particles create a detection zone, the sample liquid that potentially includes an analyte may be flowed through the device. To do so, the sample liquid may be applied to the device at the application zone. The device may then be rotated, creating a centrifugal force on the sample liquid and thus causing the liquid to move laterally along the porous carrier matrix. When the sample liquid reaches the detection zone, analyte in the sample liquid may bind with the analyte- specifϊc binding partner held in the detection zone because of its attachment to the magnetic particles. As such, the analyte may be separated from the sample liquid and held in the detection zone.

[00113] hi an alternative embodiment, a reagent-impregnated matrix or other nonmagnetic testing device may be used instead of a matrix with a magnetically created detection zone. In such an embodiment, flowing the carrier liquid including the magnetic particles through the detection zone may be unnecessary. Rather, the sample may be flowed through the test strip, without the initial step of flowing magnetic particles through the test strip.

[00114] After a sample flows through the porous carrier matrix, the presence and/or amount of an analyte may be determined by flowing a conjugate reagent through the porous carrier matrix. The conjugate reagent may include a conjugate analyte-specifϊc binding partner and a label. Again, the carrier matrix may be rotated, creating a centrifugal force on the conjugate reagent. As the conjugate reagent passes through conjugate analyte-specific binding partner may bind to the analyte captured in the detection zone, hi addition, the label may be bound to the binding partner and thus captured in the detection zone as well. The label may then be used to determine the presence and/or amount of an analyte in the detection zone.

[00115] Figure 5 shows the results of an experiment for determining the amount of time necessary for liquids to travel along a porous carrier matrix of an ultra high molecular weight polyethylene sheet (UHMW-PE) material made from fusing spherical particles of the material by sintering. The resulting a porous structure has an average pore size of eight microns. The polyethylene surface is treated with an oxygen plasma and then coated with alternating layers of polyethylene imine (PEI) and poly acrylic acid (PAA) to create surfactant-free hydrophilic surface, hi this experiment, a liquid sample took 420 seconds to travel 100 mm through the matrix in a static condition. However, when the matrix is mounted on a rotor operating at approximately 100 rotations per minute (RPM) to apply centrifugal force in the direction of lateral flow, the same sample took only 90 seconds to travel through the matrix.

[00116] Figure 6 shows the results of an experiment for detecting canine pancreatic lipase (cPL) according to a method of the invention. A first mouse monoclonal antibody specific for cPL attached to a magnetic particle was mixed with sample and with a second cPL-specific mouse monoclonal antibody conjugated to horse radish peroxidase. The mixture was applied to the application zone of the carrier matrix and centrifuged at 100 RPM for 30 seconds. The centrifuge was stopped and a chemiluminescent substrate was added to the application zone and allowed to migrate to the detection zone without centrifugation. The light signal was read using a PMT in 2-second intervals.

[00117] In various embodiments of the invention, sample, reagents including one or more of the particulate reagent and conjugate reagents may be mixed prior to contact with the device, depending upon the format of the assay. For example, in a sandwich-type assay, the sample may be mixed with either the particulate reagent, or the conjugate reagent before the sample is added to the device.

[00118] Figure 7 shows another example of an apparatus 700 for detecting analytes in one or more liquid samples. Generally, apparatus 700 operates similarly to apparatus 400 of Figure 4, with differences in at least the arrangement and mobility of certain components. In particular, the rotor 702 and detection instrument 704 are fixedly mounted. Further, the pipetting system 706 is movable along the X axis and the Z axis.

[00119] Although various specific embodiments of the present invention have been described herein, it is to be understood that the invention is not limited to those precise embodiments and that various changes or modifications can be affected therein by one skilled in the art without departing from the scope and spirit of the invention. All references cited herein are incorporated by reference in their entirety.

Claims

What is Claimed Is:

1. A method for affecting the lateral movement of at least one of a liquid sample and a reagent along a porous carrier matrix, the method comprising contacting the matrix with the at least one of the sample and the reagent and exposing the at least one of the sample and the reagent to a centrifugal force, thereby affecting the lateral movement of the at least one of the sample and the reagent along the porous carrier matrix.

2. The method of claim 1 wherein the porous carrier matrix is associated with a lateral flow immunoassay device.

3. A method for determining the presence or amount of an analyte in a liquid sample comprising contacting the liquid sample with an application zone of a porous carrier matrix, wherein the porous carrier matrix further comprises a detection zone laterally spaced from the application zone, providing centrifugal force to move or assist in the movement of the liquid sample from the application zone to the detection zone, and determining, at the detection zone, the presence or amount of the analyte in the liquid sample.

4. The method of claim 3, wherein the determining the presence or amount of the analyte in the liquid sample comprises separating the analyte from the liquid sample.

5. The method of claim 3, further comprising populating the detection zone with an analyte binding partner prior to contacting the liquid sample with the sample application zone.

6. The method of claim 3, wherein the detection zone comprises a binding partner for the analyte immobilized in the detection zone.

7. The method of claim 5, wherein populating the detection zone with an analyte binding partner comprises contacting a reagent with the application zone and using centrifugal force to move or assist in the movement of the reagent from the application zone to the detection zone, wherein the reagent comprises the analyte binding partner bound to magnetic particles and the detection zone is associated with a magnet.

8. An apparatus for detecting analytes in liquid samples comprising a rotary system arranged to provide centrifugal force to a liquid sample on a porous carrier matrix of a lateral flow device and a detection instrument that detects a signal generated by a label associated with the analyte or a reagent in a detection zone of the matrix.

9. The apparatus of claim 8 wherein the detection instrument includes at least one of a photodetector for measuring a fluorescent signal from the detection zone, and a spectrophotometer for measuring a chemiluminescent signal from the detection zone.

10. A method for determining the presence or amount of an analyte in a liquid sample comprising:

(a) providing a device comprising a porous carrier matrix having an application zone for the application of at least one of the sample and a reagent, and detection zone comprising an analyte analog or a first binding partner for the analyte;

(b) adding the at least one of the sample and the reagent to the one or more application zone;

(c) using centrifugal force to affect the movement of the at least one of the sample and the reagent from the application zone to the detection zone; and

(d) measuring a signal from the detection zone and relating the signal to the presence or amount of analyte in the sample.

11. The method of claim 10 wherein the reagent comprises a second binding partner for the analyte conjugated to a label.

12. The method of claim 10 wherein the reagent comprises an analyte analog conjugated to a label.

13. The method of claim 10 wherein the sample and reagent are mixed prior to being contacted with the application zone.

14. The method of claim 10 wherein the analyte-analog or the binding partner for the analyte are bound to one or more magnetic nanoparticles.

15. The method of claim 14 wherein the analyte-analog or the binding partner for the analyte are retained in the detection zone with a magnet.

16. The method of claim 10, further comprising reversing the orientation of the porous carrier with respect to centrifugal force and using centrifugal force to oppose the movement of the at least one of the sample and the reagent from the application zone to the detection zone, thereby decreasing, stopping, or reversing the movement of the at least one of the sample and the reagent from the application zone to the detection zone.

17. A method for determining the presence or amount of an analyte in a liquid sample comprising: forming a mixture comprising the sample and the reagent comprising a complex comprising a magnetic nanoparticle bound to a metal colloid wherein at least one of the nanoparticle or the colloid comprises an analyte-specific binding partner, and the colloid further comprises a first label attached to the colloid; applying the mixture to a device comprising a porous carrier matrix comprising a detection zone comprising a magnet, wherein the porous carrier has an average pore size that allows for the substantially unimpeded lateral flow of the magnetic particles; contacting the matrix with a conjugate reagent comprising a second label bound to a second analyte-specific binding partner or an analog of the analyte; exposing, either simultaneously or sequentially, at least one of the mixture and the conjugate reagent to centrifugal force to affect the movement of the at least one of the mixture and the conjugate reagent from one or more zones for the application of the at least one of the mixture and the reagent to the detection zone. simultaneously or sequentially detecting a signal from the first label and the second label in the detection zone to determine the presence or amount of the analyte in the sample.

18. The method of claim 17 wherein the determination of the amount of the analyte comprises comparing an amount the signal from the first label to an amount of the signal from the second label.

19. An apparatus for detecting analytes in one or more liquid samples, the apparatus comprising:

(a) a rotor for mounting and rotating one or more lateral flow devices each comprising a porous carrier matrix, an application zone, and a detection zone;

(b) a detection instrument operable to detect a signal generated by a label associated with the analyte or a reagent in the detection zone.

20. The apparatus of claim 19, wherein each porous carrier matrix is rotatable independent from rotation of the rotor, such that the orientation of the application and the detection zone can be substantially reversed.

21. The apparatus of claim 19 further comprising a pipetting system for contacting the liquid sample to the porous carrier matrix at the application zone;

22. The apparatus of claim 21 wherein the label associated with the analyte or the reagent is chemoluminescent or fluorescent.

23. The apparatus of claim 22 wherein the detection instrument detects a chemoluminescence or fluorescence signal.

24. A method for lyophilizing antibody functionalized paramagnetic particles comprising:

(a) freezing the paramagnetic particles at approximately -40 degrees Celsius; and

(b) drying the paramagnetic particles at approximately 40 to 45 degrees Celsius, wherein the drying time is at least about 72 hours thereby producing stable calces of lyophilized paramagnetic particles.

25. The method of claim 24, further comprising, before freezing the paramagnetic particles, mixing the paramagnetic particles with a approximately 30% sucrose solution in about a 1:1 ratio.