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WO1996012962A9 - Titrage a l'aide de particules de diffraction - Google Patents

Titrage a l'aide de particules de diffraction

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Publication number
WO1996012962A9
WO1996012962A9 PCT/US1995/013410 US9513410W WO9612962A9 WO 1996012962 A9 WO1996012962 A9 WO 1996012962A9 US 9513410 W US9513410 W US 9513410W WO 9612962 A9 WO9612962 A9 WO 9612962A9
Authority
WO
WIPO (PCT)
Prior art keywords
particles
analyte
diffraction
diffraction pattern
specific binding
Prior art date
Application number
PCT/US1995/013410
Other languages
English (en)
Other versions
WO1996012962A1 (fr
Filing date
Publication date
Application filed filed Critical
Publication of WO1996012962A1 publication Critical patent/WO1996012962A1/fr
Publication of WO1996012962A9 publication Critical patent/WO1996012962A9/fr

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Definitions

  • the technical field of this invention is the detection of analytes.
  • analytes In a variety of applications, there continues to be a need to detect and measure a wide variety of analytes.
  • a large industry has developed associated with the detection of analytes in a variety of situations: measuring drugs of abuse; monitoring therapeutic dosage levels; measuring levels of naturally occurring substances having physiological significance, such as cholesterol, chorionic gonadotrophin, high density lipoprotein, thyroxin, etc.; detecting the presence of pathogens, including both viruses and microorganisms; detecting the presence of surface membrane markers or circulating markers for neoplasia, autoimmune diseases, or the like; detecting DNA or RNA sequences for forensic medicine; diagnosing of genetic diseases; detecting receptors, such as T-cell receptors, growth factor receptors, and the like as well as other conditions, situations, and analytes of interest.
  • the market sector consisting of doctor's offices, homes and some laboratories in third world countries need assays which are simple to use by untrained operators.
  • the assays must be reproducible and reliable in detecting small amounts of analyte.
  • the assay should utilize a device that is more sensitive than the human eye and easy to quantitate.
  • the assay should also be capable of accepting whole blood samples.
  • the assay should provide improved convenience, sensitivity, reliability, greater simplicity in protocol, and be compatible with whole blood.
  • Patents involving the use of microbeads and various optical or other methods for measurement include U.S. Patent Nos: 4,080,264; 4,115,535; 4,164,558; 4,174,952; 4,340,564; 4,351,824; 4,521,521; 4,568,644; 4,582, 810; 4,690,906; 4,695,537; 4,711,841; 4,716,123; 4,738,534.
  • U.S. Patent No. 4,806,015 discusses an assay for measuring agglutination of particles by detecting the attenuation of a diffraction limited spot as it passes through a layer of the agglutinated particles.
  • U.S. Patent No. 5,132,097 describes analyte detection through detection of shadows caused by specific bindings pairs on the surface of a support.
  • U.S. Patent No. 5,086,002 and International Publication No. WO 91/04492 describe agglutination assays with cross-linking reagents for erythrocytes.
  • analyte, particles unless the analyte is the particle, and specific binding pair members are combined in an assay medium with a solid transparent surface.
  • the assay medium is incubated such that analyte, any additional particles, and specific binding pair members form a random coplanar distribution of diffraction particles.
  • irradiating light is diffracted by the diffraction particles into a diffraction pattern.
  • the spacing and amplitude of the diffraction pattern varies with the amount of analyte in the sample.
  • the particles cross-link in an amount proportional to the amount of analyte in the sample.
  • the particles may contact the solid surface or may remain mobile in a layer of fluid above the surface.
  • the surface comprises randomly distributed members of a binding pair providing for specific binding of the diffraction particles to the surface.
  • the random coplanar distribution of particles is then irradiated with coherent light resulting in a diffraction pattern.
  • the amplitude and spacing of the resultant diffraction pattern is measured. Using a logic means, the measured amplitude and spacing is related to the presence of analyte in the sample.
  • BRIEF DESCRIPTION OF THF DRAWTNfiS Fig. 1 is a schematic of the device of the subject invention where the diffraction particles specifically bind to the support surface.
  • Fig. 2 is a perspective view of a surface used in the detection of a plurality analytes with different sized diffraction particles.
  • Fig. 3 is a diagram of an exemplary circuit for detecting and measuring the diffraction pattern.
  • Fig. 4 is a schematic of the device of the subject invention where the diffraction particles non-specifically bind to the support surface.
  • Fig. 5a is a graphical comparison of the radial cross-sections derived from diffraction patterns from a negative control and increasing amounts of analyte.
  • Fig. 5b is an expanded view of the graph in FIG. 5a.
  • an assay medium is prepared by combining analyte, binding members, and particles in the presence of a solid transparent surface.
  • the analyte is the particle and the particle is not added as a separate component to the assay medium.
  • the analyte, specific binding pair members, and particles are allowed to bind to the complementary specific binding pair members such that diffraction particles are formed in random and coplanar distribution within the assay medium. Irradiation of the random coplanar diffraction particles results in a diffraction pattern.
  • the diffraction pattern is related to the presence of analyte in the sample. By comparing the diffraction pattern of interest to the diffraction pattern of a controlled amount of analyte, the amount of analyte in the sample is determined. By providing different reagents and protocols for the formation of diffraction particles, the diffraction pattern can be changed.
  • the particles are cross-linked in an amount related to the amount of sample by a cross-linking reagent.
  • the cross-linked particle may be mobile in fluid covering the surface or by settling on the surface or immobile by being specifically bound to the surface. Alternatively, the particles do not cross-link, but bind to the surface through specific binding pair members on the surface.
  • the first component in the assay member is the analyte in a sample.
  • the subject assay may be used to detect the presence of a wide variety of analytes. In some assays, analytes will be members of specific binding pairs, e.g. ligands or receptors, where "ligands and receptors" includes polymeric molecules that specifically complex, such as nucleic acids.
  • ligand and receptor are used in a broad sense, where ligand is arbitrarily selected, in many instances being the smaller molecule as compared to its complementary receptor.
  • Analytes of interest may be both naturally occurring and synthetic.
  • Naturally occurring analytes of interest include lipids, bacteria, hormones, cytokines, growth factors, irregular-shaped cells, red blood cells, cholesterol, surface membrane proteins, nucleic acids, endorphins, blood proteins, lipoproteins, plant proteins, alkaloids, polysaccharides, narcotics, digitalis etc.
  • Synthetic analytes of interest include particularly drugs, including drugs of abuse and therapeutic drugs.
  • Other analytes of interest include pesticides, pollutants, and the like.
  • Analytes of interest which are ligands include haptens, hapten conjugates and antigens.
  • Receptors of interest include surface membrane proteins, immunoglobulins and specific binding fragments thereof, e.g. Fab and F(ab) 2 , and the like.
  • Samples may be obtained from any convenient source, including physiological sources such as blood, serum, plasma, nasopharyngeal aspirates, urine, cervical swab, saliva, spinal fluid, bone marrow, vitreous or the like. Blood supplies can be measured for bacterial infections. Samples may also be obtained from the environment, such as air, water, soil, minerals, petroleum or the like, where one is interested in the presence of pollutants, organisms, pesticides, etc. Samples further can be a bacterial culture on a transparent agarose medium, or other such transparent media. One may also obtain samples from various commercial sources, such as food processing plants and the like, where one is interested in the presence of trace contaminants.
  • Samples may be assayed directly or, if appropriate, pre-treated prior to assay. Pre-treatments which may be employed include extraction, dilution, chromatography, electrophoresis, HPLC, molecular weight or density separation. For example, if a sample is initially dry or viscous, it may be extracted, diluted or dissolved.
  • the assay medium further comprises particles.
  • the particles may be synthetic or naturally occurring particles. Synthetic particles, e.g. plastic beads, may be fabricated from a variety of materials which may be functionalized for linking specific binding pair members to the particle surface. Materials of interest include various plastics, such as acrylics and polystyrene, liposomes, polymerized liposomes, or other polymerized particles, e.g.
  • polydiacetylenic materials paramagnetic or magnetic materials such as iron oxide particles, glass such as controlled pore glass, silica, innert particles, latex, starburst dendrimers, colloidal metals, zeolites and graphite.
  • Naturally occurring particles include blood cells, immune cells, tissue cells, bacteria, eukarytoic cells, and the like, particularly red blood cells (erythrocytes).
  • the number of particles used in an assay will depend on the binding capacity of the particle, i.e. the specific binding pair on the binding surface, and will typically be the number of particles required for binding and for signal detection above background. Normally, between about 1000 and 10 6 particles will be sufficient for a particular assay. However, where specific binding occurs, fewer than 100 particles may be used. In principle, a single particle could be detected where a detector was sufficiently sensitive to distinguish the resultant diffraction pattern from the background noise.
  • the particles will typically range in size from about 1.0 to 100 ⁇ m, more usually from about 2 to 5 ⁇ m.
  • the individual particles or the cross-linked particles will have particular characteristics including optical densities, sizes, shapes, refractive indexes, or colors which differ from that of various components of the sample.
  • the diffraction patterns formed from the particles will be distinguishable from the diffraction patterns of the sample components.
  • the assay may be performed directly on the sample and various sample components need not be separated from the sample.
  • particles may be added directly to blood.
  • the characteristics of the particles can be altered by various methods.
  • the particles can be labeled prior to the assay, e.g. label red blood cells with colloidal gold.
  • the particles may be induced to change shape by exposure to various events, such as analyte binding, cross-linking or environmental changes, e.g. receptor binding or osmotic pressure. Examples of shape changing type particles include liposomes, cells and cancer cells.
  • more than one type of particle can be used in an assay to detect more than one type of analyte.
  • a first particle such as a latex particle
  • red blood cells can be used as a second particle.
  • the various types of particles must create distinct diffraction patterns so that each type of diffraction pattern represents a particular analyte.
  • reagents e.g. aldehydes to fix red blood cells. Stabilization of particles may assist in preserving the cross-linked particles, where cross-linking is included in the assay, especially during mixing or separation steps. It may further be necessary to functionalize the particles in order to provide for a member of a specific binding pair member on the particle surface. Functionalization techniques are adequately described in the literature cited supra and need not be exemplified here.
  • the number of functional groups on each particle to bind the binding pair member will be at least one and may be as many as ten or more functional groups per particle.
  • Techniques may be used to provide for a predetermined average number of functional groups capable of binding to the specific binding pair member and controlling the average number of specific binding pair members bound to the particles.
  • Techniques such as site-specific illumination for photoactivation, controlled kinetics, and chemical blocking, can be used for providing for a relatively sharp distribution of the ratio of the specific binding pair members to particle.
  • the assay medium also includes a solid transparent surface.
  • the solid surface is selected to provide for transparency in the wavelength range of interest, planarity and minimization of the amount of randomly scattered light.
  • Various smooth, flat surfaces may be employed, such as molded polystyrene, glass, acrylics, etc. Any transparent surface which is solid or porous which may include other beam geometries and which does not interfere optically with the optical diffraction process can also be used.
  • the solid surface may or may not include members of a specific binding pair which are randomly distributed on the surface. These spdm's provide for specific binding of the particles and/or analyte to the surface. Where the solid surface comprises randomly distributed sbpm, the materials selected for the solid surface should provide for the convenience of specific binding and ease of removal of non-specificaUy bound particles. Typically, the surface will be modified to provide for the randomly distributed sbpm's.
  • any convenient technique may be employed to modify the solid surface with the sbpm. In some instances, it may be sufficient to contact the solid surface to a solution comprising the sbpm, e.g. where the sbpm is a protein, such as an antibody or fragment thereof. Methods of stable coating glass and plastic surfaces are well known. See Harlowe & Lane, Antibodies: A laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. (1988). In many instances, where the binding member is not a protein, it may be conjugated to a protein leaving the binding site available for binding to the complementary member. For example, haptens may be conjugated to a protein which will not interfere with the assay and the protein conjugate used in the binding solution.
  • a non-binding analyte or mimetic thereof may be directly bound to the solid surface without having to functionalize the surface to provide covalent binding of the non-binding analyte or mimetic thereof.
  • the surface may be activated or functionalized to provide for covalent binding of the binding member to the surface.
  • the particular technique used in treating the solid surface will depend on the composition of the surface and the sbpm, e.g. the functional groups available on the sbpm for reaction. For treating the surfaces of glass having a functional alkylsilyl group, silanization may find use, where the sbpm's comprise suitable functional groups, e.g.
  • the surface may be functionalized to provide for reactive amino, carboxy, thio, sulfonyl, hydroxy or other functional groups, by acylation, nitration and reduction, oxidation with ozone, chlorosulfonation, and the like.
  • the specific functional group provided on the solid support will depend on the sbpm. If the binding member does not naturally comprise a useful available functional group, the binding member may be modified, so as to provide for a functional group that will react with the activated surface, e.g. amino with carboxy, thiol with activated olefin, hydroxy with an activated halogen, and the like.
  • the sbpm's may be a cross-linking agent.
  • the cross-linking agent provides for cross-linking of the particles in an amount proportional to the amount of analyte in the sample.
  • the particular cross-linking agent included in the assay medium will depend on the type of particles used and the analyte to be detected, as well as the particular binding format desired.
  • the cross-linking agent will comprise at least one particle binding moiety coupled to at least one analyte binding moiety.
  • the cross-linking agent can bind a plurality of one type of particle to a singular particle of another type, e.g. several red blood cells bound to a single latex particle, or several colloidal gold particles bound to a single red blood cell.
  • the cross- linking agent can also bind several of the same type of particles together.
  • the particle binding moiety may be an antibody or binding fragment thereof, e.g. Fab or F(ab) 2 , lectins, peptides with specific affinity for the particle surface and the like.
  • the particular analyte binding moiety of the cross-linking agent will depend on the analyte of interest and may be antibody or specific binding fragment thereof, antigen, hapten, lectin, enzyme or other binding protein or substance.
  • Exemplary cross-linking agent include antibodies or haptens immobilized on the particles, as well as the erythrocyte agglutination reagents comprising erythrocyte binding molecules coupled to analyte binding molecules disclosed in U.S. Patent No. 5,086,002 and International Patent Publication No. WO 91/04492, the disclosures of which are specifically incorporated by reference.
  • the amount of cross-linking agent included in the assay medium will not be critical, so long as the amount included is not a limiting amount with respect to cross-linking interactions which may occur in the assay medium.
  • preparation of the assay medium may also involve incubations.
  • the incubation conditions will be chosen to promote the complexing between complementary binding moieties in the assay medium, e.g. complexes between sbpm's, cross-linking of particles, displacements of one binding pair member by another binding pair member, and the like.
  • the incubation conditions also promote the random distribution of the resulting diffraction particles in a plane which is parallel to the solid transparent surface.
  • incubations will be carried out at temperatures ranging from about
  • aqueous media will be employed, although polar organic solvents may be used to varying degrees.
  • the solutions may be buffered in the range of about pH 3 to pH 10, more usually pH 6 to pH 9, with a wide variety of buffers, including phosphate, borate, Tris, HEPES, carbonate and the like.
  • concentration of the buffers will be sufficient to maintain the pH, generally ranging from about 25 to 500 mM.
  • Other components in the medium may include salt, generally ranging from about 0.01 to 0.2 M, stabilizing agents, biocides, e.g. azide, and the like.
  • agitate the assay medium so that components of the assay medium are evenly distributed, e.g. where cross-linking agents are included in the assay medium.
  • Agitation may be accomplished using any suitable means, including shaking, mixing, sonication, microwaves, rocking , pulsating as with a suction device, stirring as with an impeller device, rolling, vibrating, and the like.
  • the various elements may be combined simultaneously or sequentially.
  • the sample, diffraction particles, and reagents may be combined in a first step and then combined with the solid surface in a second step.
  • Stepwise preparation of the assay medium may be used where it is desirable to incubate the sample, particles and cross-linking agent prior to combination with the support.
  • the assay medium a variety of interactions may take place between the analyte, particles (where the analyte is not the particle), and the specific binding pair members, depending on the protocol and nature of the analyte.
  • Different combinations of reagent will be employed and different methods of creating the coplanar and randomly distributed diffraction particles.
  • the particular interactions will influence the nature of the observed diffraction pattern.
  • the interactions may involve competitive binding or non-competitive binding.
  • the protocols may be conveniently divided into two broad categories based on whether the diffraction particles are mobile by being either in a liquid layer covering the surface or in contact with the solid surface, i.e. settling on the surface, or are immobilized by being bound to sbpm's on the surface.
  • the assay may include the cross- linking or removal of cross-links from individual diffraction particles through interaction with analyte.
  • particles are conjugated to a molecule which will result in binding to a polyepitopic analyte.
  • the analyte acts as a bridge to bring two particles together.
  • the linked particles provides for a different diffraction pattern from that obtained solely from unlinked particles.
  • the assay procedure could include the bridging of the particles with a polyepitopic molecule, and the subsequent displacement of the bridging by the analyte. The analyte would cause the linked particles to separate into individual particles.
  • the diffraction pattern obtained from the unlinked particles would be different from the diffraction pattern obtained from the linked particles.
  • non-competitive assays may further include the use of more than one type of particle to alter the diffraction pattern in relation to the amount of analyte.
  • a first particle has a molecule which reacts with the analyte.
  • a second molecule is conjugated to a second particle.
  • the analyte acts as a bridge between the first particle and a second particle, e.g. the first particle is a red blood cell and the second particle is colloidal gold, a polymerized vesicle, or a magnetic bead.
  • Multiple second particles can attach to a single first particle.
  • the second particle can have characteristics, e.g. size, shape, optical density, refractive index, or color, which contrast with the first particle.
  • the diffraction pattern of the linked particles would differ from the unlinked particles in proportion to the amount of analyte.
  • a complementary binding member is attached to a second particle, so that the analyte in the sample and the second particle would compete for the complementary member on the first particle surface.
  • the number of particles capable of binding to the second particle would be inversely proportional to the number of molecules of analyte in the sample.
  • the solid surface comprises specific binding pair members, thereby providing for specific binding of the diffraction particles to the surface.
  • the interaction between the analyte and the diffraction particles may be as a result of direct binding or displacement of a member of a specific binding pair complex.
  • a simple competitive assay one could provide for a complementary binding member on the substrate surface.
  • the surface binding pair member would compete with the analyte for the member on the particle.
  • the number of particles capable of binding to the surface within a predetermined time would be inversely proportional to the amount of analyte.
  • analyte is polyepitopic
  • the number of particles bound to the surface would be directly proportional to the number of molecules of analyte in the sample medium.
  • the cross-reactive member is bound to another member of a specific binding pair.
  • the cross-reactive member could be bound to biotin, where streptavidin or avidin is bound to the surface.
  • the analyte displaces the conjugate of the cross-reactive member and biotin, the particle will no longer be able to bind to the surface.
  • the number of particles which bind to the surface will be inversely proportional to the number of molecules of analyte in the sample.
  • Another protocol would provide for analyte facilitating the cross-linking of particles or the separation of linked particles. For example, one could provide for particles which are conjugated to a molecule which will result in binding to the surface. A second molecule reacts with a polyepitopic analyte, so that the analyte connects together the two particles. Similarly, the analyte could displace a polyepitopic molecule which connects the two particles together. Thus separate individual particles would be created.
  • the diffraction particles is iteself the analyte in the sample.
  • This protocol requires that the particles create a diffraction pattern that is distinct from other components in the sample.
  • the particles is bacteria
  • colonization of the bacteria can be determined and certain resistant strains distinguished from non- resistant strains.
  • the bacteria can be cultured in a medium that induces replication of the bacteria, e.g. agarose gel.
  • the bacteria can be bound to the surface of the plate or can be suspended in the agarose gel.
  • the particle are deformed red blood cells, neoplastic cells or other deformed particles, e.g. vesicles, polymerized vesicles, beads, etc.
  • the particles are distinguished from normal cells by the particular diffraction pattern that is created.
  • removal of unreacted particles may be accomplished by a number of methods, including mechanical methods, e.g. gyration, shaking, etc., by gravity, field flow fractionation, use of filters, use of surface geometries * or combinations thereof.
  • mechanical methods e.g. gyration, shaking, etc.
  • field flow fractionation e.g., gyration, shaking, etc.
  • filters e.g., gyration, etc.
  • surface geometries * or combinations thereof e.g., a magnetic field may be used. Centrifugation or gravity may be used with particles with a specific gravity different from that of the surrounding medium. A gentle, continuous flow such as capillary flow, may also be used.
  • the particles must be in randomly distributed in a single plane which is substantially parallel to the surface.
  • the amount of diffraction particles should be sufficient to form this single plane.
  • the coplanar diffraction particles will cover .001 to less than 100% of the plane, more usually from about 1.0 to less than 100% of the plane and typically from about 10 to 50% of the plane.
  • the solid surface and assay medium may by tilted such that the diffraction particles spread along the plane.
  • a properly sized diffraction chamber may be employed to contain the diffraction particles.
  • the surface to volume ratio in the diffraction chamber should be large enough to allow for flow of solution, but narrow enough to permit only a monolayer of particles to form. Further techniques may include centrifugation by spin coating the surface.
  • the surface is irradiated with coherent light to provide for passage of light through the diffraction particles to produce the diffraction pattern.
  • the amplitude and spacing of the resultant diffraction pattern is first detected and measured. The amplitude and spacing of the pattern can be detected in regular intervals over a given period of time, in order to determine the rate of formation or change of the diffraction pattern. It may be desirable to take the measurements during the early stages of the assay, prior to massive agglutination of the dffraction particles, often during the first 5 minutes, more usually during the first 2 minutes.
  • a data reduction means is then employed to relate the measured amplitude spacing of the diffraction pattern to the presence of analyte in the sample.
  • any light source that provides the desired coherence, intensity and wavelength of light may be employed, such as a laser.
  • the wavelength will be chosen to be, at most, not greater than twice the diameter of the diffraction particles.
  • the wavelength of the irradiating light will generally not be greater than the diameter of the particles, and usually will not exceed the radius of the maximum sized diffraction particles.
  • One or more coherent light sources may be employed, so as to interrogate different test and control zones on the support surface, or means may be provided for moving the light beam or source so as to irradiate each assay site independently.
  • the particular choice of irradiation source will be one of convenience and economics, and is not critical to this invention.
  • red solid state lasers can be employed for low cost instrumentations.
  • a beam expander may be employed, where the beam expander will expand the beam to cover an area of from about 0.05 mm to 2 cm 2 , more usually 0.5 mm 2 to 1.0 cm 2 . Integration over a large area may be advantageous in providing a large number of particles to average and thus resulting in improved precision.
  • the beam may be focused and 2-D scanning techniques may be employed.
  • the diffraction pattern resulting from interaction of the irradiating light with the coplanar diffraction particles may be detected using a variety of detectors. Where one is measuring the diffraction pattern from single sized particles, a simple detector comprising two photodiodes may be sufficient. One of the photodiodes would be located at a diffraction maximum and the other would be located at a diffraction minimum. The difference between the current generated at these two photodiodes would be a measure of the diffraction pattern. To achieve a better signal to noise ratio, a linear diode array detector may be used to measure a section of the diffraction profile.
  • a linear diode array detector would also allow one to measure the diffraction pattern from different sized diffraction particles.
  • An array detector such as a CCD or CID array may also be employed.
  • a rotating single photodiode in a semi-circular pattern through the diffraction rings. This detection would yield a scattering intensity profile as a function of angle and would provide for the measurement of particle size distribution and number above background.
  • a data reduction and analysis computer form is used to relate the measured spacing and amplitude to the presence of analyte in the sample.
  • Data reduction means which relate the observed diffraction pattern to the presence of analyte are readily devisable.
  • the data reduction computer form may comprise an algorithm which converts the amplitude and spacing of the observed diffraction pattern to a radial cross-section which is representative of the diffraction pattern, e.g. a plot of intensity as a function of distance from the center of the pattern.
  • the radial cross-section may then be compared with a radial cross-section of a diffraction pattern from a known diffraction particle profile, e.g. a control or standard, using a variety of techniques.
  • the logic means could compare the radial cross sections at a predetermined distance from the center of the profile, with a difference in values indicating a change in the diffraction pattern as a result of the presence of analyte in the sample.
  • one could integrate the radial cross section of a diffraction pattern of a sample over defined intervals and compare the resultant integral to an integral obtained from the diffraction pattern of a standard or control sample.
  • a ratio scheme analogous to those used in spectroscopy could be employed to compare the values.
  • a Fourier transform analysis can be employed to pick periodic features in the diffraction pattern analysis.
  • a quantitative, or at least semi-quantitative, value for the presence of analyte is also possible to obtain a quantitative, or at least semi-quantitative, value for the presence of analyte.
  • the radial cross-section derived from the diffraction pattern is compared to radial cross-sections derived from calibration samples having known concentration of analyte, where the conditions of the assay sample and calibration samples are identical.
  • T e rate of formation of the diffraction pattern or change in diffraction pattern can be measured by taking periodic readings of amplitude and spacing of the diffraction patterns.
  • the subject method is simple, can be readily automated, and provides for an accurate means of qualitative and quantitative measuring.
  • devices which can be employed may range from hand-held devices which may be used in the field to highly sophisticated devices for use in clinical laboratories.
  • the devices should facilitate analyte detection by enhancing control and reproducibility over reagent interaction in the assay.
  • the devices can promote the formation of particle monolayers, cross-linking of particles and separation of assay components, such as separating linked particles from unlinked particles.
  • the design can be such that a multitude of analytes can be detected and several assays can be simultaneously performed including tests and control assays.
  • the device can also provide for control over temperature of the assay components.
  • the device comprises at least one reaction well, a means to add sample and reagents, and a means to form random coplanar distributed diffraction particles.
  • At least one of the wells can be used for a control, and the remainder wells can be used for tests.
  • the wells should have a hydrophilic surface and be of a dimension suitable to hold the volume of sample and reagents.
  • the device further includes a means for adding sample and reagents to the wells. Examples of such means for addition include channels or ports connected to the wells.
  • the wells may also have an assessable open top section for pipetting directly into the well. Dry assay reagents including specific binding pair members may be dired into appropriate test or control zones.
  • the device includes a means to form a single plane of diffraction particles.
  • This means may include a means to tilt the reaction wells, either manually or by an automated instrument, or a diffraction chamber of the appropriate surface to solution depth ratio for producing a particle layer.
  • Another embodiment of the device comprises a housing with a bottom plate and a top plate. The plates are sealed together to reduce evaporation during performance of the assay.
  • the device contains openings for sample application, reagent application, venting of gasses and/or waste removal.
  • a sample application port is typically located in the top plate of this embodiment.
  • the sample application port is in fluid communication with a main channel which provides for capillary flow.
  • the main channel may contain a separation means to separate diffraction particles form the sample or to separate components from the sample. Suitable separation means can include filters, porous material, and magnetic or paramagnetic systems.
  • the main channel can further comprise a capillary valve to stop flow for a desired period of time.
  • the sample may be held by the capillary valve while the diffraction particles first react with a conjugate. After a given period of time, the sample is allowed to pass through the main channel to react with the diffraction particles.
  • Capillary valves are described in PCT application Ser. No. PCT/US94/01623, entitled, "Disposable Device in Diagnostic Assays," filed on February 14, 1994, which is assigned to the assignee of the present application.
  • This embodiment includes a reaction area which is in fluid communication with the main channel.
  • the reaction area is of a dimension suitable for the volume of sample and reagents.
  • the opposite walls of the reaction area are optical clear windows which are transparent to a particular wavelength of visible coherent light.
  • the walls are further hydrophilic.
  • Located on one wall is a transparent support to which the diffraction particles may bind or settle if the particle assay so requires. Diffusely or non-diffusely bound to one or both walls can be dry reagents.
  • Cross-linking of particles and separation of cross-linked particles can be controlled through use of a variety of means.
  • the solid surface which is in the reaction area may comprise hydrophilic areas surrounded by hydrophobic barriers to attract and isolate the assay solution onto areas of the platform.
  • the reagent area is in the form of a U-shape with the surface being at the bottom of the U- shape. The U-shape promotes concentration of particles on the support and thus cross- linking of particles.
  • the device design can allow for centrifuging to further concentrate the diffraction particles.
  • the reagent area in the device may comprise an agitation means where the reagents, diffraction particles and/or sample can be mixed.
  • the agitation means may be mechanical tilting of the platform, rotating with an impeller or magnetic beads, pulsating with a suction device, or the like. Agitation may employ manual or automated means.
  • the device may further include a sub-channel which is smaller than the main channel.
  • the sub-channel is in fluid communication with the main channel at one end and the reaction area at the other end. If a separation means is present in the main channel, at one end the sub channel is located upstream from the separation means.
  • the sub-channel allows for a small portion of the sample with diffraction particles to exit the main channel through an opening in the main channel. The diffraction particles are allowed to enter the reaction area at the other end and react with a conjugate and the sample.
  • the device further provides for a separation means in the main channel, the naturally occurring diffraction particles may be separated from the sample and allowed to enter the reaction area via the sub- channel. In this manner, the ratio of diffraction particles to analyte in the sample can be controlled to improve the sensitivity of the assay.
  • the reaction area of the device may be in fluid connection with a waste area.
  • the waste area serves to receive the sample, diffraction particles, and/or wash fluids that flow through the reaction area.
  • Coherent light source 12 provides light beam 14 which is expanded by beam expander 16 and passes through transparent support 18. Randomly distributed on the surface of the support 18 is binding member 22. Diffraction particles 28 comprise complementary binding member 30 on their surface. In the assay, analyte competes with the surface binding member 22 for binding to the particle binding members 30. Light passing through the support is diffracted by the particles 28, filtered by the diffraction grating 24 and measured by the detector 26.
  • Fig. 2 is a perspective view of the support where more than one analyte is assayed simultaneously using a uniquely sized diffraction panicle for each analyte.
  • the surface has different binding members 22 (indicated by different geometric shapes) which compete with the different analytes for binding to the different sized particles 28.
  • square binding member competes with one class of analyte in binding to larger particles while triangle binding member competes with another class of analyte in binding to the smaller particles. From the amplitude and spacing of the resultant diffraction pattern arising from the different sized particles, the diffraction pattern can be related to the presence of both analytes in the sample.
  • Fig. 3. provides a simple circuit for detecting the diffraction pattern.
  • Two photodiodes are employed, one at a diffraction maximum 34 and the other at a diffraction minimum 36 for detecting the diffracted light from support 18.
  • the difference in current generated by these photodiodes is a measure of the diffraction.
  • the placement of these photodiodes would be limited to measurement of a single particle size.
  • the signal difference between the diffraction peak and node from support 18 is differentially amplified using amplifiers 62, feedback capacitors and resistors 50 and 54, respectively, and a differential amplifier 44.
  • the circuit is grounded at 60 and the voltage output 46 gives a measure of the signal. Fig.
  • Coherent light source (a He/Ne laser) 70 provides a coherent light beam 72 which passes through beam expander 74, neutral density filter 76 and is redirected at mirror 78.
  • Redirected beam 80 passes through an adjustable aperture 82 to irradiate the floor 86 of diffraction chamber 84.
  • Settled on the floor of the diffraction chamber is a monolayer of diffraction particles 88. Light passing through the particle monolayer is diffracted into a diffraction pattern on a projection screen 90 which is imaged by a CCD camera 92.
  • Sample solution was prepared by diluting freshly drawn blood in a 1/12 dilution ratio with 85 % saline solution. 10 ⁇ l samples of this dilution were added to a plurality of microtitre wells. To half of the microtitre wells were added 25 ⁇ l of a cross-linking reagent solution comprising a bifunctional antibody, 5 mg/ml BSA. lOOmM phosphate and 0.05% azide (obtained from Agen Biomedical Ltd., Australia). To the remaining wells were added 25 ⁇ l of a negative control comprising .9% NaCl and 0.05% azide (also obtained from Agen Biomedical Ltd., Australia). The contents of each well were agitated and incubated for 2 min. Agitation was performed on a plate shaker with the speed adjusted to maintain the erythrocytes in suspension.
  • serial dilutions of increasing concentrations of analyte were prepared and tested.
  • the serial dilutions were prepared as follows. Analyte solution comprising D-dimer standard plus buffer reagents ( D-dimer solution from Agen Biomedical Ltd., Australia) was diluted in increasing amounts of PBS buffer, pH 7.2 to obtain dilutions having the following ratios of solution to buffer: 1/2400, 1/1200, 1/960, 1/480, 1/240. 25 ⁇ l of each dilution were added to a positive and negative well. Each well was incubated and agitated for 15 min. The contents of each well were visually checked for agglutination every five minutes.
  • a 26 ⁇ l sample from each microtiter well was placed in a diffraction chamber and incubated for 2 min. to allow the cells present in the sample to settle on the surface.
  • the dimensions of the diffraction chamber were chosen so that the erythrocytes would randomly settle in a single plane on the bottom surface of the chamber. Because the blood in the assay was diluted in a ratio of 1/72 following addition of the various reagents, a diffraction chamber having a volume to area ratio of 0.030 cm 3 / 1.00 cm 2 was used. The diffraction chamber was placed in the instrument shown in Fig. 4.
  • the bottom surface of the diffraction chamber was then irradiated with a laser beam from a 1 mW helium-neon laser which had been passed through a neutral density filter (3.0 OD) and a 5x beam expander.
  • light diffracted by the monolayer of cells on the surface of the diffraction chamber floor was projected onto a white translucent screen (high grade paper taped to a glass slide).
  • the projection on the screen was imaged using a cooled CCD camera (Photometries, Ltd., Arlington, AZ).
  • the diffraction pattern was converted to a radial cross section or plot of the intensity as a function of distance from the center beam.
  • the radial cross sections obtained for each dilution were then compared with a radial cross section observed from a known, negative control where the cells were not agglutinated.
  • the diffraction assay provided positive indications of analyte where analyte concentrations equaled, or exceeded, the analyte concentration found in the 1/960 dilution.
  • visual agglutination assays were only able to provide positive results where the concentration of analyte equaled, or exceeded, the concentrations found in the 1/480 dilution.

Abstract

Des procédés et appareils permettent de détecter un analyte dans un échantillon grâce à des motifs de diffraction. Selon un tel procédé, un échantillon supposé contenir un analyte, et des particules, si l'analyte n'est pas la particule recherchée, ainsi que des éléments d'une paire à liaison spécifique, sont combinés dans un milieu de titrage avec une surface solide transparente à une gamme de longueurs d'onde utile. Dans une variante, un agent de réticulation est inclus dans ce milieu de titrage, où les particules se réticulent proportionellement à la quantité d'analyte présente dans l'échantillon. Tandis que les particules interagissent avec un analyte et des éléments d'une paire à liaison spécifique, il se forme des particules de diffraction de plus grosse taille qui se répartissent de manière aléatoire dans un seul plan du milieu de titrage. Ces particules de diffraction peuvent rester mobiles et en solution ou se déposer sur la surface solide. Dans une autre variante, la surface solide comprend des éléments d'une paire à liaison spécifique qui sont répartis de manière aléatoire et permettent la liaison spécifique des particules de diffraction avec la surface. Ces particules de diffraction sont alors éclairées avec une lumière cohérente qui est diffractée selon un motif de diffraction par lesdites particules. L'espacement et l'amplitude du motif de diffraction sont détectés et mesurés. Un dispositif logique permet de corréler cet espacement et cette amplitude à la présence de l'analyte dans l'échantillon.
PCT/US1995/013410 1994-10-21 1995-10-19 Titrage a l'aide de particules de diffraction WO1996012962A1 (fr)

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