WO2015008287A1

WO2015008287A1 - Device and method for analysis of multiple analytes

Info

Publication number: WO2015008287A1
Application number: PCT/IL2014/050647
Authority: WO
Inventors: Yury Yuzefovitch VENGEROV
Original assignee: Tatakoto Company Ltd.; LOTAN, Mirit
Priority date: 2013-07-18
Filing date: 2014-07-17
Publication date: 2015-01-22
Also published as: EP3055694A1; IL227524A0

Abstract

The invention relates to point-of-care devices and methods for simultaneous diagnosis and analysis of multiple analytes in a liquid sample.

Description

DEVICE AND METHOD FOR ANALYSIS OF MULTIPLE ANALYTES

TECHNOLOGICAL FIELD

The present invention relates to diagnosis and analysis of multiple analytes in a liquid sample.

PRIOR ART

References considered to be relevant as background to the presently disclosed subject matter are listed below:

[1] US 2005/0203353

[2] WO 2000/31539

[3] US 7,858,396

[4] WO 2003/062824

[5] WO 2012/037369

[6] Taranova et al, Microchim Acta 2013, DOI 10.1007/s00604-013- 1043-2

Acknowledgement of the above references herein is not to be inferred as meaning these are in any way relevant to the patentability of the presently disclosed subject matter.

BACKGROUND

Diagnostic apparatuses and methods for quantitative and qualitative analyses of substances (analytes) in liquid samples of various origins, such as urine, blood, saliva and environmental samples have been developed over the past 30 years (for example, see references [1-6]). Among the more common methods used today for detecting polypeptide analytes are RIA (radio-immunological assay) and ELISA (enzyme linked immunosorbent assay), which were modified for analysis of a plurality of analytes. However, RIA and ELISA, as well as other immunodiagnostic or nucleic acid based detection methods, typically involve multiple steps and often require professional skill, expensive reagents, and complex and stationary machinery. As such, these methods and apparatuses cannot be readily used in small-scale hospitals, field-labs and home testing (i.e. point-of-care applications).

Therefore, there is a need for generic diagnostic assay methods, which may provide simple and rapid detection and quantification of multiple analytes in point-of- care settings.

GENERAL DESCRIPTION

The present invention provides a planar, porous matrix for use in the detection of a plurality of analytes in a liquid sample. Other aspects of the invention comprise devices, a diagnostic system and methods of using such device.

Thus, in one of its aspects, the invention provides a planar, porous matrix, comprising: at least one first array of first spots and at least one counterpart second array of second spots spaced apart along a proximally-distally extending axis, wherein the second array being distal to said first array, each first spot having a counterpart second spot, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis; each of said first spots comprises a recognition element capable of specifically binding to an analyte in a sample, thereby forming a complex with (i) said specific analyte or (ii) an analyte-comprising species, at least some of the first spots comprise a recognition element that differs from recognition elements in other first spots; the complex being capable of migrating along said line with a liquid advancing in the direction of said axis; and each of said second spots comprises an immobilized capturing agent capable of binding said complex.

Another aspect of the invention provides a planar, porous matrix, comprising at least one first array of first spots and at least one counterpart second array of second spots spaced apart along a proximally-distally extending axis, wherein the second array being distal to said first array, each first spot having a counterpart second spot, each pair of first spot and corresponding second spot being situated on a line that links the spots and is parallel to said axis; each of said first spots comprises a conjugate, the conjugate comprising a label conjugated to a binding moiety for binding to a specific analyte in a sample thereby forming a complex with said analyte, at least some of the first spots comprise conjugates that differ from those in other first spots; the complex being capable of migrating along said line with a liquid advancing in the direction of said axis; each of said second spots comprises an immobilized capturing agent capable of binding (i) to the analyte in the complex, or (ii) to the binding moiety in the complex.

A further aspect of the invention provides a planar, porous matrix, comprising at least one first array of first spots and at least one counterpart second array of second spots, the arrays being spaced apart along a proximally-distally extending axis, wherein the second array being distal to said first array, each first spot having a counterpart second spot, each pair of first spot and corresponding second spot being situated on a line that links the spots and is parallel to said axis; each of said first spots comprises an antigen associated with a first member of an affinity couple, the antigen being capable of specifically binding to a labeled analyte -comprising species, thereby forming a complex capable of migrating along said line with a liquid advancing in the direction of said axis; and each of said second spots comprises an immobilized second member of said affinity couple for binding to the first member of the affinity couple.

In another one of its aspect, the invention provides a device for detection of analytes in a liquid sample, comprising a sample -receiving zone for receiving the liquid sample and a planar, porous matrix having a reaction zone, the reaction zone comprising at least one first array of first spots and at least one counterpart second array of second spots, the arrays being spaced apart along a proximally-distally extending axis, said first array being distal to the sample -receiving zone and the second array being distal to said first array, each first spot having a counterpart second spot, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis; each of said first spots comprises a conjugate, the conjugate comprises a label conjugated to a binding moiety for binding a specific analyte in the sample thereby forming a complex, at least some of the first spots comprise conjugates with a binding moiety that differs from that in other first spots, the complex being capable of migrating along said line with a liquid advancing in the direction of said axis, and each of said second spots comprises an immobilized capturing agent for binding (i) to the analyte in the complex, or (ii) to the binding moiety in the complex.

In yet another aspect, the invention provides a device for detecting immunoglobulins in a liquid sample, a sample-receiving zone for receiving the liquid sample and a planar, porous matrix having a reaction zone, the sample-receiving zone comprising a conjugate of a label and a binding moiety, the binding moiety capable of binding an immunoglobulin analyte in said sample, to thereby form a labeled analyte - comprising species; and a reaction zone comprising: at least one first array of first spots and at least one counterpart second array of second spots, the arrays being spaced apart along a proximally-distally extending axis, said first array being distal to the sample- receiving zone and the second array being distal to said first array, each first spot having a counterpart second spot, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis; each of said first spots comprises an antigen for binding a specific species of immunoglobulins, to thereby form a complex with said labeled analyte-comprising species, the antigens being associated with a first member of an affinity couple, the complex being capable of migrating along said line with a liquid advancing in the direction of said axis; and each of said second spots comprises an immobilized second member of said affinity couple for binding to the first member of the affinity couple.

According to another aspect of the invention there is provided a diagnostic system for detecting analytes in a sample, comprising a device of the invention as herein described, and an image capturing device for capturing an image formed in said reaction zone.

According to a further aspect, the invention provides a method for detecting a plurality of analytes in a liquid sample, the method comprising: (a) bringing the liquid sample into contact with a sample receiving zone associated with a planar porous matrix, such that the sample transfers from the sample receiving zone to the planar porous matrix, (b) allowing the sample to migrate in a direction parallel to said axis, and (c) detecting the label in the spots of said second array, presence of the label being indicative of presence of an analyte in the sample. The matrix comprises a reaction zone that is spaced apart and distal to the sample receiving zone along a proximally-distally extending axis. The reaction zone has at least one first array of first spots and at least one counterpart second array of second spots. The at least one first array of spots comprise each a recognition element capable of specifically binding to a specific analyte in the sample, to thereby form a labeled complex with said specific analyte; the labeled complex comprising a label-comprising species originally present in (i) the sample-receiving zone, or (ii) each of said first spots. The at least one second array of second spots is spaced apart along said axis from and is distal to said first array. Each first spot in the first array has a counterpart second spot in the second array, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis. Each of the second spots comprises an immobilized capturing agent for binding the labeled complex. The labeled complex is capable of migrating along said line with a liquid advancing in the direction of said axis.

By one embodiment of the aforementioned aspect, the matrix comprises a reaction zone spaced apart and distal to the sample receiving zone along a proximally- distally extending axis, the reaction zone comprises at least one first array of first spots and at least one counterpart second array of second spots. At least some of the first spots comprise a conjugate that has a label and a binding moiety for binding a specific analyte in the sample to thereby form a labeled complex. At least some of the first spots comprise a conjugate with a binding moiety that differs from that in other first spots. The at least one counterpart second array of second spots is spaced apart along said axis and is distal to said first array. Each first spot in the first array has a counterpart second spot in the second array, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis. Each of said second spots comprises an immobilized capturing agent for binding to (i) the analyte in the labeled complex, or (ii) the binding moiety of the labeled complex. The labeled complex is capable of migrating along said line with a liquid that advances in the direction of said axis;

By another embodiment, the sample receiving zone comprises a conjugate of a label and a binding moiety, the binding moiety being capable of binding an immunoglobulin analyte in said sample, to thereby form a labeled analyte-comprising species. The matrix comprises a reaction zone spaced apart and distal to the sample receiving zone along a proximally-distally extending axis. The reaction zone has at least one first array of first spots and at least one counterpart second array of second spots, the arrays being spaced apart along a proximally-distally extending axis. Said first array is distal to the sample-receiving zone and proximal to said second array. Each first spot in the first array has a counterpart second spot in the second array, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis. Each of said first spots comprises an antigen for binding a specific species of immunoglobulins in the sample to form a labeled complex with said labeled analyte- comprising species. The antigens are associated with a first member of an affinity couple. The labeled complex is capable of migrating along said line with a liquid advancing in the direction of said axis. Each of said second spots comprises an immobilized second member of said affinity couple for binding to the first member of the affinity couple.

DETAILED DESCRIPTION OF THE INVENTION

The arrangement of arrays on the matrices of the invention comprises discrete spots for binding and capturing different analytes, enabling analysis of multiple analytes in a single sample and a single assay run (i.e. multiplex detection).

The term analyte is used herein to denote a chemical or biological species, which may be present in a tested sample. Non-limiting examples of such chemical or biological species are infectious disease markers, diagnostic serum markers, biomarkers of animal or plant diseases, drugs and substances of abuse, hormones, immunoglobulins, environmental analytes, toxins, and others. In certain embodiments, the analyte is composed of amino acids (i.e. is a peptide, polypeptide or protein). In other embodiments, the analyte is composed of nucleic acids.

Infectious diseases markers are molecular determinants of a host or a pathogen, usually proteins. Non-limiting examples of such markers are Hepatitis B virus (HBV) surface antigen (HBs), antibodies to HBV, Human Immunodeficiency virus (HIV), Hepatitis C virus (HCV), Herpes Simplex viruses (HSV) and other virus antigens. Pro- calcitonin (PCT) is one of the known markers of bacterial infection (sepsis), especially in critical care.

Prognostic or diagnostic serum markers of mammalian diseases are molecular indicators that reflect the presence and/or severity of a disease state. Examples include troponin I, MB-type creatine kinase (CK-MB), brain natriuretic peptide (BNP) and myoglobin as markers of cardiac disease; prostate specific antigen (PSA); cancer antigen 125 (CA-125 or MUC16); cancer-embryonic antigen (CEA); D-dimer (a marker of coagulation and thrombocytopenia) and others.

Drugs and substances of abuse are smaller molecules having mood or mind altering effects, usually consumed for non-medicinal purposes, and may lead to physical and mental damage, dependence and addiction. Examples include marijuana, ***e, amphetamine and others, the presence of which is usually tested in urine. Hormones are protein or steroid molecules that partake in intercellular communication and signal transduction in a multicellular organism. The levels of specific hormones in the blood or urine are indicative of certain physiological states, such as human chorionic gonadotropin (hCG) used in pregnancy testing, Luteinizing (LH) and Follicle-stimulating (FSH) hormones used in determination of maturation and function of the reproductive system, and others.

Immunoglobulins (Ig) generally refer to antibodies of various types which may be indicative of various abnormal physiological conditions, such as allergies associated with elevated levels of IgE-type or IgG-type antibodies targeted to specific allergens or elevated levels of specific IgG-type antibodies in autoimmune diseases.

Environmental analytes are chemical or biological substances indicative of environmental pollution in food, water, etc, which may include antibiotics, toxins and other pollutants.

In the context of the present disclosure, nucleic acids will refer to nuclear and mitochondrial DNA, as well as RNA, which by the rule of homologous complementation may be used for the detection of infectious pathogens (bacterial or viral or other microorganism) in the host. For non-infectious diseases, nucleic acid- based diagnostics may be used to detect a specific gene variant or expression levels of a specific gene that may be indicative of certain diseases or disease conditions, such as genetic aberrations associated with congenital disorders, Philadelphia chromosomal translocation associated with chronic myeloid leukemia (CML) and others.

In the context of the invention, the liquid sample may be any sample for analysis in a liquid form. The sample may be originally in liquid form, or may be a solid sample solubilized in a carrier liquid, typically water or a suitable buffer. The sample may be of a biological source, such as blood, urine, saliva, cerebrospinal fluid or other medical or veterinary samples; a liquid fraction of any of these; or solids from such a source that are solubilized or suspended in a liquid. However other types of samples are also contemplated within the scope of the present invention (non-limiting examples of such samples are water or beverage samples, food samples, plant extracts, cell or bacterial cultural fluids, etc.).

One characteristic feature of the matrices of the invention is the presence of at least two arrays of spots on the matrix, that comprise at least one array referred to herein as a first array, and at least one other array referred to herein as a second array. Each spot in the second array (referred to as second spot) corresponds to a spot (i.e. first spot) in the first array.

Each of the first spots in the first array comprises a recognition element, which may be analyte-specific conjugates or antigens associated with a member of a binding couple, as will be further explained below. The recognition element is specific to analytes in the sample. Therefore, the recognition element in each of the first spots may differ from that in other first spots of the first array, thereby enabling association with different analytes in the sample. The term complex will denote, in the context of this disclosure, the species formed by the association of the recognition elements in the first spots with the analyte or analyte-comprising species originating from the sample, as will be further explained below.

The second array includes capturing agents for capturing the complex, which immobilize the complex to allow its ensued detection. The selection of the capturing agents is consistent with the nature of the complex to be captured, such that complementary couples are formed. In cases where the same capturing agent may be used for capturing all relevant formed complexes, the capturing agent may be placed onto the matrix in discrete spots or in a consecutive line. In the specific embodiment wherein the capturing agent is placed in a consecutive line, discrete spots will form (and therefore identified later on) where the complex has been immobilized by the capturing agent.

In some embodiments, each of the recognition elements is a conjugate. The conjugate comprises a label conjugated to, i.e. chemically associated with, a binding moiety for specifically binding the analyte in the sample to form the complex. In such a case, the capturing agent may bind either to the analyte in the complex, or to the binding moiety in the complex. Namely, in the so-called "sandwich type assay", the capturing agent may be the same or different binding moiety that binds specifically to the analyte in the complex. Alternatively, the capturing agent may be identical to the analyte, or may be another substance having the same specificity as analyte in which case the complex will be captured in the absence of analyte (so-called "competition assay").

The binding moiety may be a member of a binding couple, capable of specific interaction, consisting of antibody-antigen, complementary nucleic acid sequences, sugar-lectin, and receptor-ligand, where one of the binding couple is the binding moiety and the other is the analyte.

A specific example of a binding moiety, as well as of a capturing agent, are molecular entities that comprise binding sites of an antibody, such entities being antibodies or a variety of constructs which include the binding sites as known per se.

In another case, the capturing agent has the same specificity as the analyte and, therefore, binds to the binding moiety itself. In such a case, there will be an inverse correlation between the capturing of the complex and the detection signal obtained. Non-limiting examples of the capturing agent in accordance with this embodiment are immobilized analytes, or a conjugate of analyte with carrier proteins in case of low molecular weight analytes.

In such embodiments, the capturing agent may comprise a low molecular weight molecule conjuga ted to a larger molecule, usually a protein (i.e. a carrier molecule) for immobilization. Such selection of a capturing agent is typically suitable for capturing low molecular weight analytes, such as drugs of abuse.

At least some of the first spots comprise a conjugate with a binding moiety that differs from other first spots and hence capable of binding to a different analyte. The conjugates are loosely associated with the matrix and accordingly are capable of dissociating from the matrix and migrating with liquid that flows along the matrix along the said axis.

In other embodiments, each of the recognition elements is an antigen associated with a first member of an affinity couple. The recognition elements are capable of forming the complex with analyte-comprising species. The complex is immobilized by an immobilized capturing agent, which is a second member of said affinity couple that binds to the first member of the affinity couple.

The term analyte-comprising species denotes a multi-component molecular entity, which may comprise a label conjugated to an anti-immunoglobulin that binds to a specific immunoglobulin (i.e. the analyte) in the sample. The analyte-comprising species associate with the specific recognition elements in the first spots, to thereby form the complexes. In such embodiments, the capturing agent may be avidin, in which case the recognition element will comprise biotinilated antigens (i.e. antigens conjugated to biotin), where the biotin/avidin binding will be the basis for capturing the complex in the second spot. A specific example of such an antigen is an allergen.

The detection of an analyte in a sample is based on the detection and visualization of the captured complex once immobilized in the second spots. For this purpose, the complex is labeled by a label, which is present either in the first spots (as part of the conjugates) or in the analyte-comprising species, as defined above.

The label is typically optically detectable and encompasses any detectable label known in the art, typically optically detectable labels such as, but not limited to, fluorescent labels or colloidal particles. In some embodiments of the present invention, colloidal gold particles are used as a label which can act as an indicator of presence or absence of an analyte in a liquid sample. In other embodiments the conjugate may be labeled using a fluorescent material, such as, but are not limited to, fluorescent particles, quantum dots, lanthanide chelates, and fluorochromes, such as FITC, Rhodamine, etc.

In some embodiments the label is a nanoparticle, typically a gold nanoparticle. Such nanoparticles are those having at least one of their dimensions in the nanometric range, typically 15 nm to 80 nm in length or diameter. The use of a nanoparticle label further enables control of the flow-migration properties of the conjugate across the matrix. The term "averaged diameter", as used herein, whether relating to the nanoparticle labels or to the spots of the arrays, refers to the arithmetic mean of measured diameters, wherein the diameters range ±25% of the mean.

The arrays are located on the matrix in a spaced apart arrangement along a proximally-distally oriented axis. The terms proximal and distal (or any lingual variation thereof) will be used to denote positions or directions on the matrix relative to the point of placement of the liquid sample (i.e. the point where the sample comes into contact with the matrix).

This proximally-distally oriented axis, as will also become clear from the description below, thus defines the direction of migration of the liquid sample placed onto (or that is brought into contact with) the matrix towards the arrays of spots that are located distally to the sample placement point. In accordance with the invention, when a liquid sample migrates through the porous matrix in a linear direction, molecular entities that exist in discrete spots in the first array migrate in a linear direction with the migrating liquid, substantially along said proximally-distally oriented axis.

The matrix is formed of a material that permits uniform flow, such that a liquid sample placed on the matrix proximal to the first array, migrates substantially linearly along the proximally-distally oriented axis, whereby molecular entities that exist in the spots of the first array migrate with the flow of liquid, along discrete lines parallel to the axis, to the counterpart spot in the second array.

The matrix is formed of a material that permits uniform flow, such that a liquid sample placed on the matrix proximal to the first array, migrates substantially linearly along the proximally-distally oriented axis.

Thus, the fluid-assisted movement of the molecular entities or particles from the first spot towards the second spot and onwards defines a so-called flow channel. The term "channel" may at times be used herein to denote this linear flow along a substantially straight line virtually linking between counterpart spots. As will also be described below, the liquid which forms these flow channels may be focused by the use of flow focusing agents that are added to substances used in the spots in the first array.

The arrays are arranged such that the second array is more distal to the first array along said axis (i.e. spaced apart). Each first spot has a counterpart second spot corresponding to (or located on) the same flow channel and the two counterpart spots are arranged in a line parallel to said axis.

The terms counterpart or corresponding are used herein interchangeably to denote spots of the different arrays that are placed on the same flow channel, i.e. are located on discrete lines parallel to the proximally-distally extending axis. In other words, each spot in the second array lies on a line parallel to the proximally-distally oriented axis that passes through the corresponding first spot, thereby linking each first spot with its counterpart second spot. Therefore, the complexes formed at the first spots migrate along these lines to the counterpart second spots, forming virtual flow channels.

In some embodiments, the matrix may also comprise at least one array of spots that serves for control, which will be referred to herein as a third array. The third array comprises third spots, in which each of the third spots counterparts a first and a second spot in the first and second arrays, respectively, and comprising a control capturing agent for binding the recognition element.

Such control spots are used to verify the viability of the conjugate or the analyte- comprising species, and consists of capturing agents that are specific to the conjugate or the analyte-comprising species. In cases when the conjugate is not intact, no label will be detected at the control spots, indicating that the test is invalid. In cases where the same control capturing agent may be used for providing control signals for all relevant species on the matrix, the control capturing agent may be placed onto the matrix in discrete spots or in a consecutive line. In the specific embodiment wherein the control capturing agent is placed in a consecutive line, discrete spots will form (and therefore identified later on) where the relevant species have been immobilized by the capturing agent.

Each spot in the third array lies on the line parallel to the proximally-distally oriented axis that passes through its corresponding first and second spots.

In some embodiments of the invention, there may be more than two arrays. For example, there may be a plurality of pairs of first and second arrays; or there may be several second arrays or third arrays for each first array; etc. Examples of such embodiments will be described in somewhat more detail in the specific description below.

The spots may have a variety of shapes which may typically be circular, elongated, or rectangular. The spots may be mini-spots having a largest diameter not exceeding 2 mm, typically not exceeding 0.5 mm and at times even not exceeding 0.1 mm. As will be appreciated, the invention is not limited by the size, shape or manner of formation of such spots. For example, the first spots may be formed by a plurality of closely located or converged smaller spots.

The spots in the arrays are arranged such that each of said first spots is substantially equally distant from its counterpart second and third spots. In this arrangement, the spots in each of the first, second and third array are arranged along a first, second and third lines, respectively, the first, second and third lines being perpendicular to said axis. In some embodiments, the matrix is a lateral flow assay strip. The strip may have any elongated shape, provided that longitudinal capillary flow is enabled, e.g. a rectangular shape. The thickness of the porous matrix (e.g. the strip) is typically 0.1 to 2 mm, more typically 0.15 to 1 mm, preferably 0.2 to 0.7 mm, although any strip that allows capillary flow is suitable for use in accordance with the invention.

The width of the strip is typically less than 20 mm, preferably less than 10 mm, e.g. in the range of about 3 mm and 8 mm. The width of the strip can be determined depending on the amount of analytes to be tested and the type of housing encasing the strip. However, the dimensions of the strip may vary and should not be considered as limiting the invention in any manner.

Similarly, the strip may have any desired length provided that the capillary flow is enabled. The length is therefore determined depending on various requirements, e.g. the amount of analytes to be tested, the type of housing encasing the strip, the presence and number of control spots, preferred speed of reaction etc. The length of the strip is typically between about 2 cm and 10 cm, e.g. in the range of about 5 cm and 8 cm.

As noted above, by some embodiments, agents are added or conditions are provided to ensure that the flow of the liquid comprising the complex is focused, i.e. directed, in order to facilitate migration along the line in the direction of the axis. The flow focusing agents may be used for minimizing the thickness (i.e. the dimension perpendicular to the flow direction) of the flow channel, thereby preventing interaction between individual flow channels and minimizing side diffusion. The flow focusing agents may be agents that increase the viscosity or slow down dissolving of reagents in the spots by the migrating liquid. Alternatively, these may be agents that do not dissolve in the liquid, thereby forming a physical barrier in the first spot, forcing the liquid to flow circumferentially around the perimeter of the first spot, thereby slowing-down and focusing the flow of the liquid. Typically, the flow focusing agent is added to the reagents which form first spots. Examples of flow focusing agents are sucrose, glucose, glycerol, and others.

In accordance with one embodiment, each of the first spots comprises a detergent that facilitates dissociation between the conjugates and matrix to permit their migration with the migrating liquid.

In one of its aspects, the invention provides a planar, porous matrix, comprising: at least one first array of first spots and at least one counterpart second array of second spots spaced apart along a proximally-distally extending axis, wherein the second array being distal to said first array, each first spot having a counterpart second spot, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis;

each of said first spots comprises a recognition element capable of specifically binding to an analyte in a sample, thereby forming a complex with (i) said specific analyte or (ii) an analyte-comprising conjugate,

at least some of the first spots comprise a recognition element that differ from recognition elements in other first spots; the complex being capable of migrating along said line with a liquid advancing in the direction of said axis; and

each of said second spots comprises an immobilized capturing agent capable of binding said complex.

In another aspect, the invention provides a planar, porous matrix, comprising: at least one first array of first spots and at least one counterpart second array of second spots spaced apart along a proximally-distally extending axis, wherein the second array being distal to said first array, each first spot having a counterpart second spot, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis;

each of said first spots comprises a conjugate, the conjugate comprising a label conjugated to a binding moiety for binding to a specific analyte in a sample thereby forming a complex with said analyte, at least some of the first spots comprise conjugates that differ from those in other first spots; the complex being capable of migrating along said line with a liquid advancing in the direction of said axis;

each of said second spots comprises an immobilized capturing agent capable of binding (i) to the analyte in the complex, or (ii) to the binding moiety in the complex, as described above.

In a further aspect, the invention provides a planar, porous matrix, comprising: at least one first array of first spots and at least one counterpart second array of second spots spaced apart along a proximally-distally extending axis, wherein the second array being distal to said first array, each first spot having a counterpart second spot, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis;

each of said first spots comprises an antigen associated with a first member of an affinity couple, the antigen being capable of specifically binding to a labeled analyte - comprising conjugate, thereby forming a complex capable of migrating along said line with a liquid advancing in the direction of said axis; and

each of said second spots comprises an immobilized second member of said affinity couple for binding to the first member of the affinity couple.

As noted above, the invention further provides a device comprising the matrix of the invention for detection of analytes in a liquid sample. A specific embodiment of the matrix and of a device comprising it is one intended to be used for the detection of immunoglobulins in a liquid sample.

Therefore, in another aspect, there is provided a device for detection of analytes in a liquid sample, comprising:

a sample-receiving zone for receiving the liquid sample and a planar, porous matrix having a reaction zone, the reaction zone comprising:

at least one first array of first spots and at least one counterpart second array of second spots, the arrays being spaced apart along a proximally-distally extending axis, said first array being distal to the sample-receiving zone and the second array being distal to said first array, each first spot having a counterpart second spot, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis,

each of said first spots comprises a conjugate, the conjugate comprises a label conjugated to a binding moiety for binding a specific analyte in the sample thereby forming a complex, at least some of the first spots comprise conjugates with a binding moiety that differs from that in other first spots, the complex being capable of migrating along said line with a liquid advancing in the direction of said axis; and

each of said second spots comprises an immobilized capturing agent for binding (i) to the analyte in the complex, or (ii) to the binding moiety in the complex. In another aspect, the invention provides a device for detecting immunoglobulins in a liquid sample, a sample-receiving zone for receiving the liquid sample and a planar, porous matrix having a reaction zone,

the sample -receiving zone comprising a conjugate of a label and a binding moiety, the binding moiety capable of binding an immunoglobulin analyte in said sample, to thereby form a labeled analyte-comprising conjugate;

and a reaction zone comprising:

each of said first spots comprises an antigen for binding a specific species of immunoglobulins, to thereby form a complex with said labeled analyte- comprising conjugate, the antigens being associated with a first member of an affinity couple, the complex being capable of migrating along said line with a liquid advancing in the direction of said axis; and

In a specific embodiment where the immobilized second member of said affinity couple is placed in a consecutive line perpendicular to the liquid flow, discrete spots will form (and therefore identified later on) indicating the presence of biotinilated antigen originating from the first spots.

In the devices of the invention, the area in which the arrays are located on the matrix is defined as the reaction zone, while the area in which the sample is placed is defined as the sample-receiving zone. The sample receiving zone may constitute a part of the matrix on which the liquid sample is placed, or may be a separate matrix or pad, which is associated with the matrix via physical contact. The arrangement of the device is such that proximally-distally oriented axis extends from the sample-receiving zone to the reaction zone. The device may comprise a sample receptacle in association with the sample- receiving zone such that at least a portion of the liquid sample introduced into the sample receptacle transfers to the sample -receiving zone, and thereafter to the reaction zone.

The device may also comprise, by some embodiments, a diluent compartment that is capable of releasing a diluent onto the sample receiving zone (a diluent may, for example, be a buffer such as 0.1M NaCl in 0.05 M phosphate buffer, pH 7.4 (PBS). The diluent compartment is typically made to be rupturable, for releasing the diluent onto the matrix upon rupturing of the compartment.

In some embodiments the device has two parts, e.g. a main body housing the matrix, and a cap which may house the diluent compartment. In this case the arrangement may be such that, once combined with the matrix-containing body, the diluents compartment is automatically ruptured to thereby release its contents.

The device also typically comprises a window over the reaction zone to permit visualization of the reaction.

By another aspect, the invention provides a diagnostic system for detecting analytes in a sample that comprises a device of the kind disclosed herein, and an image capturing device for capturing an image formed on said matrix.

In some embodiments, the system comprises a computerized device linked to said image capturing device for image analysis.

By another aspect, the invention provides a method for detecting a plurality of analytes in a liquid sample. The method comprises bringing the liquid sample into contact with a sample receiving zone associated with a planar porous matrix such that the sample transfers from the sample receiving zone to the planar porous matrix, the matrix comprising a reaction zone, being spaced apart and distal to the sample receiving zone along a proximally-distally extending axis. The reaction zone comprises at least one first array of first spots comprising a recognition element capable of specifically binding to an analyte in the sample, thereby forming a labeled complex with said specific analyte, the labeled complex comprising a label-comprising species present in (i) the sample -receiving zone, or (ii) each of said first spots. The reaction zone further comprises at least one counterpart second array of second spots spaced apart along said axis and being distal to said first array such that each first spot in the first array having a counterpart second spot in the second array, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis, each of said second spots comprises an immobilized capturing agent for binding the labeled complex. The labeled complex is capable of migrating along said line with a liquid advancing in the direction of said axis. The sample is allowed to migrate in a direction parallel to said axis. Then, the label is detected in the spots of the second array, presence of the label being indicative of presence of an analyte in the sample.

In some embodiments, the label is detected continuously or at discrete time points.

In other embodiments, the label is visualized through the use of an image capturing module, and the image is optionally further processed by image analysis software.

The label-comprising species are said to be located in either (i) the sample- receiving zone, or (ii) each of said first spots.

According to some embodiments, the species of recognition elements is said label-comprising species in the first spots, being a conjugate comprising the label and a binding moiety for specifically binding the analyte in the sample to form the labeled complex; and the immobilized capturing agent binds (i) to the analyte in the complex, or (ii) to the binding moiety in the complex.

According to other embodiments, the label-comprising species is present in the sample-receiving zone, the labeled-comprising species being a conjugate of a label and a binding moiety, the binding moiety capable of binding an immunoglobulin analyte in said sample, to thereby form the labeled analyte-comprising species. In such embodiments, the recognition element is an antigen associated with a first member of an affinity couple, the recognition element capable of forming said labeled complex with said label-comprising species, and the immobilized capturing agent is a second member of said affinity couple for binding to the first member of the affinity couple.

In embodiments where the reaction comprises a at least one third, control array of third spots, the method further comprises detecting presence of a label in said third spots. BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. 1 shows a schematic representation of a device in accordance with an embodiment of the invention.

Fig. 2A shows a schematic top view of a matrix in accordance with an embodiment of the invention comprising first and second arrays of spots.

Figs. 2B and 2E shows a schematic top view of a matrix in accordance with another embodiment of the invention comprising first, second and third arrays of spots.

Figs. 2C and 2D shows a schematic top view of a matrix in accordance with another embodiment of the invention comprising several first and second arrays and one third array of spots.

Fig. 3 shows a device in accordance with an embodiment of the invention.

Figs. 4A-4E show images of test results taken in time sequence of analysis a sample containing the hepatitis B surface antigen (HBs antigen) at the concentration of 5ng/ml, using immunological reagents as labeled conjugates [Fig. 4A - 15 seconds; Fig. 4B - 20 seconds; Fig. 4C - 30 seconds; Fig. 4D - 40 seconds; Fig. 4E - 50 seconds].

Figs. 5A-5E show images of test results taken in time sequence of exemplary analysis of two samples: (i) no ***e; (ii) containing 100 ng/ml of ***e [Fig. 5A - 15 seconds; Fig. 5B - 20 seconds; Fig. 5C - 30 seconds; Fig. 5D - 40 seconds; Fig. 5E - 50 seconds].

DETAILED DESCRIPTION OF EMBODIMENTS

Fig. 1 is a schematic representation of an exemplary device in accordance with an embodiment of the invention. The device 100 comprises a planar base 102, typically made from a polymer, on-top of which a planar, porous matrix 104 is placed. The matrix is made of a porous, absorbent material, e.g. a nitrocellulose membrane. The matrix is associated in one of its ends with a sample application pad (forming the sample-receiving zone) 106. The other end of the matrix is associated with a liquid driving pad 108, promoting capillary flow of the liquid through the matrix. A directional axis extending between the sample application pad and the liquid driving pad defines the proximally-distally oriented flow axis (represented by arrow 110) of the sample in the matrix, namely, a liquid sample will migrate along this axis from the sample application pad to the liquid driving pad through the matrix. The liquid migration through the matrix is typically caused by capillary forces.

A top view of the device of Fig. 1 is presented in Figs. 2A-2E, each representing a device in accordance with an embodiment of the invention.

In the device of Fig. 2A, the matrix carries two parallel arrays of spots. A first array 112 comprises a plurality of first, discrete spots. In accordance with this exemplary embodiment, the array comprises 4 spots. Each of the spots in the first array comprises a conjugate, which may be different in each spot of the first array, thereby allowing the analysis of several different analytes on a single matrix using a single sample. The conjugates are associated with the porous matrix, in a manner that enables their dissociation and migration upon contact with a liquid across the matrix along the axis 110. Such dissociation may be assisted by detergent molecules embedded in each of the first spots.

A second array 114, positioned distal to the first array along the axis 110, comprises a plurality of second spots. Each of the first spots of array 112 has a counterpart second spot on a line parallel to the axis 110 in the second array 114. In this configuration a so-called "channel" is formed between each pair of first and second spots, parallel to the direction of the axis 110 and further along the axis. Each of said second spots comprises an immobilized capturing agent.

A liquid sample placed on sample receiving pad 106 migrates on the matrix along direction 110. Once the sample reaches array 112, analytes to be captured bind to the conjugate via the binding-moiety of the conjugate, and the so-formed analyte- conjugate complex migrates with the liquid flow in the direction 110. The immobilized capturing agents in array 114 then bind to the analyte-conjugate complex via either the analyte itself or via the binding moiety of the conjugate.

The channeled movement of the analyte-conjugate complex assures the formation of appropriate spot couples (i.e. first spot and corresponding second spot), whereby in each "channel" a single analyte is tested and the results for different analytes are thereby isolated one from the other.

As noted above, the matrix may carry a third array 116 of third spots, as shown in Figs. 2B-2C, which are used as control spots. The third spots typically comprise a capture agent capable of capturing the conjugates and thereby demonstrate their intact state. In one embodiment, the third spots comprise secondary anti-species antibodies directed to the antibodies used as primary binding moieties in the conjugates.

In another embodiment, the matrix may carry a plurality of first arrays and second arrays. As can be seen in Figs. 2C-2D, two first arrays 112, 212 and two second arrays 114, 214 are shown, as well as a third array 116. In the configuration of Fig. 2C, first arrays 112 and 212 are identical to each other, and similarly second arrays 114 and 214 are identical. The third array 116, therefore, provides a common control for both sets of arrays. This allows for repetitive analysis of the analyte while using a single sample.

It is possible to increase the number of analysis points for different analytes by using the configuration shown in Fig. 2D. In this configuration, first arrays 112, 212 each comprise different conjugates, and correspondingly, second arrays 114, 214 each comprise different capturing agents. The pair of arrays 114, 214 is perpendicularly shifted from the pair 112, 212, thereby allowing 8 distinct channels to form (rather then the 4 channels of Fig. 2C). For effective control, it can be seen that the number of control points in array 116 has been doubled to correspond to the new number of channels.

In the device of Fig. 2E, the sample receiving zone 220 comprises conjugates of label and anti-immunoglobulin. Once the sample is places on the sample receiving zone 220, labeled analyte-comprising species are formed between the conjugates and the immunoglobulins in the sample. The sample then flows along the axis 110 towards the first array 222, in which each of the first spots comprise a different antigen. All of the antigens are biotinilated by a biotin moiety. The antigens bind to the immunoglobulin in the labeled analyte-comprising species to form the labeled complexes.

The second array 224, positioned distal to the first array along the axis 110, comprises a plurality of second spots, each spot comprises avidin to enable immobilization of the complexes by association of the biotin-avidin pair. As noted above, the matrix may carry a third array 226 of third spots, which are used as control spots.

A device in accordance with an embodiment of the invention is shown in Fig. 3. The device comprises a housing 302 and a cap 304. The housing houses the matrix, and comprises a clear window 306 positioned above the reaction zone of the matrix for ease of inspection of the test results. The housing also comprises a sample collection pad 308, protruding out of the housing, and associated with the sample receiving zone of the matrix.

The cap 304 may house a diluents chamber (not shown) for providing a diluent, typically a buffer, to facilitate flow of the sample through the matrix. The diluent chamber may be made of a rupturable material, such as a thin polymeric sheet.

When in use, a user places a drop of liquid sample, which may be (although not limited to) a bodily fluid such as blood, urine or saliva, onto the sample collection pad. The housing 302 and the cap 304 are then coupled, causing rupture of the diluent chamber and release of the diluent onto the sample collection pad. The diluted sample flows from the sample collection pad to the matrix for analysis. Once the analysis is complete, the results may be viewed through window 306 and further analyzed.

Example 1

The analysis was carried out using an arrangement of arrays of spots as in Fig. 2B. 100 μL· of a serum sample containing HBs antigen at a concentration of 5ng/ml was tested by the device of the invention. The test strip used has a width of 5mm, prepared of a plastic support, a working nitrocellulose membrane CNPF with a 10 μπι pore size, GFB-R4 separation membrane (sample receiving zone), and AP045 adsorption- membrane (liquid driving pad). All components of the strip were manufactured by company Advanced Microdevices, Ambala Cantt, (Mdi Easypack kits).

Colloidal gold conjugates with mouse monoclonal antibodies were synthesized according to a known procedure [Hermanson GT (2008), Bioconjugate Techniques, Elsevier, Amsterdam, 2nd ed., pp. 596, 924-931]. The conjugates in concentration corresponding to OD₅₂₀ = 8.0 in PBS buffer with 0.25% BSA, 0.1% Tween 20 and 2% glucose, were applied in the first array of spots by touching the membrane with a steel pin driven by a program-controlled manipulator. The diameter of the pin was 200 μπι, the drop volume was 20 nL. To achieve appropriate amount of conjugate in the spots, each spot was obtained by 10 successive applications of conjugate droplets by the pin.

The second array comprised of second spots formed by single pin application of a solution of anti-HBs antigen goat antibodies, with concentration of 1 mg/ml in Tris buffer pH 7.5. An array of anti-mouse IgG rabbit antibodies applied in same way as the third array was used as control. The spots had a diameter of 0.15-0.2 mm and were spaced apart across a distance of 1.5-2 mm along the proximal-distal axis (total distance of about 5 mm for the whole run). The selection of species was based upon desired formation of a so-called "sandwich" complex at the second array, i.e. the analyte is sandwiched at the capturing point and immobilized between the two anti-HBs antibodies.

The assay was performed at room temperature. 100 μΐ. of a liquid serum sample containing HBs antigen at a concentration of 5ng/ml was applied to the sample receiving zone of the strip. The strip was immediately placed in the image capturing device, employed for capturing sequential images of the nitrocellulose matrix. The contact of the sample receiving zone of the strip with the sample initiates the movement of reactants along the matrix, followed by immunochemical reactions and the formation of immune complexes in the discrete spots.

The image capturing device allows following the process of antigen detection in a real-time.

Figs. 4A-4E show images of the matrix taken during a period of analysis of 1 minute, starting from placement of the sample onto the sample -receiving zone.

The results show a unidirectional, rapid movement (less than 1 min) of the analyte/labeled antibody conjugate complex along discrete virtual flow channels from the first spots to corresponding second and third spots. (Fig. 4D, taken after 40 sec). Once the focusing agent (which resides in the first conjugate spots) is dissolved into the liquid, the effect of flow focusing is demonstrated (fig. 4B, taken after 10 sec). No significant changes in the spot diameter and limited traversal diffusion were observed, thereby showing no convergence of flow channels. At the test endpoint (images taken after 50 sec, Fig. 4E), discreet and discernible spots at the second array and the control (third) array were observed. Once interpreted and analyzed against known standards using image analysis software, these results may be converted into semi-quantitative values of an analyte concentration in the liquid sample. Furthermore, each part of matrix along the proximally-distally extending axis could be considered as an individual test of a distinct analyte, thus enabling to perform a plurality of analyses in a single liquid sample (multiplex assay).

Example 2

Results of another test are shown in Fig. 5. In this example, the first array comprised colloidal gold conjugate, prepared and applied to the device as in example 1, with mouse monoclonal antibodies specific to the analyte (in this case being ***e), but without addition of glucose. The second array comprised immobilized capturing agent-analyte (***e) conjugates with bovine serum albumin (BSA) as a carrier protein applied in concentration of 0.5 mg/ml in Tris buffer pH 7.5. Both monoclonal antibodies and BSA-***e conjugates used were manufactured by Arista Biologicals (Allentown, PA, USA). In such a selection of species, the analyte in a liquid sample competes with the capturing agent (present in the spots of the second array) for binding to the antibodies of the conjugates in the first array. This is known as the "competition method". This method may be used for analysis of low molecular weight analytes, such as drugs.

The assay was performed at room temperature. 100 μL· of a sample was applied to the sample receiving zone of the strip. The strip was immediately placed in the image capturing device, employed for capturing sequential images of the nitrocellulose matrix. The contact of the sample receiving zone of the strip with the sample initiates the movement of reactants along membrane followed by immunochemical reactions and the formation of immune complexes in specific zones.

Figs. 5A-5E show analysis of two samples: (i) PBS not containing ***e (left column), and (ii) a sample containing lOOng/ml of ***e in PBS (images in the right column). The configuration used was that of Fig. 2B, with a distance between spots within the arrays of 1.5 mm and distance between channels of 1 mm. The results demonstrate that the antibody conjugate contained in first spots was captured by the immobilized capturing agent of the spots in the second array with production of discrete visible spots within less than 1 minute. The captured images may, in turn, be subjected to further image analysis for comparing spot intensities to known standard controls.

Similarly to the results of Example 1, despite not using flow focusing agent there was no significant traversal diffusion observed without any converging of channels suggesting that plurality of analytes could be detected on the same strip.

Further of note that at the reaction endpoint, clearly stained spots were detectable in an analyte absence (Fig. 5E(i)), while evident diminishment of spot intensity in the presence of analyte (Fig. 5E(ii)), suggests specificity and validity of the obtained signals and possibility for quantification of results.

Claims

CLAIMS:

1. A planar, porous matrix, comprising:

at least one first array of first spots and at least one counterpart second array of second spots spaced apart along a proximally-distally extending axis, wherein the second array being distal to said first array, each first spot having a counterpart second spot, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis;

each of said first spots comprises a recognition element capable of specifically binding to an analyte in a sample, thereby forming a complex with (i) said specific analyte or (ii) an analyte-comprising species,

2. The matrix of claim 1, wherein said complex comprises a detectable label.

3. The matrix of claim 2, wherein the label is optically detectable.

4. The matrix of claim 3, wherein the label is colloidal gold.

5. The matrix of any one of claims 1-4, wherein each of the recognition elements is a conjugate, the conjugate comprising a label conjugated to a binding moiety for specifically binding the analyte in the sample to form the complex; and wherein the immobilized capturing agent binds (i) to the analyte in the complex, or (ii) to the binding moiety in the complex.

6. The matrix of claim 5, wherein the binding moiety is a member of a binding couple.

7. The matrix of claim 6, wherein the binding couple is selected from the group consisting of antibody-antigen, complementary nucleic acid sequences, sugar-lectin, and receptor-ligand, wherein one of the binding couple is the binding moiety and the other is the analyte.

8. The matrix of any one of claims 5-7, wherein the binding moiety and the capturing agent are molecular entities that comprise a binding site of an antibody.

9. The matrix of any one of claims 1-4, wherein each of the recognition elements is an antigen associated with a first member of an affinity couple, the recognition elements capable of forming said complex with said analyte-comprising species; and wherein the immobilized capturing agent is a second member of said affinity couple for binding to the first member of the affinity couple.

10. The matrix of claim 9, wherein said analyte-comprising species is labeled.

11. The matrix of claim 9 or 10, wherein the first member of the affinity couple is a biotinilated antigen and the second member of the affinity couple is avidin.

12. The matrix of any one of claims 9-11, wherein the antigen is an allergen.

13. The matrix of any one of the preceding claims, comprising at least one third array of third spots, in which each of said third spots counterparts a first and a second spot in the first and second arrays, respectively, and comprising a control capturing agent for binding the recognition element.

14. The matrix of any one of the preceding claims, wherein each of said first spots is equally distant from its counterpart second spot.

15. The matrix of claim 14, wherein the spots in each of the first, second and third array are arranged along a first, second and third lines, respectively, being perpendicular to said axis.

16. The matrix of any one of the preceding claims, wherein the complex migrates along said line in the direction of said axis with limited side diffusion.

17. The matrix of any one of the preceding claims, wherein each of said first spots comprises a flow focusing agent, as to focus the migration of the complex along said line with less side diffusion as in the absence of said flow focusing agent.

18. The matrix of claim 17, wherein the flow focusing agent is selected from sucrose, glucose, and glycerol.

19. A planar, porous matrix, comprising:

each of said second spots comprises an immobilized capturing agent capable of binding (i) to the analyte in the complex, or (ii) to the binding moiety in the complex.

20. A planar, porous matrix, comprising:

each of said first spots comprises an antigen associated with a first member of an affinity couple, the antigen being capable of specifically binding to a labeled analyte - comprising species, thereby forming a complex capable of migrating along said line with a liquid advancing in the direction of said axis; and

21. A device for detection of analytes in a liquid sample, comprising:

at least one first array of first spots and at least one counterpart second array of second spots, the arrays being spaced apart along a proximally-distally extending axis, said first array being distal to the sample -receiving zone and the second array being distal to said first array, each first spot having a counterpart second spot, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis,

each of said first spots comprises a conjugate, the conjugate comprises a label conjugated to a binding moiety for binding a specific analyte in the sample thereby forming a complex, at least some of the first spots comprise conjugates with a binding moiety that differs from that in other first spots, the complex being capable of migrating along said line with a liquid advancing in the direction of said axis, and each of said second spots comprises an immobilized capturing agent for binding (i) to the analyte in the complex, or (ii) to the binding moiety in the complex.

22. The device of claim 21, wherein the binding moiety is a member of a binding couple.

23. The device of claim 22, wherein the binding couple is selected from the group consisting of antibody-antigen, complementary nucleic acid sequences, sugar-lectin, and receptor-ligand, wherein one of the binding couple is the binding moiety and the other is the analyte.

24. The device of any one of claims 21-23, wherein the binding moiety and the capturing agent are molecular entities that comprise a binding site of an antibody.

25. A device for detecting immunoglobulins in a liquid sample, a sample-receiving zone for receiving the liquid sample and a planar, porous matrix having a reaction zone, the sample -receiving zone comprising a conjugate of a label and a binding moiety, the binding moiety capable of binding an immunoglobulin analyte in said sample, to thereby form a labeled analyte-comprising species;

and a reaction zone comprising:

each of said first spots comprises an antigen for binding a specific species of immunoglobulins, to thereby form a complex with said labeled analyte-comprising species, the antigens being associated with a first member of an affinity couple, the complex being capable of migrating along said line with a liquid advancing in the direction of said axis; and

26. The device of any one of claims 21-25, comprising a sample receptacle in association with the sample-receiving zone such that at least a portion of the liquid sample introduced into the sample receptacle transfers to the sample -receiving zone.

27. The device of any one of claims 21-26-, comprising a diluent compartment capable of releasing a diluent onto the sample -receiving zone.

28. The device of claim 27, wherein the diluent compartment can be ruptured to cause the release of the diluent.

29. The device of any one of claims 21-28, wherein the reaction zone comprises at least one third array of third spots, in which each of said third spots counterparts a first and a second spot in the first and second arrays, respectively, and comprising a control capturing agent for binding the conjugate.

30. The device of claim 29, the spots in each of the first, second and third array are arranged along a first, second and third lines, respectively, being perpendicular to said axis.

31. The device of any one of claims 21-30, wherein the complex migrates along said line in the direction of said axis with limited side diffusion.

32. The device of any one claims 21-31, wherein each of said first spots comprises a flow focusing agent, a detergent or a mixture thereof.

33. The device of any one of claims 21-32, wherein the label is optically detectable.

34. The device of claim 33, wherein the label is colloidal gold.

35. The device of any one of claims 21-34, comprising a housing that houses the absorbent carrier, the housing having an opening over said second array.

36. A diagnostic system for detecting analytes in a sample, comprising a device of any one of claims 21-35 and an image capturing device for capturing an image formed in said reaction zone.

37. The system of claim 36, comprising a computerized device linked to said image capturing device for image analysis.

38. A method for detecting a plurality of analytes in a liquid sample, the method comprising:

(a) bringing the liquid sample into contact with a sample receiving zone associated with a planar porous matrix such that the sample transfers from the sample receiving zone to the planar porous matrix,

the matrix comprising a reaction zone, being spaced apart and distal to the sample receiving zone along a proximally-distally extending axis, the reaction zone comprising: at least one first array of first spots comprising a recognition element capable of specifically binding to an analyte in the sample, thereby forming a labeled complex with said specific analyte, the labeled complex comprising a label-comprising species present in (i) the sample-receiving zone, or (ii) each of said first spots, and at least one counterpart second array of second spots spaced apart along said axis and being distal to said first array such that each first spot in the first array having a counterpart second spot in the second array, each of said second spots comprises an immobilized capturing agent for binding the labeled complex, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis; the labeled complex being capable of migrating along said line with a liquid advancing in the direction of said axis;

(b) allowing the sample to migrate in a direction parallel to said axis; and

(c) detecting the label in the spots of said second array, presence of the label being indicative of presence of an analyte in the sample.

39. The method of claim 38, wherein the label is detected continuously or at discrete time points.

40. The method of claim 38 or 39, wherein the label is visualized through the use of an image capturing module, and optionally further processed by image analysis software.

41. The method of any one of claims 38-40, wherein

the species of recognition elements is said label-comprising species in the first spots, being a conjugate comprising the label and a binding moiety for specifically binding the analyte in the sample to form the labeled complex; and

wherein the immobilized capturing agent binds (i) to the analyte in the complex, or (ii) to the binding moiety in the complex.

42. The method of any one of claims 38-41, wherein

said label-comprising species is present in the sample -receiving zone, the labeled-comprising species being a conjugate of a label and a binding moiety, the binding moiety capable of binding an immunoglobulin analyte in said sample, to thereby form the labeled-comprising species; said recognition element is an antigen associated with a first member of an affinity couple, the recognition element capable of forming said labeled complex with said label-comprising species, and

the immobilized capturing agent is a second member of said affinity couple for binding to the first member of the affinity couple.

43. The method of claim 42, wherein the first member of the affinity couple is a biotinilated antigen and the second member of the affinity couple is avidin.

44. The method of claim 42 or 43, wherein the antigen is an allergen.

45. The method of any one of claims 38-44, wherein the reaction zone comprises at least one third, control array of third spots, each of said third spots counterparts a first and a second spot in the first and second arrays, respectively, and comprising a control capturing agent for binding the recognition element; and wherein said detecting comprises detecting presence of a label in said third spots.

46. The method of any one of claims 45, wherein the spots in each of the first, second and third array are arranged along a first, second and third lines, respectively, being perpendicular to said axis.

47. The method of any one of claims 38-46, wherein each of said first spots comprises a flow focusing agent, a detergent or a mixture thereof.

48. The method of any one of claims 38-47, wherein

the label is optically detectable; and

the method comprises optically detecting the label.

49. The method of claim 48, wherein the label is colloidal gold.

50. A method for detecting a plurality of analytes in a liquid sample, the method comprising:

(a) bringing the liquid sample into contact with a sample receiving zone associated with a planar porous matrix, such that the sample transfers from the sample receiving zone to the planar porous matrix,

the matrix comprising a reaction zone spaced apart and distal to the sample receiving zone along a proximally-distally extending axis,

the reaction zone comprising:

at least one first array of first spots, at least some of the spots comprise a conjugate, the conjugate comprising a label and a binding moiety for binding a specific analyte in the sample, thereby forming a labeled complex; at least some of the first spots comprise a conjugate with a binding moiety that differs from that in other first spots, and at least one counterpart second array of second spots spaced apart along said axis and being distal to said first array, such that each first spot in the first array having a counterpart second spot in the second array, each of said second spots comprises an immobilized capturing agent for binding to (i) the analyte in the labeled complex, or (ii) the binding moiety of the labeled complex, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis;

the labeled complex being capable of migrating along said line with a liquid advancing in the direction of said axis;

(b) allowing the sample to migrate in a direction parallel to said axis; and

51. A method for detecting immunoglobulins in a sample, the method comprising:

(a) bringing a liquid sample into contact with a sample receiving zone associated with a planar porous matrix, such that the sample transfers from the sample receiving zone to the planar porous matrix,

the sample receiving zone comprising a conjugate of a label and a binding moiety, the binding moiety capable of binding an immunoglobulin analyte in said sample, to thereby form a labeled analyte-comprising species,

the reaction zone comprising:

at least one first array of first spots and at least one counterpart second array of second spots, the arrays being spaced apart along a proximally- distally extending axis, said first array being distal to the sample- receiving zone and proximal to said second array, such that each first spot in the first array having a counterpart second spot in the second array, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis; each of said first spots comprises an antigen for binding a specific species of immunoglobulins in the sample to form a labeled complex with said labeled analyte-comprising species, the antigens being associated with a first member of an affinity couple, the labeled complex being capable of migrating along said line with a liquid advancing in the direction of said axis; and

each of said second spots comprises an immobilized second member of said affinity couple for binding to the first member of the affinity couple;

(b) allowing the sample to migrate in a direction parallel to said axis; and

(c) detecting label in the spots of said second array, presence of a label being indicative of presence of an immunoglobulins in a sample.