WO2008007359A2

WO2008007359A2 - Rapid diagnostic devices based on molecular imprinted polymers

Info

Publication number: WO2008007359A2
Application number: PCT/IL2007/000851
Authority: WO
Inventors: Raphael Levi; Ido Margalit; Yarden Dloomy
Original assignee: Infigo Diagnostics Ltd.
Priority date: 2006-07-09
Filing date: 2007-07-08
Publication date: 2008-01-17
Also published as: EP2041567A2; EP2041567A4; WO2008007359A3

Abstract

The invention pertains generally to sensitive diagnostic assay devices, methods and kits for determining the presence, absence and/or amount of analytes in fluid samples The devices (figures 3a & 3b) are based on molecularly imprinted polymers (MlPs) for use in real¬ time measurements of levels of analytes, particularly small molecules, in fluid samples One such device is a device for determination of B12 (cobalamin) deficiency or methylmalonic aciduria by measuring methylmalonic acid (MVA) in either blood or urine.

Description

RAPID DIAGNOSTIC DEVICES BASED ON MOLECULAR IMPRINTED POLYMERS

FIELD OF THE INVENTION

The present invention relates generally to rapid and sensitive lateral-flow, dipstick and flow- through diagnostic devices, methods, and kits for determining the presence, absence and/or amount of analytes in fluid samples. The devices include molecularly imprinted polymers (MIPs) for use in realtime measurement of levels of analytes, particularly small molecules, in fluid samples. BACKGROUND

Methods and devices for efficient and accurate detection and quantification of the level of analytes, particularly small molecules, in liquid samples are of particular interest for use in a wide range of applications. Currently, detection and measurement of various small molecules of interest in many fields rely on sophisticated instrumentation (such as HPLC and GC/MS) available only at central laboratories. There is an increasing need for analytical capabilities in doctors' offices, in the home, in the field, in production lines to identify or monitor physiological and pathological conditions using different biological samples, for analysis of environmental samples, as well as to monitor food production lines to detect the presence of contaminants. The health, food, and environmental industries (to name a few) call for a rapid, real-time, and simple analysis of various molecules of interest in liquid samples, which is not available with current technologies.

Numerous approaches and analytical tests have been developed to fill this need. Many of these tests are based on the highly specific interactions between specific binding pairs, such as biotin/avidin, antigen/antibody, lectin/carbohydrate, enzyme/substrate, cellulose/cellulose binding protein, proteins A and G/immunoglobulins and apoprotein/lipid. Furthermore, many of these tests involve devices (e.g., dipstick, lateral-flow, flow-through, and migratory devices) with one or more of the members of a binding pair attached to a mobile or immobile solid phase material such as latex beads, glass fibers, glass beads, cellulose strips or nitrocellulose membranes (U.S. Pat. Nos. 4,703,017; 4,743,560; 5,073,484).

Dipstick devices, such as disclosed by Hochstrasser (U.S. Pat. 4,059,407) are designed to be immersed in a fluid biological sample and to a give a semi-quantitative estimation of the target analyte in the fluid. Dipsticks are essentially lateral flow devices whose application method involves immersing the device in the liquid sample. Also of interest in the area of dipstick devices are U.S. Pat. Nos. 3,802,842, 3,915,639 and 4,689,309.

Lateral flow devices (see U.S. Pat. Nos. 5,075,078; 5,096,837; 5,354,692 and 5,229,073) generally comprise a porous matrix containing the relevant specific reagents, which is layered on a solid strip, such as plastic. Instead of vertically wicking the samples up the "dipstick," the lateral flow format allows a sample to flow laterally across the porous, solid phase material by capillary action, across one or more reagents that interact with the analyte (if it is present in the sample). A visual signal (produced by colored beads, enzymatic reaction or other color-forming reactions) indicates the presence of the analyte.

In flow-through type devices, the applied test sample flows through a porous material, bringing the analyte in the sample in contact with the specific reagents contained in the porous material, eventually producing a visible signal on the porous material that provides an indication of the presence of target analyte in the sample. Such devices often require various action be taken by users, such as extraction steps, addition and incubation of reagents, washing steps, etc. These devices are therefore more time consuming, require a knowledgeable user, and increase the chances for erroneous results.

Visible detection of test results without the need to add external reagents is achieved in migration assay devices by incorporating reagents that have been coupled to colored labels (i.e., conjugates), thereby permitting visible detection of the assay results without addition of further substances. See, for example, Bernstein, U.S. Pat. No. 4,770,853. Such labels include, for example, gold sol particles as described by Leuvering (U.S. Pat. No. 4,313,734) dye sol particles (Gribnau et al., U.S. Pat. No. 4,373,932) and dyed latex (May et al., WO 88/08534). In an attempt to improve the performance of migratory assays, May et al. (U.S. Pat. 7,109,042) disclose diagnostic test devices that have two distinct pathways for the sample and the conjugated reagent.

There are a number of commercially available lateral-flow and flow-through type tests and patents disclosing methods for the detection of large analytes (MW greater than 1,000 Daltons).

A quantitative chromatographic test strip is described by Deutsch et al. in U.S. PatNos. 4,094,647, 4,235,601 and 4,361,537. The device consists of a strip that moves the sample solution by capillary action to zones in the strip containing the reagents that in the presence of the analyte produce a detectable signal.

Many variations on the Deutsch et al. device have been disclosed. For example, Tom et al. (U.S. Pat. No. 4,366,241) disclose a device with an immunosorbing zone to which the test sample is applied. Grubb et al. (U.S. Pat. No. 4,168,146) describe the use of a "sandwich" formation of antibody- antigen-antibody, whereas second antibody is conjugated to a reporter to allow quantitative determination of the antigen (the analyte) in the sample by measuring the length of the strip that contains bound antigen. Rosenstein (U.S. Pat. No. 5,591,645) discloses a chromatographic test strip comprising a solid support having two portions that permit capillary flow that is useful in a variety of immunoassays. The first portion includes a movable tracer and the second portion includes an immobilized binder capable of binding to the analyte.

Reagent-impregnated test strips have been used in various specific binding assays, such as immunoassays, for quite some time. The sample is applied to one portion of the test strip and migrates through the porous strip material, in some cases with the aid of an eluting solvent such as water. The sample advances into or through a detection zone where a specific binding reagent for the examined analyte is immobilized. Analyte present in the sample is then entrapped within the detection zone. The amount of bound analyte is determined usually by using labeled reagents incorporated in the test strip or applied subsequently. A variety of labels, such as radiolabels, chromophores, colored particles (gold, latex), enzymes, and fluorescent labels may be used in these assays. In most cases, the detecting binding agents are analyte-specific antibodies.

The present invention is concerned with improving the known approaches to rapid binding assays, in the aspects of implementation of MIPs as the detecting agents, improved signal generation and results interpretation. The introduction of MIPs into these types of assays allows analysis of molecules that were not ideal candidates for antibodies production, especially, but not only, small molecules. Molecular Imprinted Polymers

In contrast to most rapid diagnostic devices in which the sensing element is antibodies specific to the analyte, the present invention utilizes the specific recognition properties of molecular imprinted polymers ("MIPs"). A "molecular imprint polymer" is a polymer which is prepared by polymerizing functional monomers around a template or "print" molecule, which is then removed from the polymer by extraction or other means so that the polymer will selectively absorb the template or print molecule upon re-exposure to the print molecule. U.S. Pat. Nos. 5,821,311; 5,872,198, and 5,959,050 to Mosbach, et al. describe certain MIP polymers, a polymerization process, and symmetrical beads produced by suspension polymerization from functional monomers for use as chromatographic media.

MIPs hold several advantages over antibodies for diagnostics and sample analysis, particularly for small molecules, due to their controlled synthesis and remarkable stability. Molecular imprinting originates from the concept of creating tailor-made recognition sites in polymers by template polymerization (Mosbach K. et al., Bio/Technology, 1996, 14, 163-170; Ansell R. J. et al., Curr. Opin. Biotechnol., 1996, 7, 89-94; Wulff G. Angew. Chem. Int. Ed. Engl., 1995, 34, 1812-32; Vidyasankar S. et al., Curr. Opin. Biotechnol., 1995, 6, 218-224; and Shea K. J, Trends In Polymer Science, 1994, 2, 166-173). Molecularly imprinted polymers demonstrated remarkable recognition properties that were applied in various fields such as drug separation (Fischer L., et al., J. Am. Chem. Soc, 1991, 113, 9358- 9360; Kempe M, et al., J. Chromatogr., 1994, 664, 276-279; Nilsson K., et al., J. Chromatogr., 1994, 680, 57-61), receptor mimics (Ramstrom O., et al., Tetrahedron: Asymmetry, 1994, 5, 649-656; Ramstrom O., et al., J. MoI. Recogn., 1996, 9, 691-696; Andersson L. L, et al., Proc. Natl. Acad. Sci., 1995, 92, 4788-4792; Andersson L. L, Anal. Chem., 1996, 68, 111-117) bio-mimetic sensors (Kriz D., et al., Anal. Chem., 1995, 67, 2142-2144], antibody mimics (Vlatakis G., et al., Nature, 1993, 361, 645- 647), template-assisted synthesis (Bystrom S. E., et al, J. Am. Chem. Soc, 1993, 115, 2081-2083) and catalysis (Muller R., et al., Makromol. Chem., 1993, 14, 637-641; Beach J. V., et al., J. Am. Chem. Soc, 1994, Vol. 116, 379-380).

The great potential embodied in MIPs resulted in numerous inventions for analytical devices and methods of detection of various analytes, based on molecular imprinting, reviewed by Ye and Haupt

(Anal. Bioanal. Chem. 2004, 378,1887-1897). One of the major challenges is obtaining an apparent signal from the polymer-analyte binding event. A variety of approaches were brought forward, yet the great majority of those involve sophisticated methods and machinery. Some examples of MIP-based sensors are as follows:

Yan et al. (U.S. Pat. No. 5,587,273) present an invention of sensors employing molecularly imprinted film and measuring the capacitance or the light characteristics of the film after the exposing step or analyzing the film spectroscopically. MIP-based devices for detecting, analyzing and quantifying macromolecules are disclosed by Huang (U.S. Pat. No. 6,680,210). Detection is performed by dissociating the analyte molecules from the polymer after the binding and then analyzing them.

Williams et al. (U.S. Pat. 6,807,842) disclose a molecular recognition sensor system for detecting the presence and concentration of an analyte including a resistive sensor having a semiconductive polymer film which swells when exposed to the analyte.

Kroeger et al. (G.B. Pat. No. 2,337,332) disclose an electrode that has a surface modified with an imprinted synthetic polymer that specifically recognizes, binds and concentrates the analyte, in close proximity to the electrode surface. Either the bound analyte itself or an electrochemically active derivative pre-incubated with the electrode or added to the sample (competition/displacement assay) is quantified electrochemically (for example by differential pulse or square wave voltametry) directly at the electrode surface.

Penelle (U.S. Pat. No. 6,890,486) discloses an MIP-based sensor for a specific molecule, namely, hexachlorobenzene, at concentrations consistent with applications in pollution control by a process of combining MIP techniques with quartz crystal microbalance (QCM).

Green et al. (U.S. Pat. No. 6,638,498) disclose devices utilizing MIPs with specific binding capacity for particular bile acids and/or salts, such as DCA and CDCA. The detection is performed by displacement of radioactive-labeled CDCA by CDCA in the sample. In a modification of the above, a fluorescent derivative of cholic acid is used as the assay substance.

Lawrence et al. (U.S. Pat. No. 6,833,274) disclose a Cortisol fiber optic sensor using a cortisol- imprinted polymer and displacement of a cortisol-fluorescent chromophore conjugate.

Schwartz et al. (U.S. Pat. No. 6,967,103) disclose an explosive detector utilizing an array of MIP-coated, bifurcated fiber optic cables. Individual sensor fiber assemblies, each with a calibrated airflow, are used to expose the fibers to the target molecule. The detector energizes a dedicated excitation light source for each fiber and, through a detector comprising a filter and photodiode, simultaneously reads and processes the intensity of the resulting fluorescence that is indicative of the concentration of the target molecules.

Pectu et al. (WO Pat. No. 0200737) disclose a method for detection and measurement of phenols using phenol-specific MIPs. In one embodiment, the detection method is similar to radioimmunoassay, where radiolabeled phenol is incorporated into a sample, and binding of the radioactive phenol to the polymer is inversely related to the amount of relevant phenol present in the sample. Another proposed method for analyzing the sample involves incorporation of the polymer into a biosensor comprising an amperometric probe with an electrode of MIP coated platinum mesh.

Catania, et al. (U.S. Pat. No. 6,461,873) discloses a MIP-based analytical device for detecting caffeine that comprises a paper chromatographic technique visual color signal. The MIP selectively absorbs caffeine and chromogenic reagents which provide colorimetric visualization. The concentration of caffeine in the sample is determined by the height or distance that the caffeine migrates up a paper strip.

Rittenburg (Patent Nos. 5,710,005 and 6,140,134) discloses methods for determining the concentration of an analyte in a sample in which an analyte gradient is established. The sample contacts an indicator zone where the analyte associates with a mobile binding member, which may comprise a molecular imprinted polymer. Thus, if a MIP is used, it is not fixed to the device in the indicator zone, but instead is mobile, i.e., able to move out of that zone. In addition, the mobile binding member (i.e., MIP) may be associated with a signaling substance. This contrasts to the present invention where the MIP is fixed and the analyte analog is attached to a reporter molecule. As the analtye analog in Rittenburg is not attached to a reporter molecule, its displacement and binding in the test zone may be used only for some kind of competition assay and not for determining the signal directly from the analog itself, as proposed in the instant invention.

Using MIPs combined with displacement of analyte-marker conjugate was shown to be practical in several laboratories (Vlatakis G. et al., Nature, 1993, 361, 645-647, Levi et al., 1997, Anal. Chem. 69. 2017-2021; Nathaniel T. et al., J. Am. Chem. Soc. 2005, 127, 5695-5700; Nicholls C. et al, Biosens. Bioelec, 2006, 21, 1171-1177). However, the prior art does not disclose methods, devices and kits using MIPs combined with a single displacement approach on rapid flow devices, without the need for auxiliary instrumentation, or detection methods, devices and kits that use a novel double displacement approach in combination with MIPs on rapid flow devices, as disclosed herein. This approach holds several advantages over the typical displacement methods, as set forth below. Detection of MMA

Cobalamin (CbI or vitamin Bn) is a water-soluble vitamin that is part of the vitamin B complex found in red meat. It is exclusively synthesized by bacteria and is found primarily in meat, eggs and dairy products. It acts in-vivo as a co-enzyme for enzymes that are known to catalyze intra-molecular rearrangements and methylations. CbI deficiency may also disturb cytokine and growth factor regulation (See Miller, Nutrition Reviews 60: 142-144 (2002) and Scalabrino et al., J. Neuroimmunology 1:37-42(2002)). Early diagnosis of vitamin B12 deficiency is essential due to the latent nature of this disorder and the possible risk of irreversible neurological damage damage (Carmel, Am. J Hematol. 34: 108-14 (1990), Healton et al., Medicine; 70: 229-45 (1991) and Martin et al., J Am Geriatr Soc, 40: 168-72 (1992)). A simple, reliable and generally accepted method for determination of vitamin Bj₂ deficiency is not available. Plasma vitamin Bi₂ concentration does not reliably rule out vitamin B₁₂ deficiency. (Lindenbaum et al., Am J Hematol. 34: 99-107 (1990), England and Linnell, Lancet, 2. 1072-3 (1980) and Herbert et al., J Lab Clin Med; 104: 829-41 (1984)). Elevated methylmalonic acid (MMA) and total homocysteine (tHcy) in plasma are sensitive metabolic markers for vitamin B12 deficiency (Schilling, Proceedings of the Association of American Physicians; 108: 68-70 (1996), Carmel, Annu Rev Med; 51: 357-75 (2000) and Hvas et al., Clin Chem; 47:1396-404 (2001)).

Vitamin B₎₂ assays were initially microbiologic. Later, radio-assays which used competition for binding to intrinsic factor between radio-labeled vitamin B_]2 and vitamin Bi₂ in the sample were developed. Intrinsic factor facilitates the physiological uptake of vitamin Bi₂ and possesses high specificity only to biologically active forms OfBi₂. Pourfarzaneh et al. (WO 91/00519) describe immunoassays for vitamin B_]2 based on monoclonal antibodies specific to the intrinsic factoπvitamin Bi₂ complex and to the vitamin B_!2 binding site on intrinsic factor. Competitive assays of this type are slow and lack sensitivity. Most of the immunoassays now in use employ radioisotopes and require R- factor free intrinsic factor or vitamin Bi₂ analogues. Examples of such radio-assays are described in U.S. Pat. Nos. 4,188,189 and 4,426,455.

Newman et al (U.S. Pat. No. 6,942,977) disclose an immunoassay for the determination of vitamin B]₂ by utilizing two classes of antibodies to intrinsic factor. The antibodies from one class bind to intrinsic factor competitively with vitamin Bi₂., i.e., they bind only in the absence of vitamin Bi₂ and are dissociated in the presence, and upon binding, of vitamin B_n to intrinsic factor. Antibodies of the second class bind only to the complex of intrinsic factor and vitamin Bi₂.

In plasma, the majority of total cobalamin (vitamin B12) is bound to two carrier proteins, transcobalamin (TC) and haptocorrin (HC). TC is essential for the transport of cobalamin from the intestine and into most cells of the body. TC carries one-third of the circulating cobalamin (holo-TC), but the major portion of this protein is present in its unsaturated form (apo-TC). Lars et al. (published U.S. Pat. Application 20060240570) disclose a method for determination of CbI concentration using monoclonal antibodies to HoIoTC, having specificity for holoTC over apoTC of at least 40-fold.

Methylmalonic acid (MMA) is a metabolite whose concentration is directly proportional to the amount of active vitamin Bl 2 in the blood and urine and, thus, can be used to detect B12 deficiency. Increased MMA levels are also associated with a type of organic acid disorder called "methylmalonic acidemia". People with this disorder have problems breaking down and using certain amino acids and fatty acids from the food they eat. In infants, if not treated, methylmalonic acidemia can even lead to death.

Detection of MMA is considered a preferred assay for determination OfBi₂ deficiency over direct measurement OfBi₂ itself (Elin and Winter, 2001, Arch. Pathol. Lab. Med. 125, 824-827). MMA analysis in biological fluids is typically performed by GC/MS (gas chromatography/mass spectrometry) methods, such as described by Allen et al. (U.S. Pat. No. 5,438,017). GC/MS is available only at central laboratories and it is costly and requires trained personnel.

Accordingly, there is a continuing need for a sensitive, rapid and single step method to detect and quantify large (MW greater than ten thousand Daltons), medium, and small analytes (about MW 100-500 Daltons).

SUMMARY QF THE INVENTION

The invention provides devices, methods and kits for rapid and simple determination of target analytes in liquid samples, that are capable of real-time measurement of target molecules in fluid samples, particularly, but not limited to, small molecules and that are highly selective, highly sensitive, simple to operate, low cost, and portable. The devices, methods and kits are suitable for use by untrained personnel without the need for uncommon and sophisticated equipment. When the kits of the invention optionally require auxiliary equipment, it is portable, hand held and simple to use.

The invention provides a rapid and simple to use assay device, method and kit for determination of target analytes in liquid samples, particularly small analytes, designed for use in the home, clinic, doctor's office, hospital bedside, factory or field. The assay devices achieve greater sensitivity than conventional rapid test assays, without compromising specificity, leading to stronger and/or more stable visual signals than those produced by conventional devices, easier interpretation of results, and reduced occurrence of indeterminate results. The devices may be used for detecting analytes in a variety of biological, environmental and industrial samples in a short amount of time without the need for complicated sample preparation procedures, and thus are suitable for use by untrained personnel even in field conditions. It is contemplated that the devices of the invention will provide a "real-time" measurement, i.e., a relatively rapid detection time, preferably of about 15 minutes, and more preferably less than 15 minutes. Alternatively, depending Alternatively, depending on the detectable reporter used, auxilliary instrumentation such as readers, scanners, etc. may be used to interpret the results.

The present invention provides sensitive, rapid methods and devices for determining the presence, absence and/or amount of small and/or large analytes of interest in a fluid sample. An analyte- specific molecular imprinted polymer (MIP) is used to detect the presence of a target analyte in a sample based on binding of the analyte by the MIP. An exemplified embodiment of the present invention is directed to methods and devices for the rapid detection and quantitation of MMA or Atrazine. Results from the methods and devices disclosed herein can be read directly from the assay device by visual inspection or using an instrument, such as an appropriate reader or scanner. For instance, such reader devices may detect color or fluorescence from the readout area.

The present invention combines the advantages of the lateral and flow through devices (rapid, relatively cheap, and simple to use) with the advantages of MIPs for diagnostics (specific, controlled production, and very stable) and a novel double displacement approach.

The target analyte may be any molecule of interest. Any known analyte to which an appropriate analyte-specific MIP may be prepared may be easily detected and/or quantified using the disclosed methods and devices. The methods and devices described herein can be used in the context of basic scientific research, the practice of medicine, including veterinary and dental medicine, forensic analysis, environmental protection monitoring and studies, industrial or chemical manufacturing, and the development and testing of pharmaceutical, food, and cosmetic products.

The analyte detected may include, but is not limited to, analytes selected from the group consisting of a protein, a hormone, an enzyme, a biomarker, a natural or synthetic toxin, a steroid, a drug, a drug metabolite, a drug-protein conjugate, a drug metabolite-protein conjugate, a vitamin, a drug of abuse, a chemical or biological warfare agent, a vitamin, antibodies to a drug, antibodies to infectious agents, an environmental pollutant, a lipoprotein, a polysaccharide, an immunoglobulin, a lymphokine, a cytokine, a soluble cancer antigen, a hapten, an oligonucleotide, an oligonucleotide that binds specifically with a protein, a growth factor, a neurotransmitter, a molecule indicating the safety or quality of a foodstuff, a process chemical (i.e., chemicals used in water systems such as flocculating polymers, biocides, corrosion inhibitors, and anti-sealants), a byproduct of a production process, a pesticide, an insecticide, a herbicide, a fertilizer, a surfactant, an adhesive, and an agent used in the manufacture of food, industrial agents or chemical products.

The analyte may include, but is not limited to, a biomarker (both for humans and for animals) selected from the group consisting of MMA, homocysteine, glucose, cholesterol, citrozine, hormones, BNP, FSH, and vitamin K. Other suitable biomarkers include TSH, free T3 T4, PSA, testosterone, androstenedione, dihydrotestosterone, dehydroepiandrosterone, anabolic steroids, estradiol, diethylstilbestrol, progesterone, and progestins.

For instance, the analyte may comprise an environmental pollutant selected from the group consisting of 2,4-Dichlorophenoxyacetic acid (2-4,D), alachlor, atrazine, cyanazine, metolachlor, and simazine. The analyte may comprise a therapeutic drug or antibiotic selected from the group consisting of cumadine, Acetaminophen, Amikacin, Aminophylline, Amitriptyline:, Carbamazepine, Chloramphenicol, Desipramine, Digitoxin, Digoxin, Disopyramide, Ethosuximide, Flecainide, Gentamicin, Imipramine, Kanamycin, Lidocaine, Lithium, Methotrexate, Nortriptyline, Phenobarbital, Phenytoin, Primidone, Procainamide, Propranolol, Quinidine, Dalicylate, Theophylline, Tobramycin and Valproic acid.

The analyte may comprise a drug of abuse selected from the group consisting of hashish, marijuana, barbiturates, benzodiazepines, fiunitrazepam, GHB, methaqualone, ketamine, PCP and analogs, LSD, mescaline, psilocybin, codeine, fentanyl and fentanyl analogs, heroin, morphine, opium, oxycodone HCL, hydrocodone, bitartrate, acetaminophen, amphetamine, cocaine, MDMA (methylenedioxy-methamphetarnine, methamphetamine and nicotine. The analyte may comprise a biological and chemical warfare agent selected from the group consisting of Tabun, Sarin, Soman, Cyclosarin, Methylphosphonothioic Acid, Sulfur Mustard, Nitrogen Mustard, Lewisite, Phosgene Oximine, Phosgene, Diphosgene, Chloropicrin, staphylococcal enterotoxin, staphylococcal enterotoxin B, ricin, botulinum toxin, mycotoxin, and trichothecene mycotoxins.

The analyte may comprise a foodstuff pollutant selected from the group consisting of aflatoxins, ciguatoxin, patulin, scombrotoxins, furans, polychlorinated biphenyls, fumonisins, ochratoxins, patulins, tricothecenes, zearalenone, Polycyclic aromatic hydrocarbons (PAHs), for example Benzo(a)pyrene, Benzanthracene, Benzo(b)fluoranthene, Fluoranthene and Naphthalene. The analyte may comprise a poison selected from the group consisting of Aconite, Antimony, Arsenic, Cyanide, Hemlock, Hyoscine and Strychnine. The analyte may comprise an anabolic steroid selected from the group consisting of Boldenone, Dromostanolone, Dihydrotestosterone, Methenolone, Norethandrolone, Oxymetholone, Clostebol, DHEA, Fluoxymesterone, Androstenedione, Mesterolone, Methyltestosterone, Oxandrolone, Dehydrochlormethyl-Testosterone, Epitestosterone, Methandienone, Nandrolone, Oxymesterone and Stanozolol.

At this time, the analyte is preferably selected from the group consisting of MMA, homocysteine, troponin sub-units I or T, CKMB, LDH and GOT/SGOT, citrozine, Acetaminophen, Carbamazepine, Chloramphenicol, Digitoxin, Digoxin, Gentamicin, Kanamycin, Lidocaine, Lithium, Methotrexate, Phenobarbital, Propranolol, Quinidine, Theophylline, BNP, FSH, cumadine, vitamin K, aflatoxins, ricin, 2,4-Dichlorophenoxyacetic acid (2-4,D), atrazine, Alachlor, cyanazine, metolachlor, simazine, epinephrine, amphetamine, TSH, free T3 T4, PSA, steroids, estradiol, progesterone, testosterone, estrogen, Arsenic, Cyanide, and Strychnine.

Any liquid suspected of containing a specific analyte can be used as a sample. These liquids include biological fluids, environment samples, dyes, foodstuffs, drugs, toxins, industrial samples and byproducts of industrial^' production procedures. The biological fluid is selected from the group consisting of body fluids, liquid obtained from breath, tissue homogenates, and process fluids.

The liquid sample may be an environmental sample selected from the group consisting of liquids, such as water, oil, liquid waste, or liquid extracted from solids, such as liquid extracted from solid waste, soil, plants, and gas dissolved in solution.

The sample can be used as obtained directly from the source or following a pretreatment so as to modify its character. Pretreatment may involve separating plasma from blood, diluting viscous fluids, or the like. Methods of treatment can involve filtration, distillation, concentration, inactivation of interfering components, and the addition of reagents. For example, the sample may be centrifuged or filtered to remove particulate matter, or may be dissolved in or supplemented by a buffer or surfactants to provide a suitable medium in order to allow more efficient detection of the analyte. Suitable buffers include any of those known to skilled artisans, such as a 1-1,000 mM solution of Tris (TRIZMA, Sigma Chemical Co., St. Louis, Mo.), or 1-1,000 mM TRIS (2-Amino-2-(hydroxy-methyl)-l, 3-propanediol) or 0.05-0.5 % of the surfactant Polysorbate 20 (commercially also known as Tween^® 20). Other buffers include phosphate buffered saline (PBS), citrate buffer, or bicarbonate buffer.

The invention may also be used to detect analytes that are initially contained within solid-phase samples. These analytes would simply be extracted and suspended or dissolved in liquid prior to analysis using appropriate reagents or compositions for extracting or processing the sample to elute the analyte. The extraction process could be as simple as shaking a solid sample in a diluent or buffer such as those listed above, which could then be applied to the solid support. Optionally, equipment such as a manual press, mortar and pestle, homogenizer, juicer, mixer or food processor may be used to pre-treat or extract a sample to bring the target analyte into liquid form suitable for testing in the device. Solid samples may include biological tissues (obtained, for example, in the process of performing a biopsy), soil, or foliage.

The analyte-specific molecular imprinted polymer (MIP) may be prepared in accordance with any technique known to those skilled in the art. These methods include covalent imprinting (WuIIfG, 1982, Pure & Appl.Chem., 54, 2093-2102) whereby the monomers are covalently attached to the analyte and polymerized using a cross-linker. Subsequently, the analyte is cleaved from the polymer leaving analyte-specific binding cavities. Alternatively, a non-covalent imprinting method such as disclosed by Mosbach (U.S. Pat. No. 5,110,833) may be used, whereby the monomers interact with the target molecule by non-covalent forces and are then connected via a cross-linker to form target specific binding sites after removal of the target molecule. Variations on these methods may be used to construct thin molecularly imprinted films and membranes (Hong et al. 1998 Chem. Mater., 10, 1029- 1033); imprinting on the surface of solid supports (Blanco-Lόpez, et al, 2004, Anal. Bioanal. Chem., 378, 1922-1928; Sulitzky C. et al, 2002 Macromolecules, 35, 79-91); and microspheres (Ye et al., 2000, Macromolecules, 33, 8239 -8245).

Preferably, the molecular imprint polymer comprises a polymer polymerized from monomers cross-linked with cross-linker in the presence of the target analyte and a porogen, the polymer having a capacity for selectively binding the target analyte. In the preferred embodiment of an MMA-specific MIP, the molecular imprinted polymer comprises a polymer polymerized from diethylaminoethyl methacrylate monomer cross-linked with ethylene glycol dimethacrylate (EGDMA), in the presence of methylmalonic acid (MMA) and toluene as a porogen, the polymer having a capacity for selectively binding MMA.

The detectable reporter is selected from the group consisting of HABA, dyes, fluorescers, fluorescent dyes, radiolabels, magnetic particles, metallic particles, colored particles, metal sols, enzyme substrates, enzymes, chemiluminescers, photosensitizers and suspendable particles.

As evident from the preceding list, the detectable reporter may be a visible substance, such as a colored latex bead, or it may participate in a reaction by which a colored product is produced. The reaction product may be visible when viewed with the naked eye, or may be apparent, for example, when exposed to a specialized light source, such as ultraviolet light. Although it is expected that viewing the result zone (either directly or indirectly) will be the primary way in which the test result is obtained, other methods, for example where the analyte is associated with a fluorescent substance that is detected by subsequent exposure to a scanner, are also considered within the scope of the invention.

The concentration of analyte in the sample will be indicated by how much of the detectable reporter subsequently becomes associated with the results zone. A detectable signal that indicates the concentration of the analyte is produced in the results zone.

Optionally, the devices of the invention may include a reference zone positioned downstream of the results zone, and a control zone, wherein a detectable signal that is independent of the analyte is produced. Thus, the control zone may serve as an indication that the device is functioning properly. Optionally, the devices of the invention may include one or more absorbent pads that are positioned to facilitate the flow of the analyte through the results zone, the reference zone, and/or the control zone.

The method of the invention involves introducing a liquid sample (which is suspected of containing the analyte to be tested for) on to the support, and permitting the sample to migrate along the support by capillary action. If the analyte is present in the sample, it will bind to the analtye-specific MIP, and occupy binding sites that previously contained an analyte analog-.reporter conjugate. Hence the displaced reporter conjugate will migrate along or through the solid support to the results zone, where it is bound to the reporter conjugate binding element, where the presence and/or intensity of the detectable signal positively indicates an amount of analyte in the sample.

Thus, in one aspect, the invention is directed to a single displacement diagnostic device for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample. The device comprises a solid support capable of conveying a liquid sample therethrough, the sample being movable along or through the solid support in the path of liquid flow by capillary action. The support comprises: a. a defined sample application area for applying the sample to the device and bringing it in contact with the solid support; b. a defined MIP-conjugate zone downstream of the sample application area comprising an analyte-specific MIP fixed to the solid support on the flow path of the sample. The MIP has analyte- specific binding sites saturated with a releasable analyte analog:reporter conjugate in a dry state. The affinity of the analyte to the binding sites of the analyte-specific MIP is greater than the affinity of the analyte analog:reporter conjugate to the binding sites of the analyte-specific MIP. The MIP, when contacted with a liquid sample containing the analyte, is capable of binding the analyte and displacing the analyte analog:reporter conjugate in an amount directly proportional to the concentration of the specific analyte, causing the displaced analyte analog:reporter conjugate to flow downstream in the path of liquid flow; c. a defined results zone comprising an analyte analog:reporter conjugate binding element fixed to the solid support on the flow path of the sample downstream of the MIP-conjugate zone. The reporter conjugate binding element is capable of binding the analyte analog: reporter conjugate displaced from the MIP-conjugate zone when a liquid sample containing the analyte flows in the flow path zone for providing a detectable signal that indicates the presence or concentration of the analyte in a sample;

Optionally, the solid support further comprises a reference zone for establishing a reference point in determining the presence or semi-quantification of an analyte in the tested sample, wherein the reference zone is not capable of capturing by specific binding any compound in said sample. The support may also optionally include a positive control zone comprising means for generating a positive control confirming the proper flow and binding of the analyte analog:reporter conjugate to the results zone to thereby determine that a test is working. Additionally, the support may optionally include an absorbent zone comprising a pad of absorbent material in fluid communication with the solid support when the pad and solid support are wet, the pad having sufficient porosity and capacity to absorb excess liquid.

In another aspect, the invention is directed to a double displacement diagnostic device for directly detecting and determining the presence, absence or concentration of a target analyte in a liquid sample, the device comprising a solid support capable of conveying a liquid sample therethrough, the sample being movable along or through the solid support in the path of liquid flow by capillary action, the support comprising: a. a defined sample application area for applying the sample to the device and bringing it in contact with the solid support; b. a defined MlP-conjugate zone downstream of the sample application area comprising an analyte-specific MIP fixed to the solid support on the flow path of the sample. The MIP has analyte- specific binding sites saturated with a releasable first binding agent:analyte conjugate in a dry state, wherein the affinity of the analyte to the binding sites of the analyte-specific MIP is greater than the affinity of the first binding agentanalyte conjugate to the binding sites of the analyte-specific MIP. The MIP when contacted with a liquid sample containing the analyte capable of binding the analyte and displacing the first binding agentanalyte conjugate in an amount directly proportional to the concentration of the specific analyte, causing the displaced first binding agentanalyte conjugate to flow downstream in the path of liquid flow; c. a defined reporter-conjugate binding zone comprising a first binding area of reporter- conjugate binding element immobilized to the solid support on the flow path of the sample downstream of the MlP-conjugate zone. The reporter-conjugate binding element has binding sites saturated with a detectable, releasable second binding agentreporter conjugate in a dry state. The affinity of the first binding agentanalyte conjugate to the binding sites of the reporter-conjugate binding element is greater than the affinity of the second binding agentreporter conjugate to the binding sites of the reporter- conjugate binding element. The first binding area of reporter-conjugate binding element is capable of binding the first binding agentanaltye conjugate and displacing second binding agentreporter conjugate in an amount directly proportional to the concentration of the specific analyte in the sample, causing the displaced second binding agentreporter conjugate to continue to flow downstream in the path of liquid flow; d. a defined results zone comprising a second binding area of reporter-conjugate binding element immobilized to the solid support on the flow path of the sample downstream of the reporter- conjugate binding zone. The second binding area of reporter-conjugate-binding element is capable of binding displaced second binding agentreporter conjugate for providing a detectable signal that indicates the presence or concentration of said analyte in a sample; The device may optionally include a reference zone, a positive control zone and an absorbent zone to absorb excess liquid.

In yet another aspect, the invention is directed to a device comprising a solid support including a first component comprising an analyte-specific MIP saturated with an analyte analog-conjugate comprising the analyte conjugated to biotin, optionally through a spacer; a second component comprising a first biotin binding element saturated with reporter-conjugate comprising a biotin analog having lower affinity to the biotin binding element than biotin conjugated to a reporter, and a third component comprising a second biotin binding element.

In a further aspect, the invention is directed to a method for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample by an assay comprising the steps of applying a sample suspected of containing the analyte to the sample application area of the single displacement diagnostic device of the invention and allowing the sample to flow along or through the solid support and contact the MlP-conjugate zone so that, if analyte is present in the sample, analyte binds to the binding sites of the MIP, displacing analyte analog:reporter conjugate which flows to the results zone where it is captured by the analyte analog:reporter conjugate binding element, producing a detectable signal that indicates the presence or amount of the analyte in the sample.

In yet another aspect, the invention is directed to a method for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample by an assay, the method comprising the steps of:

(a) applying a sample suspected of containing the analyte to the sample application area of the double displacement diagnostic device of the invention; and

(b) allowing the sample to flow along or through the solid support and contact the MIP- conjugate zone so that, if analyte is present in the sample, analyte binds to the binding sites of the MIP displacing first binding agentanalyte conjugate which flows to the reporter-conjugate binding zone, the first binding area of reporter-conjugate binding element binding the displaced first binding agentanalyte conjugate and thereby displacing second binding agentreporter conjugate which continues along the path of liquid flow to the results zone and is captured by the second binding area of reporter-conjugate binding element, producing a detectable signal that indicates the presence or amount of the analyte in the sample, wherein the amount of second binding agent:reporter conjugate captured is proportional to the concentration of said target analyte in the sample.

In a further aspect, the invention is directed to a method for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample by an assay, the method comprising the steps of: (a) fixing MIP specific to a target analyte to a solid support;

(b) saturating the binding sites on the analyte-specific MIP with a releasable first binding agentanalyte conjugate to form an MIP-conjugate zone;

(c) immobilizing a first reporter-conjugate binding element to the solid support downstream of said MIP-conjugate zone;

(d) saturating the binding sites of the reporter-conjugate binding element with a detectable, releasable second binding agentreporter conjugate to form an immobilized reporter-conjugate binding zone;

(e) immobilizing a second reporter-conjugate binding element to the solid support downstream of the reporter-conjugate binding zone;

(f) introducing a sample of liquid to be analyzed for the presence of the target analyte onto a sample application area that contacts the solid support to generate a flow of the liquid sample along or through the solid support;

(g) allowing an analyte, if present in the sample, to displace the first binding agent:analyte conjugate from the analyte-specific binding sites on the MIP in an amount directly proportional to the amount of analyte present in the sample;

(h) allowing the first binding agent:analyte conjugate to displace the second binding agent:reporter conjugate from the binding sites of the reporter-conjugate binding element in an amount directly proportional to the amount of analyte present in the sample; wherein the affinity of the first binding agentanalyte conjugate to the analyte-specific MIP is lower than the affinity of the analyte to the analyte-specific MIP, and wherein the affinity of the second binding agent:reporter conjugate to the reporter-conjugate binding element is lower than the affinity of the first binding agentanalyte conjugate to the reporter-conjugate binding element;

(i) allowing the displaced second binding agentreporter conjugate to flow freely until it is captured by said second reporter-conjugate binding element; and

(J) detecting the signal displayed by the second reporter-conjugate binding element wherein the concentration of displaced second binding agentreporter conjugate corresponds to the concentration of the analyte in the sample.

In yet further aspects, the method includes the step of comparing the intensity of the signal in the results zone with the intensity of the signal in a reference zone to determine the concentration of the analyte in the sample.

In still further aspects, the results zone includes a scale parallel to the immobilized reporter- conjugate binding element calibrated to correspond to the analyte concentration in the liquid sample being analyzed and wherein the presence and concentration of analyte in the liquid is determined by the area covered by the reporter-conjugate along and through the reporter-conjugate binding element in the results zone.

In yet another aspect, the invention is directed to a method of detecting MMA present in a liquid sample, the method comprising the steps of: a) contacting the sample with a single or double displacement device according to the invention, and b) detecting, in the results zone, the amount of reporter conjugate bound to the reporter- conjugate binding element, wherein the amount of the reporter-conjugate is indicative of the presence or amount of MMA in the sample; c) diagnosing a vitamin B12 deficiency based upon the amount of MMA detected in the sample; and d) optionally, diagnosing methylmalonic aciduria disease based upon the amount of MMA detected in the sample.

In another aspect, the devices of the invention may be packaged together in a diagnostic kit with any or all of the diluents or reagents for extracting or processing the sample to elute the analyte and to detect a given analyte.

In yet other aspects, the diagnostic kit further includes packaging material and instructions for performance of the quantitative analysis on at least one type of liquid sample.

The foregoing and other features and advantages of the invention will be apparent from the following description which proceeds with reference to the accompanying Figures and claims.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings and the following detailed description, it being understood that the particulars shown are by way of example and illustrative discussion only, and are presented to provide what is believed to be the most useful and readily understood description of the embodiments of the invention. No attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. In the drawings:

FIG. IA is a side view illustration of a first embodiment of a lateral flow device with single displacement and vertical visualization as described in Example 1. FIG. IB is a a side view illustration of lateral flow device with single displacement and horizontal visualization as described in Example 2.

FIG. 2A is a side view illustration of lateral flow device with double displacement and vertical visualization as described in Example 3.

FIG. 2B is a side view illustration of lateral flow device with double displacement and horizontal visualization as described in Example 4.

FIG. 3 A is a side view illustration of flow through device with single displacement and vertical visualization as described in Example 5.

FIG. 3B is a side view illustration of flow through device with single displacement and horizontal visualization as described in Example 6.

FIG. 4A is a side view illustration of flow through device with double displacement and vertical visualization as described in Example 7.

FIG. 4B is a side view illustration of flow through device with double displacement and horizontal visualization as described in Example 8.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The diagnostic methods, devices and kits according to the present invention may be better understood with reference to the accompanying figures, examples, and description. It is contemplated that the invention is not limited in its application to the details set forth in the following description or drawings, or exemplified by the Examples. The invention may be practiced in various other ways and is capable of other embodiments. Also, it is contemplated that the phraseology and terminology used herein are for purposes of description and should not be regarded as limiting.

Before describing particular methods and devices of the invention, the general features of the invention will be described. These methods and devices provide the means to move the sample along or through one or more zones so that the concentration of a target analyte in a liquid sample may be determined.

For a clearer understanding of the present invention, a glossary defining key terms related to this invention is presented.

I. Glossary

ANALYTE - an atom, molecule, group of molecules or compound of natural or synthetic origin sought to be detected or measured, or an analogue or derivative thereof, that is capable of binding specifically to at least one binding partner (e.g., MIP). Analogues or derivatives may be used when they participate in an assay, as one member of a binding pair, in a manner that is substantially equivalent to that of the analyte itself.

The methods of this invention may be practiced with assays for virtually any analyte. Analytes vary in size and may range from small molecule analytes, such as MMA or atrazine, having a molecular weight of less than about 500 Daltons, to large analytes such as TSH (about 26kD) CK-MB (about 4IkD) or immunoglobulin analytes such as IgG, which is about 16OkD.

ANALYTE ANALOG - a modified analyte that has structural similarity to the natural form of the analyte and can bind to at least one analyte binding partner. In certain embodiments of the invention, the analyte analog is a detectable analyte analog-reporter conjugate. For instance, an analyte analog- conjugate may comprise the analyte conjugated to biotin.

ANALYTE ANALOG:REPORTER CONJUGATE - The target analyte or its analog conjugated to a reporter molecule from the group of molecules discussed below. The analyte analog:reporter conjugate has high binding affinity to the analyte-specific MIP, but lower than the affinity of the unmodified analyte. The conjugated molecules remain attached to the binding cavities of the analyte-specific MIP even in a moist state unless they are displaced, in a dose-dependent manner, by the target analyte in the tested liquid sample. When displaced from the polymer the analyte analog:reporter conjugate is freely mobile within the moist solid support. These elements could be, but are not limited to, biotin binding elements (Avidin, StrepAvidin and NeutrAvidin), reporter-specific antibodies, enzymes, substrates and MIP prepared specifically against the analyte analog:reporter conjugate.

ANALYTE ANALOG:REPORTER CONJUGATE BINDING ELEMENT- A zone on the device comprising element(s) that possess high affinity to the ANALYTE ANALOG :REPORTER CONJUGATE molecule.

ANALYTE-SPECIFIC - Refers to a binding reaction which is determinative of the presence of the analyte in the presence of a heterogeneous population of molecules.

BINDING AFFINITY- A term that refers to the strength of binding of one molecule to another. If a particular molecule will bind to or specifically associate with another particular molecule, these two molecules are said to exhibit binding affinity for each other. Binding affinity is related to the association constant and dissociation constant for a pair of molecules, but it is not critical to the invention that these constants be measured or determined. Rather, affinities as used herein to describe interactions between molecules of the described methods and devices are generally apparent affinities (unless otherwise specified) observed in empirical studies, which can be used to compare the relative strength with which one molecule (e.g., a MIP or other specific binding partner) will bind two other molecules (e.g., an analyte and an analyte-reporter conjugate). The concepts of binding affinity, association constant, and dissociation constant are well known.

BINDING AGENT: ANALYTE CONJUGATE - The target analyte of the test, conjugated to a binding agent (preferably biotin or a derivative of biotin) directly or via a spacer. The binding agent has specific affinity to its corresponding binding partner. Where the binding agent is biotin and its derivatives, the corresponding binding partner or agent is Avidin, StrepAvidin, NeutrAvidin and similar biotin binding agents. The binding agenfcanalyte conjugate has high binding affinity to the analyte- specific MIP, but lower than the affinity of the unmodified analyte. The binding agent: analyte conjugate molecules remain attached to the binding cavities of the analyte-specific MIP even in a moist state unless they are displaced, in a dose-dependent manner, by the target analyte in the tested liquid sample. When displaced from the polymer, the binding agentanaltye conjugate is freely mobile within the moist solid support.

BINDING AGENT:REPORTER CONJUGATE - Any reporter molecule, as described for the analyte analog:reporter conjugate, conjugated to a binding agent capable of specific binding to the same binding element as the BINDING AGENT:ANALYTE CONJUGATE. This binding agent possesses high affinity to this binding element, but lower than the affinity of the BINDING AGENT:ANALYTE CONJUGATE that are employed in the device. When biotin, for example, is employed in the device as the binding agent in the BINDING AGENT: ANALYTE CONJUGATE, a derivative of biotin or other molecules (such as HABA) having lower affinity to biotin binding elements, such as Avidin, StrepAvidin and NeutrAvidin than biotin will be used in the construction of the BINDING AGENT:REPORTER CONJUGATE.

BINDING ELEMENT - a molecular structure able to specifically bind its respective binding partner with sufficient affinity. Neither the specific sequences nor the specific boundaries of such elements are critical, so long as binding activity is exhibited. Binding characteristics necessarily includes a range of affinities, avidities and specificities, and combinations thereof, so long as binding activity is exhibited.

BINDING PARTNER OR BINDING AGENT - Any molecule or composition capable of recognizing and binding to a specific structural aspect of another molecule or composition. Examples of such binding partners and corresponding molecule or composition include biotin/avidin, antigen/antibody, hapten/antibody, hormone/receptor, nucleic acid strand/complementary nucleic acid strand, substrate/enzyme, inhibitor/enzyme, protein A (or G)/immunoglobulins, carbohydrate/lectin, virus/cellular receptor and apoprotein/lipid.

The releasable first binding agent is selected from the group consisting of biotin, a biotin analog, a biotin derivative, an antigen, Protein A and Protein G, cellulose binding protein, hormones, toxins, lipids, fatty acids, nucleic acids, glycoconjugates, lectins, substrates and ligands.

The releasable second binding agent may be selected from the group consisting of a biotin analog, a biotin derivative, HABA, an antigen, Protein A and Protein G, cellulose binding protein, liposomes, hormones, toxins, lipids, fatty acids, complementary nucleic acids, glycoconjugates, lectins, substrates and ligands or their analogs, provided that said second binding agent has lower affinity to the reporter-conjugate binding element than said first binding agent. Biotin analogs are identified in Advances in Protein Chemistry, edited by Anfinsen, Edsall and Richards, Academic Press (1975), pages 104-111.

MIP-CONJUGATE ZONE - A zone on the device comprising analyte-specific molecular imprinted polymer ("MIP") possessing specific binding affinity for the tested analyte, fixed to the solid support, either directly or indirectly. The analyte-specific MIP is saturated with molecules of either a releasable ANALYTE ANALOG:REPORTER CONJUGATE or a releasable FIRST BINDING AGENT: AN ALYTE CONJUGATE (according to the various embodiments of the device) that saturate the accessible analyte-specific binding cavities of the molecular imprinted polymer. By MIP "fixed" to the solid support is meant keeping the object in question at a fixed position, either by physical means or by chemical bonding (immobilization) of the object to the solid carrier. Immobilization refers to chemical fixation of an object to a solid support by forming bonds, covalent or non-covalent between the object and the solid support.

REFERENCE ZONE - A zone where several lines of analyte analog-reporter binding element are immobilized to the solid support, having known amounts of the analyte analog-reporter conjugate bound to them and producing a distinctive signal with an intensity proportional to the amount of the - bound reporter.

REPORTER - Any molecule or composition that is capable of being attached to an analyte, analyte analog or binding partner that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, radio-activity detection or chemical means. The reporter molecule is selected from a large variety of available molecules, according to the desired attributes of the analytical device. Some examples of such reporter molecules, but not limiting in any way, include: HABA, dyes, fluorescent dyes, radiolabels, magnetic particles, metallic particles, colored particles (such as gold and latex sols), fluorescent particles, metal sols, enzyme substrates and enzymes, chemiluminescers, photosensitizers and suspendable particles. The coupling of a compound (e.g., an analyte) to a reporter can be through covalent bonds, adsorption processes, hydrophobic and/or electrostatic bonds, as in chelates and the like, or combinations of these bonds and interactions and/or may involve a linking group.

SAMPLE APPLICATION AREA - An absorbent pad made of bibulous, porous or fibrous material capable of absorbing liquid rapidly. Its porosity may be unidirectional (i.e., with pores or fibers running wholly or predominantly parallel to an axis of the flow) or multidirectional (i.e., omnidirectional, a sponge-like structure). A porous plastic material, such as polypropylene, polyethylene (preferably of very high molecular weight), polyvinylidene flouride, ethylene vinylacetate, acrylonitrile and polytetrafluoro-ethylene may be used. Pre-treatment with a surface-active agent during manufacture may be used to reduce any inherent hydrophobicity and improve the ability to take up and transport a moist sample rapidly and efficiently. The material may also be made of paper or other cellulosic materials. The material connects an opening in the casing of the device with the solid support of the device.

A pad separating serum from whole blood, such as described in U.S. Pat. No. 5,660,798, may be used with devices that test analytes in serum. A sample applied to the sample application area is absorbed by the absorbent pad, flows freely by capillary forces and comes in contact with the solid support. Depending on the type of device, the volume of the sample that enters the device may be either random or controlled by a smart application pad that allows a precise pre-determined sample volume to enter the device (see for example U.S. Pat. No. 6,008,056).

SOLID SUPPORT - Any porous material used in the flow devices industry and having a porous structure that facilitates liquid advancement along the device by capillary forces. These materials often possess also good binding capabilities for proteins. Examples of such materials, but not limiting in any way are: Nitrocellulose (High Protein Binding) Cellulose Acetate (Low Protein Binding) Glass Fiber Membranes (Non-Protein Binding) and novel micro-fluidic systems, such as the system disclosed in U.S. Pat. Application No. 20050042766. Preferably, the solid support is selected from the group consisting of a porous material, a porous membrane, a granular material, and an absorbent material. A casing keeps the solid support in a suitable configuration in order to ensure correct functioning of the entire device. There are usually 1-4 windows in the casing. There is always a results window that serves to allow observation of the result. A control window allows for viewing of a control reaction, e.g., to confirm adequate performance of the test. A third window or opening in the casing allows application of the liquid sample to the SAMPLE APPLICATION AREA, at the sample window, either by direct placement of the device in the sample (allowing contact of the sample at the open window), or by application of the sample with a dropper or a similar device to the sample window. Additionally, there may be a reference window to allow comparison of the intensity of the results in the results zone with the intensity of indicator bands in the reference zone.

The sensitivity of particular assays is a function of the relative affinity and concentration of the various reagents, the times during which particular reagents and analyte are in contact with each other and the intensity of the signal produced by the reporting system. Those skilled in the art are familiar with methods for optimizing and characterizing the sensitivity and dose-response characteristics of conventional assays used in lateral flow and flow through devices.

In another aspect of the invention, a double displacement approach is disclosed that is designed to facilitate signal formation and interpretation and that may be applied in other apparatuses of similar nature. This approach finds particular utilization with the well-established biotin-avidin system that offers very strong binding affinity together with a multitude of analogs and derivatives that may be used. The double displacement approach is characterized by the following elements:

A. An analyte-specific MIP fixed to the solid support of the device on the flow path of the sample.

B. A conjugate of the analyte with biotin, either directly or via a spacer. This analyte conjugate possess high affinity to the analyte-specific MIP, but sufficiently lower than the affinity of the original analyte so in the presence of the analyte it is displaced from the analyte-specific binding sites in the MIP. The analyte-biotin conjugate occupies the binding cavities of the analyte-specific MIP in a dry state.

C. A biotin binding element fixed to the solid support.

D. Occupying the biotin binding sites of the second biotin binding element of item C. is a derivative of biotin covalently bound to reporter molecule(s), having high affinity to a biotin binding element, but lower than biotin itself. The reporter molecule(s) may be bound directly to the biotin derivative or through a carrier, such as a protein.

E. A second biotin binding element which may be spaced vertically or horizontally to the sample flow, at the results window.

The double displacement approach operates as follows: 1) A liquid sample applied to the device brings any molecules of the analyte in the sample in contact with the analyte-specific MIP and leads to a dose dependent displacement of the analyte-conjugate molecules.

2) The analyte conjugate molecules that were displaced by the analyte in the sample flow with the sample liquid until they reach the biotin-binding element where the biotin derivative conjugated to the reporter is bound by the biotin-binding element. The biotin of the analyte-conjugate displaces the biotin derivative from the biotin-binding element, due its superior binding capability, in a dose dependent manner.

3) The molecules of the biotin derivative conjugated to the reporter that were released by the analyte-conjugate flow freely until they reach the second biotin-binding element at the results window, where they are captured.

4) The signal of the reporter molecules conjugated to the biotin derivative that is now fixated at the results window is interpreted according to the nature of the reporter molecules.

This double displacement approach has several advantages over the common single displacement method when the reporter is directly bound to the target molecule. As biotin is a relatively small molecule, adding it to the target molecule should not interfere much with its interaction with the analyte-specific MIP. The chemistry of biotin is well established and many commercial reagents are available for the modification of the target analytes. The binding of biotin to the various biotin-binding elements is very rapid and strong, contributing to the overall performance of the devices. Since the reporter is not bound directly to the analyte, there is great flexibility in the choice of the reporter and the ability to conjugate it to the biotin-derivative. The inability or difficulties in binding a reporter molecule to certain analytes may be a limiting factor for using the displacement approach in these cases. Additionally, it allows increasing the sensitivity of the devices, since various ways of signal amplification known to those skilled in the art may be employed due to the separation between the sensing elements and the results area.

Another advantage is that for devices for various analytes that employ the same signal formation method, most of the production is similar and only the specific MIP and the analyte conjugated to biotin would need to be developed for every individual product. This makes the time to market of new products shorter and reduces development and production costs.

The double displacement approach is of special value when it is difficult to directly detect the binding of a target analyte to the sensing element, i.e., in the case of a small antigen when binding of two antibodies is impossible. In this case, competition or displacement approaches are in order. However, in some cases conjugation of a large reporter molecule is problematic, again due to interference of the reporter molecule to the binding of the conjugate to the sensing element. This problem is overcome by the double displacement approach, since the reporter molecule is not associated directly to the sensing and detection event, and thus, can be manipulated according to the specific needs of sensitivity, etc. This approach is applicable to every assay system (not just using MIPs) that uses specific recognition, where there is a capability of manipulating or obtaining analogs so as to get members with lower binding affinity to the sensing element, and, in turn, to the binding element. Thus, the double displacement approach has utility beyond MIPs to any binding pairs such as those enumerated above, including, biotin/avidin, antigen/antibody, hapten/antibody, hormone/receptor, nucleic acid strand/complementary nucleic acid strand, substrate/enzyme, inhibitor/enzyme, protein A (or G)/immunoglobulins, carbohydrate/lectin, virus/cellular receptor and apoprotein/lipid. Another important embodiment of the present invention is the method for visual monitoring and interpretation of the signal at the results window. In one variation of the monitoring system, termed the "vertical visualization method", the reporter-conjugate binding element (either biotin-binding element or analyte- reporter binding element) is placed vertical to the sample flow path to capture and concentrate as many as possible of the free-flowing reporter conjugate molecules, in a random order. The greater the amount of the reporter conjugate (which is a direct representation of the amount of the analyte in the tested sample) that binds the binding element, the stronger the visual signal obtained. Next to the results window is a reference window comprised of several indicator lines of binding element impregnated each with increasing precise amounts of the reporter conjugate. These lines will exhibit a range of color intensities, which are proportional to the known amount of the reporter conjugate bound to them. Comparison of the intensity of the signal in the results window with the intensity of the lines at the adjacent reference window enables good estimation of the amount of the analyte in the tested sample. In another variation of the monitoring system, termed the "horizontal visualization method", a stripe of well-defined reporter-conjugate binding element is accurately placed, horizontally, at the path of the sample flow. The device at this location should be manipulated in such a manner as to allow the flow of the liquid only through the area covered with the reporter-conjugate binding element and take measures to avoid sample flow around or below the binding element covered area. This can be achieved, for example, by physically cutting the porous material in such a way that only the binding element covered area remains in the flow path, or by using methods such as patterning of flow channels by photolithography [Martinez et al, 2007 Angwe. Chem. Int. 119, 1340-1342]. This ensures that all the conjugated reporter molecules displaced in response to the presence of the analyte in the sample arrive at the immobilized binding element. The displaced molecules of the reporter conjugate that reach the binding element migrate by capillary action and any reporter-conjugate molecules are captured by the binding element. However, as the absorption capacity of the binding element is limited, and as the binding sites of the binding element become filled with reporter-conjugate molecules, the binding element molecules at the front of the stripe becomes saturated and the reporter-conjugate molecules must migrate further down the stripe to find free binding sites. The higher the concentration of the analyte in the sample, the larger the area of the binding element that the reporter-conjugate will cover. Pre-calibration of the area covered by known amounts of the reporter-conjugate will enable accurate determination of the analyte concentration according to the distance covered by the reporter-conjugate along the stripe of the binding element by incorporating in the device a calibrated scale next to the immobilized binding element that allows the user to determine the amount of the analyte in the sample.

The devices may comprise more than one solid support, whereby more than one analyte can be detected in a sample.

The first binding area of reporter-conjugate binding element and the second binding area of reporter-conjugate binding element may be identical or different and each are selected from the group consisting of avidin, streptavidin, NeutrAvidin, compounds having high specific affinity to biotin, membranes, receptors, immunoglobulins, cellulose, enzymes, lectins, glycoconjugates, complementary nucleic acids and hydrophobic sites having high affinity to their respective binding partners.

The devices proposed herewith could be either lateral flow devices, which are intended to include devices with a dipstick format, or flow-through devices and the detection system may be either by single displacement and direct monitoring of the conjugated reporter or by the double displacement approach, as will be explained in the Examples below.

IL Detailed Description of the Figures

The following Examples are illustrative of the invention, and are not intended to be limiting in any way. One skilled in the art will recognize a variety of non-critical parameters that can be changed. FIGS. 1- 2 show four different embodiments of a lateral flow device of the present invention, and FIGS. 3-4 show four different embodiments of a flow through device of the present invention, each of which will be described separately in Examples 1-8.

EXAMPLE I: Embodiment of FIG. IA

There is described below the structure and operation of a lateral flow device with single displacement and vertical visualization.

FIG. IA is a side view illustration of a lateral flow device for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample. The analytical test device comprises a hollow, solid casing 10 that contains a solid support 12 in the form of a porous test strip that serves as a carrier capable of conveying a liquid sample therethrough, the sample being movable along the solid support in the path of liquid flow by capillary action. The solid support 12 has defined zones, including a sample application area comprising a sample application pad 14 for applying the sample to the device and bringing it in contact with the solid support 12. The sample application pad 14 is located adjacent to an opening or window 16 in the casing 10 for applying the liquid sample. The target analyte, if present in the liquid sample, is carried from the sample application area to a MIP- conjugate zone downstream of the sample application area comprising an analyte-specific MIP 18 fixed to the solid support 12 on the flow path of the sample. The MIP has analyte-specific binding sites saturated with a releasable analyte analog:reporter conjugate in a dry state. The affinity of the analyte to the binding sites of the analyte-specific MIP is greater than the affinity of the analyte analog:reporter conjugate to the binding sites of the analyte-specific MIP. When the liquid sample containing the analyte contacts the analyte-specific MIP, the analyte in the sample binds to the analyte-specific cavities of the MIP and thereby displaces the analyte analog-reporter conjugate which occupy these cavities in an amount directly proportional to the concentration of the specific analyte, causing the displaced analyte analog:reporter conjugate to flow downstream in the path of liquid flow.

Further downstream on the solid support 12 is an analyte analog:reporter conjugate binding element 20 immobilized to the solid support 12 on the flow path of the sample downstream of the analyte- specific MIP 18. An opening in the casing 10 is located above the analyte analog:reporter conjugate binding element 20, comprising the results window 22 of the device. The reporter conjugate binding element 20 binds the analyte analog:reporter conjugate displaced from the analyte-specific MIP 18 when a liquid sample containing the analyte flows in the flow path zone and provides a detectable signal in the results window 22 that indicates the presence or concentration of the analyte in the sample.

Further downstream on the solid support 12 are three discrete and non-overlapping indicator bands of analyte analog:reporter conjugate binding element, each of which extends longitudinally on the strip, wherein 24a represents a low concentration reference band, 24b represents a medium concentration reference band and 24c represents a high concentration reference band. The three indicator bands comprise a reference zone for establishing a reference point in determining the presence or semi- quantification of an analyte in the tested sample. A corresponding reference window 26 appears in the casing 10 above the reference bands 24a, 24b and 24c. The reference zone may comprise at least one discrete band of binding element impregnated with a known quantity of the analyte analog:reporter conjugate in the case where the presence or absence of a target analyte in the sample is to be determined. The reference zone may comprise at least two spaced indicator bands of binding element impregnated each with increasing quantities of the analyte analog:reporter conjugate and exhibiting a range of color intensities proportional to the amount of the analyte analog:reporter conjugate bound to it, in the case where the amount of a target analyte in a sample is to be determined. The user interprets the results, and determines the presence, absence or semi-quantification of the concentration of the analyte in the sample, by visually comparing the intensity of the signal in the results window 22 with the intensity of one or more bands in the reference window 26.

Downstream of the reference bands 24a, 24b and 24c is a control pad 32 impregnated with analyte analogrreporter conjugate that is stationary in a dry state but becomes freely flowing when the solid support 12 is wetted with the liquid of the sample. Further downstream is another analyte analog:reporter conjugate binding element 34 anchored to the solid support 12. There is a corresponding control window 30 in the casing above the analog:reporter conjugate binding element 34 for viewing the results. The analyte analog:reporter conjugate that is released from the control pad 32 by the fluid from the sample reaches the binding element 34 and become fixed to the solid support 12, producing a visual signal in the control window 30. The control pad and analyte analog:reporter conjugate binding element 34 together comprise a control zone for generating a positive control confirming the proper flow and binding of the analyte analog:reporter conjugate to the results zone to thereby determine that a test is working.

At the distal end of the device is an absorbent pad 36 of absorbent material in fluid communication with the solid support 12 when the pad 36 and solid support 12 are wet. The pad has sufficient porosity and capacity to absorb the surplus of the fluid and ensure continuous flow throughout the device.

EXAMPLE 2: Embodiment of FIG. IB

There is described below the structure and operation of a lateral flow device with single displacement and horizontal visualization of the test results.

The lateral flow device is the same as that described above in Example 1, with similar numerals designating similar parts except that modifications are indicated with the reference numeral and the letter "a" affixed. The modifications are as follows: There is no reference zone in the device of Fig. IB, thus, the casing 38 has fewer windows or openings, and has an enlarged results window 42. Results are read only from the results window 42 according to the distance that the analyte analog:reporter conjugate covered. The area covered by the immobilized analyte analog:reporter conjugate binding element 44 is enlarged with a scale 46 running parallel to it. The scale 46 and the area covered by the immobilized analyte analog:reporter conjugate binding element 44 are co-calibrated to correspond to the analyte concentration in the liquid sample being analyzed. The presence and concentration of analyte in the liquid is determined at the results window 42 by the area covered by the analyte analogrreporter-conjugate along and through the analyte:analog reporter-conjugate binding element 44. The results of the control signal are viewed in control window 30a. EXAMPLE 3: Embodiment of FIG. 2A

There is described below the structure and operation of a lateral flow device with double displacement and vertical visualization of the test results.

FIG. 2A is a side view illustration of a lateral flow device for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample. The device comprises a hollow, solid casing 10b that contains a solid porous support 12b in the form of a test strip capable of conveying a liquid sample therethrough, the sample being movable along the solid support in the path of liquid flow by capillary action. The solid support 12b has defined zones, including a sample application area comprising a sample application pad 14b for applying the sample to the device and bringing it in contact with the solid support 12b. The sample application pad 14b is located adjacent to an opening or window 16b in the casing 10b for applying the liquid sample.

The target analyte, if present in the liquid sample, is carried from the sample application pad 14b along and through a MlP-conjugate zone downstream of the sample application area 14b, the MIP- conjugate zone comprises an analyte-specific MIP 50 fixed to the solid support 12b on the flow path of the sample. The MIP 50 has analyte-specific binding sites saturated with a releasable first binding agentanalyte conjugate in a dry state. The affinity of the analyte to the binding sites of the analyte- specific MIP 50 is sufficiently greater than the affinity of the first binding agentanalyte conjugate to the binding sites of the analyte-specific MIP 50 to bring about the displacement of the firs binding agentanalyte conjugate from the analyte specific binding sites of the MIP 50 in the presence of the analyte. When the liquid sample containing the analyte contacts the analyte-specific MIP 50, the analyte in the sample binds to the analyte-specific cavities of the MIP and thereby displaces the first binding agentanalyte conjugate which occupy these cavities in an amount directly proportional to the concentration of the specific analyte, causing the displaced first binding agentanalyte conjugate to flow downstream in the path of liquid flow.

Further downstream on the solid support 12b is a reporter-conjugate binding zone that comprises a first binding area of reporter-conjugate binding element 52 immobilized to the solid support 12b on the flow path of the sample downstream of the analyte-specific MIP 50, comprising reporter-conjugate binding element 52 with its binding sites saturated with a detectable, releasable second binding agentreporter conjugate in a dry state. The affinity of the first binding agentanalyte conjugate to the binding sites of the reporter-conjugate binding element 52 is greater than the affinity of the second binding agentreporter conjugate to the binding sites of the reporter-conjugate binding element 52. The first binding area of reporter-conjugate binding element 52 binds the first binding agentanaltye conjugate and displaces the second binding agentreporter conjugate in an amount directly proportional to the concentration of the specific analyte in the sample, causing the displaced second binding agenfcreporter conjugate to continue to migrate downstream in the path of liquid flow.

Further downstream on the solid support 12b is a second binding area of reporter-conjugate binding element 54 immobilized to the solid support 12b on the flow path of the sample downstream of the first binding area of reporter-conjugate binding element 52. An opening in the casing 10b is located above the second binding area of reporter-conjugate binding element 54, comprising the results window 22b of the device. The second binding area of reporter-conjugate-binding element 54 binds the second binding agentreporter conjugate displaced from the first binding area of reporter-conjugate binding element 52 when a liquid sample containing the analyte flows in the flow path zone and provides a detectable signal that indicates the presence or concentration of the analyte in the sample.

As described with respect to the device in Example IA, located further downstream on the solid support 12b are three discrete and non-overlapping indicator bands of the second binding agent:reporter conjugate binding element, wherein 56a represents a low concentration reference band, 56b represents a medium concentration reference band and 56c represents a high concentration reference band. The three indicator bands comprise a reference zone for establishing a reference point in determining the presence or semi-quantification of an analyte in the tested sample, above which is a corresponding reference window 26b in casing 10b. The user interprets the results, and determines the presence, absence or semi-quantification of the concentration of the analyte in the sample, by visually comparing the intensity of the signal in the results window 22b with the intensity of one or more bands in the reference window 26b.

As described above in connection with Examples IA and IB, downstream of the reference window 26b is a control pad 58 impregnated with second binding agent:reporter conjugate that is stationary in a dry state but becomes freely flowing when the solid support 12b is wetted with the liquid of the sample. Further downstream is another reporter-conjugate binding element 64 anchored to the solid support 12b. There is a corresponding control window 30a in the casing 10b above reporter- conjugate binding element 64 for viewing the results. The second binding agent:reporter conjugate that is released from the control pad 58 by the fluid from the sample reaches the binding element 64 and become fixed to the solid support 12b, producing a visual signal, viewed in the control window 30b. The control pad 58 and reporter-conjugate binding element 64 together comprise a control zone for generating a positive control.

At the distal end of the device is an absorbent pad 36b made of absorbent material in fluid communication with the solid support 12b when the pad 36b and solid support 12b are wet. The pad has sufficient porosity and capacity to absorb the surplus of the fluid and ensure continuous flow throughout the device. EXAMPLE 4: Embodiment of FIG. 2B

There is described below the structure and operation of a lateral flow device with double displacement and horizontal visualization of the test results.

The lateral flow device is the same as that described above in Example 3, with similar numerals designating similar parts except that modifications are indicated with the reference numeral and the letter "a" affixed. The modifications are as follows: There is no reference zone in the device of Fig. 2B. Results are read only from the results window 60 according to the distance that the second binding agent:reporter conjugate covered. The second binding area of reporter-conjugate binding element 66 under the results window 60 in casing 38a is elongated and includes a scale 68 parallel to the immobilized second binding area of reporter-conjugate binding element 66 calibrated to correspond to the analyte concentration in the liquid sample being analyzed. The presence and concentration of analyte in the liquid is determined by the area covered by the second binding agentrreporter-coηjugate along the second binding area of reporter-conjugate binding element 66 as viewed in the results window 60.

EXAMPLE 5: Embodiment of FIG. 3 A

There is described below the structure and operation of a flow through device with single displacement and vertical visualization.

FIG. 3A is a side view illustration of a flow through device for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample. The device comprises a hollow, solid casing 80 containing defined layers or zones made of reagent-containing porous materials, arranged so that fluid that is applied to the top of the device flows vertically through the various layers of the device, from one layer to another, until the fluid contacts an absorbent material at the bottom of the device.

A sample application area comprising a sample application pad 82 for applying the sample to the device is located adjacent to an opening or window 84 in the casing 80 at the top of the device and leads to a MIP-conjugate zone comprising an analyte-specific MIP 86 having analyte-specific binding sites saturated with a releasable analyte analog:reporter conjugate in a dry state. The affinity of the analyte to the binding sites of the analyte-specific MIP 86 is greater than the affinity of the analyte analog:reporter conjugate to the binding sites of the analyte-specific MIP 86. The target analyte, if present in the liquid sample, migrates from the sample application pad 82 through the analyte-specific MIP 86. When the liquid sample containing the analyte contacts the analyte-specific MIP 86, the analyte in the sample binds to the analyte-specific cavities of the MIP and thereby displaces the analyte analog-reporter conjugate which occupy these cavities in an amount directly proportional to the concentration of the specific analyte in the sample, causing the displaced analyte analog:reporter conjugate to migrate further down through the device.

Next is an analyte analog:reporter conjugate binding element 90 fixated to the porous carrier or support 88 on the flow path of the sample downstream of the analyte-specific MIP 86. A clear window in the solid casing 80 is located in front of the analyte analog:reporter conjugate binding element 90, comprising the results window 92 of the device. The reporter conjugate binding element 90 binds the analyte analog:reporter conjugate displaced from the analyte-specific MIP 86 when a liquid sample containing the analyte migrates to the analyte analog:reporter conjugate binding element 90 and provides a detectable signal in the results window 92 that indicates the presence or concentration of the analyte in the sample.

The next zone is a reference zone comprising three discrete and non-overlapping indicator bands of analyte analog:reporter conjugate binding element, separated by porous carrier zones 88, wherein 94a represents a low concentration reference band, 94b represents a medium concentration reference band and 94c represents a high concentration reference band. A clear reference window 96 is located in front of the three indicator bands 94a, 94b and 94c. The user interprets the results by visually comparing the intensity of the signal in the results window 92 to the intensity of the bands in the reference window 96.

Underneath the reference zone is a porous carrier zone 88 followed by a control area 100 comprising a porous carrier impregnated with unbound analyte analog:reporter conjugate. This conjugate 100 is stationary in the dry state but becomes freely flowing when the porous carrier is wetted with the liquid of the sample. Further down, separated by another porous carrier zone 88, is another analyte analog:reporter conjugate binding element 102. A control window 104 is located in front of the reporter conjugate binding element 102. The analyte analog:reporter conjugate which is. released from the control area 100 by the fluid from the sample reaches the analyte analog:reporter conjugate binding element 102 and is bound to the carrier, producing a visual signal visible at the control window 104. At the distal end of the device is an absorbent material 106 that absorbs the surplus of the fluid and ensures continuous flow throughout the device.

EXAMPLE 6: Embodiment of FIG. 3B

There is described below the structure and operation of a flow through device with single displacement and horizontal visualization of the test results.

The flow through device is the same as that described above in Example 5, with similar numerals designating similar parts, with minor modifications as follows: There is no reference zone in the casing 108 of the device of Fig. 3B. Results are read only from the results window 114 according to the distance that the analyte analog:reporter conjugate covered. The analyte analog:reporter-conjugate binding element 110 as well as the results window 114 are elongated. A scale 112 runs parallel to the immobilized analyte analog:reporter-conjugate binding element 110, both co-calibrated to correspond to the analyte concentration in the liquid sample being analyzed. The presence and concentration of analyte in the liquid is determined by the area covered by the analyte analog:reporter-conjugate through the analyte: analog reporter-conjugate binding element that is seen in the results window 114. EXAMPLE 7: Embodiment of FIG. 4A

There is described below the structure and operation of a flow through device with double displacement and vertical visualization of the test results.

FIG. 4A is a sectional side view illustration of a flow through device for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample using double displacement. The device comprises a hollow, solid casing 80b containing defined layers or zones made of reagent-containing porous materials, arranged so that fluid that is applied to the top of the device flows vertically through the various layers of the device, from one layer to another, until the fluid contacts an absorbent material at the bottom of the device.

A sample application pad 82b for applying the sample to the device is located adjacent to an opening or window 84b in the casing 80b at the top of the device and leads to a MIP-conjugate zone comprising an analyte-specific MIP 122 having analyte-specific binding sites saturated with a releasable first binding agentanalyte conjugate in a dry state. The affinity of the analyte to the binding sites of the analyte-specific MIP 122 is greater than the affinity of the first binding agentanalyte conjugate to the binding sites of the analyte-specific MIP 122. The target analyte found in the liquid sample migrates from the sample application pad 82 through the MIP-conjugate zone where it binds to the analyte-specific cavities of the MIP 122 and thereby displaces the first binding agentanalyte conjugate which occupy these cavities in an amount directly proportional to the concentration of the specific analyte, causing the displaced first binding agentanalyte conjugate to migrate further down through the device.

The next layer is a porous carrier or support 88b, followed by a layer of a reporter-conjugate binding zone comprising a first binding area of reporter-conjugate binding element 124 having binding sites saturated with a detectable, releasable second binding agentreporter conjugate in a dry state. The affinity of the first binding agentanalyte conjugate to the binding sites of the reporter-conjugate binding element 124 is greater than the affinity of the second binding agentreporter conjugate to the binding sites of the reporter-conjugate binding element 124. The first binding area of reporter-conjugate binding element 124 in the reporter conjugate binding zone binds the first binding agentanaltye conjugate and displaces the second binding agentreporter conjugate in an amount directly proportional to the concentration of the specific analyte in the sample, causing the displaced second binding agent:reporter conjugate to continue to migrate further down the device.

Next is a second binding area of reporter-conjugate binding element 126 fixated to a porous carrier. An opening in the casing 80b is located in front of reporter-conjugate binding element 126, comprising the results window 92b of the device. The second binding area of reporter-conjugate- binding element 126 binds the second binding agent:reporter conjugate displaced from the analyte- specific MIP 122 when a liquid sample containing the analyte migrates to reporter-conjugate binding element 126, and provides a detectable signal in the results window 92b that indicates the presence or concentration of the analyte in the sample.

The next zone is the reference zone comprising three discrete and non-overlapping indicator bands of second binding agent:reporter conjugate binding element, wherein 128a represents a low concentration reference band, 128b represents a medium concentration reference band and 128c represents a high concentration reference band. A clear reference window 96b is located in front of the reference bands. The user interprets the results by visually comparing the intensity of the signal in the results window 92b to the intensity of the bands in the reference window 96b.

Underneath the reference zone is a control zone comprising a porous carrier 132 impregnated with unbound second binding agent:reporter conjugate. This conjugate is stationary in the dry state but becomes freely flowing when the porous carrier is wetted with the liquid of the sample. Further down is another reporter-conjugate binding element 134, visible at the control window 104b. The second binding agent: reporter conjugate which is released from the control pad 132 by the fluid from the sample reaches the reporter conjugate binding element 134 and is bound to the carrier, producing a visual signal at the control window 104b. At the distal end of the device is an absorbent material 106b that absorbs the surplus of the fluid and ensures continuous flow throughout the device.

EXAMPLE 8: Embodiment of FIG. 4B

There is described below the structure and operation of a flow through device with double displacement and horizontal visualization of the test results.

The flow through device is the same as that described above in Example 7, with similar numerals designating similar parts, except that modifications are indicated with the reference numeral and the letter "b" affixed. The modifications are as follows: There is no reference zone in the casing 108b of the device of Fig. 4B. Results are read only from the results window according to the distance that the second binding agentreporter conjugate covered. The immobilized second binding agent:reporter conjugate binding element 136 as well as the results window 114b are elongated. A scale 112b runs parallel to the immobilized second binding agent:reporter conjugate binding element 136 co- calibrated to correspond to the analyte concentration in the liquid sample being analyzed. The presence and concentration of analyte in the liquid is determined by the area covered by the second binding agentreporter conjugate through the second binding agent:reporter conjugate binding element 136 that is viewed in the results window 114.

EXAMPLE 9: Materials and Methods

The following materials and methods were used in the examples described below:

A. Porous, solid support: The porous, solid support used in the rapid diagnostic device of the invention is a membrane filter comprising a strongly adsorptive substance having a large surface area, such as PuraBind™ (Whatman, USA) which is 100% nitrocellulose with no post-manufacture treatments.

B. Preparation of methylmalonic acid-specific MIP: Preparation was in accordance with the methods set forth in the review by Yan and Row {Int. J. MoI. ScI 2006, 7, 155-178) as follows: The functional monomer, diethylaminoethylmethacrylate (Cat. No. 408980, Aldrich) is mixed with the target print molecule, in this case methylmalonic acid (M54058, Sigma, Israel), together with the cross-linking monomer ethylene glycol dimethacrylate (EGDMA), (Cat. No. 33568-1, Aldrich) in toluene (Cat. No. 244511, Aldrich). The mixture is degassed and purged with nitrogen for 5 min. Polymerization takes place following the addition of the initiator azobisisobutyronitrile (AIBN) (Cat. No. 441090-25G, Aldrich) and initiation of the reaction by UV, resulting after 48 hours in the formation of a rigid insoluble polymer. Methylmalonic acid-specific binding cavities are now present within the polymeric network, which are complementary in both shape and chemical functionality to the imprinted molecule. The bulk polymer is ground in a mechanical mortar and wet sieved in water through a 25 μm sieve. The print molecule is extracted by extensive washing of the particles with methanol-acetic acid (9/1, v/v), the polymer particles dried under vacuum and stored desiccated.

C. Preparation of MMA-biotin conjugate: MMA was conjugated to biotin by targeting the methyl group using Carbodiimide activation of the biotin carboxylic derivative.

D. Preparation of the MlP-Conjugate Zone: The binding cavities of the MMA-specific MIP are first saturated with the MMA-Conjugate (i.e., MMA-biotin conjugate) by incubation of the sieved MMA- specific MIP particles with a solution of the MMA-biotin conjugate for 24 hours at 37°C followed by washing to remove the excess conjugate. The washed MIP particles are then dried at 60 ⁰C in an oven and packaged inside filter paper bags, manufactured by Filtech Fabrics Ltd, India, similar to those used as tea bags. The bag containing the loaded MIP particles is attached to the nitrocellulose membrane at the MlP-coηjugate zone by pressure-sensitive adhesive-coated films (Cat. No. ARcare® 8570, Adhesives Research, U.S.).

E. Preparation of the Reporter-Conjugate: As the reporter-conjugate molecule, 4-hydroxyazobenzene- 2-carboxylic acid (HABA) (Cat. No. 54791, Fluka), conjugated to BSA (Cat. No. A3902, Sigma ) coated with colloidal gold particles, is used. The HABA is attached to BSA in accordance with the method described by Hofstetter et al., (Analytical Biochemistry (2000) 284, 354-366). HABA azo-dye binds to the biotin-binding site of avidin with an affinity constant of Kd = 10^"6 M. HABA is displaced from this binding site by biotin which has an affinity constant of Kd = 10^"15. Gold sol is available from BioAssay Works MD, USA. Loading of HABA-BSA conjugate with gold sol is performed using the coupling method described by Horrisberger and Clerc (Histochemistry, 1985, 82, 219-223A). The advantages of using gold particles are numerous. Its superior stability, sensitivity, precision and reproducibility of manufacture make gold suitable for use in rapid tests. Gold is essentially inert and forms almost perfectly spherical particles when properly manufactured. Proteins bind to the surfaces of these gold particles with enormous strength when correctly coupled, thus providing a high degree of long-term stability in both liquid and dried forms. Different sizes may be used for different applications. When accurately stabilized during manufacture, nonspecific interaction of gold conjugates can be reduced to zero. The respective loading conditions (pH, buffer, concentration of the biomolecules etc) of biomolecules with gold sols depend on the isoelectric point of the biomolecules, the MPA (minimal protecting amount) or/and the specific application of the conjugate.

F. For both biotin-binding element containing zones, NeutrAvidin™ Biotin-Binding Protein (Pierce, USA) is preferably employed. This protein is an excellent alternative to other biotin-binding proteins, such as avidin or streptavidin, when nonspecific binding must be minimized. Its immobilization was performed by direct blotting of the NeutrAvidin to the nitrocellulose membrane. 20 μg/ml NeutrAvidin in 0.5M phosphate buffer, pH 7.2 with 0.5MNaCl, was incubated O.N. at room temperature and washed once with PBS and air dried. The prepared membrane was stored desiccated at room temperature until the final construction of the device.

G. Preparation of MMA-reporter conjugate (MMA-NeutrAvidin-Gold) is accomplished by attaching NeutrAvidin to the MMA-biotin conjugate. In order to bind only one MMA-biotin conjugate to each NeutrAvidin molecule, the MMA-biotin conjugate is added stepwise to the NeutrAvidin solution. Due to the high binding affinity of biotin to NeutrAvidin, a relatively short incubation at room temperature is sufficient. The MMA-Neutr Avidin conjugate is then coated with gold sol. The coating is done as described earlier for the coating of the BSA with the gold sol. H. Preparation of MMA-reporter conjugate binding zone: The MMA-reporter conjugate binding zone is comprised of a zone on the nitrocellulose solid support having NeutrAvidin immobilized to it (as described above for the biotin-binding element zones). This zone is then saturated with a biotin dimer (EZ-Link® PEO-Biotin Dimer by PIERCE), the excess of the biotin dimer washed and the membrane dried. This zone is used to capture the MMA-NeutrAvidin-Gold conjugate via the biotin-free biotin binding sites of the NeutrAvidin.

I. Preparation of reference zone: The reference zone is preferably comprised of five identical parallel bands of MMA-reporter conjugate binding zones. Each band is applied accurately with a solution of the MMA-NeutrAvidin-Gold conjugate. Eventually, the five bands have 0.25, 0.5, 1, 2 and 4 μmoles of the conjugate bound to them, respectively. By comparing the color intensity of band in the results window with the reference bands, the user may determine the range of the amount of MMA in the tested sample.

EXAMPLE 10: Assay For MMA Using A Lateral Flow Device (double displacement)

There is described below the structure and operation of a particular displacement assay of the invention. Structure:

A device as shown in FIG. 2A is used to quantify MMA in liquid samples obtained from humans. The device comprises a housing containing windows that exposes areas of the solid support for viewing. The device includes a sample application area comprising a pad (LFl, Whatman, USA) to which a liquid sample of whole blood is introduced, bringing the sample fluid in contact with a solid support (test strip) of porous membrane (PuraBind™, Whatman, USA). The pad is designed to separate the plasma from the blood cells. The solid support comprises a defined MIP-conjugate zone comprising MMA-specific MIP saturated with MMA-biotin conjugate. Downstream of the MIP-conjugate zone is a reporter-conjugate binding zone comprising NeutrAvidin impregnated with HABA conjugated to BSA coated with gold sol. Further downstream is a results zone comprising immobilized NeutrAvidin with a scale to be used as an aid in quantifying the concentration of MMA in the sample, followed by a control pad comprising dried HABA-BSA-GOLD (reporter-conjugate) and a control zone comprising NeutrAvidin immobilized to the solid support. Operation

To initiate the assay, a drop of whole blood is applied to the sample application area. The plasma leaving the sample application area contacts the nitrocellulose membrane and flows along the device by capillary action to contact the MMA-specific MIP zone, which is impregnated with MMA- biotin conjugate. As the liquid front moves through the MlP-conjugate zone, the MMA present in the sample displaces molecules of MMA-biotin conjugate from the MIP, in an amount proportional to its concentration.

The displaced MMA-biotin conjugate molecules flow downstream in the fluid, reaching the reporter-conjugate binding zone that comprises HABA-BSA-GoId reporter-conjugate, immobilized to NeutrAvidin. The biotin of the MMA-biotin conjugate displaces the reporter- conjugate from the NeutrAvidin, in an amount proportional to the amount of the displaced MMA-biotin conjugate, due to the higher affinity of biotin.

The displaced reporter-conjugate migrates further downstream until it comes in contact with the NeutrAvidin binding element at the results zone. Parallel to the NeutrAvidin binding element is a reference scale, titrated to allow interpretation of the amount of MMA in the sample by determining the distance traveled by the reporter-conjugate along the binding element at the results window. A visual signal is evident in the results zone allowing the user to determine the amount of MMA in the blood sample. The results are determined about 15 minutes after application of the sample, which is the time required to ensure the proper functioning of all components of the device.

After passing the results zone, the fluid sample continues to move laterally across the control pad, bringing the reporter-conjugate namely HABA-BSA-GoId impregnated in the control pad into free flowing condition along the device until the NeutrAvidin band located in the control zone, where it is captured. A visual line forms across the entire control zone indicating that the assay has functioned properly. The excess liquid and reagents will continue to move laterally across the device and collect in the absorbent pad.

EXAMPLE 11 : Assay For MMA Using A Diagnostic Flow Through Device (Double Displacement) There is described below the structure and operation of a particular displacement assay of the invention (column-type assembly). Structure

A device as shown in FIG. 4B is used to quantify MMA in liquid samples obtained from humans. The device comprises a housing containing windows that exposes areas of the solid support for viewing. The device includes a sample application area comprising a pad designed to separate the plasma from the blood cells (LFl, Whatman, USA) to which a liquid sample of whole blood is introduced, bringing the fluid sample in contact with a zone of porous solid support comprising unmodified beads (Sigmacell^® Cellulose, Type 50, Cat. No S5504 Sigma).

Further downstream is a MlP-conjugate zone comprising packed MMA-specific MIP particles saturated with MMA-biotin conjugate located in physical contact underneath the solid support zone. Further downstream, in physical contact with the MIP-conjugate zone is a reporter-conjugate binding zone comprising packed cellulose biotin-immobilized sepharose beads (Cat. No VIT-H-4S, Affiland S.A. Belgium) coated with NeutrAvidin saturated with HABA-BSA-GOLD. Further downstream, in physical contact with the cellulose beads, is a results zone comprising packed biotin-immobilized sepharose beads coated with NeutrAvidin, separated by a zone of unmodified beads. The chromatographic flow of the molecules through the beads helps to form a distinct and concentrated zone of the analyte in the fluid. Parallel to the results zone is a scale, visible in a viewing window, to be used as an aid in quantifying the concentration of the MMA in the sample, followed by a control pad comprising dried HABA-BSA-GoId (reporter-conjugate) and a control zone comprising biotin- immobilized sepharose beads coated with NeutrAvidin. Sample is introduced at the sample application area and following analysis of a sample in the device, a visual signal is evident in the results zone and in the control zones, allowing the user to determine the amount of MMA in the blood sample, as well as validating the proper performance of the device. Operation:

To initiate the assay, a drop of whole blood is applied to the sample application area at the top of the device. The plasma leaving the sample application area flows along the device by capillary action to contact the MMA-specific MIP zone, which is impregnated with MMA-biotin conjugate. As the liquid front moves through the MIP-conjugate zone, the MMA present in the sample displaces molecules of MMA-biotin conjugate from the MIP, in an amount proportional to its concentration.

The displaced MMA-biotin conjugate molecules flow down the device in the fluid, reaching the reporter-conjugate binding zone. The biotin of the MMA-biotin conjugate displaces the reporter conjugate from the NeutrAvidin, in an amount proportional to the amount of the displaced MMA-biotin conjugate due to the higher affinity of biotin.

The displaced reporter conjugate migfates down the device until it comes in contact with the NeutrAvidin coated cellulose beads at the results zone. Parallel to the NeutrAvidin containing binding element is a reference scale, titrated to allow interpretation of the amount of the MMA in the sample by determining the distance traveled by the reporter-conjugate along the binding element at the results window. The results are determined about 15 minutes after the sample application, which is the time required to ensure the proper functioning of all components of the device.

After passing the results zone, the fluid sample continues to move down to the control pad bringing the un-bound reporter-conjugate, namely, HABA-BSA-GOLD impregnated in the control pad into free flowing condition down the device until the NeutrAvidin coated beads located at the control zone. A visual line forms across the entire control zone indicating that the assay has functioned properly. The excess liquid and reagents will continue to move down the device and collect in the absorbent pad.

EXAMPLE 12: Fluorescent Displacement Assay Kit for the Determination of Atrazine Structure:

This device comprises a portable detection unit. For the sake of convenience, this description will refer to the general structure of lateral flow device described in Examples 1-4, it being understood that the device may take the form of a flow-through device as described in Example 5-8, with minor modifications.

The sample application pad is comprised of CF6 absorbent material (Whatman, USA) that allows large volume absorbency with fast wicking of the device. The MIP-conjugate zone (detection zone) comprises an atrazine-specific MIP, saturated with atrazine-biotin conjugate. The reporter- conjugate is HABA-BSA-coated with fluorescent dye molecules (HABA-B S A-FLUROFOR). The reporter molecule of the reporter-conjugate is the fluorescent dye Alexa Fluor 488 (Invitrogen, USA) and determination of the results is done by means of a handheld portable fluorescent assay reader (ESE; Stockach, Germany). The device is built with a casing designed to fit into the fluorescent reader, in such a way that the area corresponding to the results zone in the visual devices is aligned with the fluorescent detection unit of the reader. Operation:

The device for detecting atrazine is used to monitor the presence and concentration of the toxic pesticide atrazine in water. Prior to application of the water sample, the device is fitted into the reader which activates the instrument and brings it to a stand-by position (only proper fitting activates the instrument or else an error message appears). Up to 0.5 ml water suspected of containing atrazine is applied to the sample application pad. The sample application triggers the instrument to perform a count-down that results in the measurement of the fluorescence intensity about 15 minutes after the sample application, which is the time required to ensure the proper function of all the components of the assay.

The atrazine in the sample displaces the atrazine-biotin from the atrazine-specific MIP and the displaced atrazine-biotin in turn displaces the HABA-BSA-fluor dye from the NeutrAvidin in the reporter-conjugate binding zone. The reporter-conjugate migrates downstream until the result zone, where it is captured by the NeutrAvidin. The amount of atrazine in the sample is determined by the fluorescent reader by comparing the signal obtained from the sample to that of an internal calibration curve. The results are displayed on the LCD of the instrument in μg/liter (with a limit of detection of 0.01 μg/liter). It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples, which are intended to illustrate but not to limit the invention.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

Claims

We claim:

1. A diagnostic device for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample, said device comprising a solid support capable of conveying a liquid sample therethrough, said sample being movable along or through the solid support in the path of liquid flow by capillary action, said support comprising: a. a defined sample application area for applying the sample to the device and bringing it in contact with the solid support; b. a defined MIP-conjugate zone downstream of the sample application area comprising an analyte- specific MIP fixed to said solid support on the flow path of the sample, said MIP having analyte- specific binding sites saturated with a releasable analyte analog:reporter conjugate in a dry state, wherein the affinity of the analyte to the binding sites of the analyte-specific MIP is greater than the affinity of the analyte analog:reporter conjugate to the binding sites of the analyte-specific MIP, said MIP, when contacted with a liquid sample containing the analyte, capable of binding said analyte and displacing the analyte analog:reporter conjugate in an amount directly proportional to the concentration of the specific analyte, causing the displaced analyte analog:reporter conjugate to flow downstream in the path of liquid flow; c. a defined results zone comprising an analyte analog:reporter conjugate binding element immobilized to said solid support on the flow path of the sample downstream of the MIP-conjugate zone, said reporter conjugate binding element capable of binding the analyte analog:reporter conjugate displaced from the MIP-conjugate zone when a liquid sample containing the analyte flows in the flow path zone for providing a detectable signal that indicates the presence or concentration of said analyte in a sample; d. optionally, a reference zone for establishing a reference point in deteπnining the presence or semi- quantification of an analyte in the tested sample, wherein said reference zone is not capable of capturing by specific binding any compound in said sample; and e. optionally, a positive control zone comprising means for generating a positive control confirming the proper flow and binding of the analyte analog:reporter conjugate to said results zone to thereby determine that a test is working; and f. optionally an absorbent zone comprising a pad of absorbent material in fluid communication with the solid support when said pad and solid support are wet, said pad having sufficient porosity and capacity to absorb excess liquid.

2. A diagnostic device for directly detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample, said device comprising a solid support capable of conveying a liquid sample therethrough, said sample being movable along or through the solid support in the path of liquid flow by capillary action, said support comprising: a. a defined sample application area for applying the sample to the device and bringing it in contact with the solid support; b. a defined MIP-conjugate zone downstream of the sample application area comprising an analyte- specific MIP fixed to said solid support on the flow path of the sample, said MIP having analyte- specific binding sites saturated with a releasable first binding agent:analyte conjugate in a dry state, wherein the affinity of the analyte to the binding sites of the analyte-specific MIP is greater than the affinity of the first binding agentanalyte conjugate to the binding sites of the analyte-specific MIP, said MIP when contacted with a liquid sample containing the analyte capable of binding said analyte and displacing said first binding agentanalyte conjugate in an amount directly proportional to the concentration of the specific analyte, causing the displaced first binding agentanalyte conjugate to flow downstream in the path of liquid flow; c) a defined reporter-conjugate binding zone comprising a first binding area of reporter-conjugate binding element immobilized to the solid support on the flow path of the sample downstream of the MIP-conjugate zone, said reporter-conjugate binding element having binding sites saturated with a detectable, releasable second binding agentreporter conjugate in a dry state, wherein the affinity of the first binding agentanalyte conjugate to the binding sites of the reporter-conjugate binding element is greater than the affinity of the second binding agentreporter conjugate to the binding sites of the reporter-conjugate binding element, said first binding area of reporter-conjugate binding element capable of binding the first binding agentanaltye conjugate and displacing second binding agentreporter conjugate in an amount directly proportional to the concentration of the specific analyte in the sample, causing said displaced second binding agentreporter conjugate to continue to flow downstream in the path of liquid flow; d) a defined results zone comprising a second binding area of reporter-conjugate binding element immobilized to the solid support on the flow path of the sample downstream of the reporter-conjugate binding zone, said second binding area of reporter-coηjugate-binding element capable of binding displaced second binding agentreporter conjugate for providing a detectable signal that indicates the presence or concentration of said analyte in a sample; e) optionally a reference zone for establishing a reference point in determining the presence or semi- quantification of an analyte in the tested sample, wherein said reference zone is not capable of capturing by specific binding any compound in said sample; f) optionally a positive control zone comprising means for generating a positive control confirming the proper flow and binding of the second binding agentreporter conjugate to the results zone to thereby determine that a test is working; and g) optionally an absorbent zone comprising a pad of absorbent material in fluid communication with the solid support when said pad and solid support are wet, said pad having sufficient porosity and capacity to absorb excess liquid.

3. A device comprising a solid support including a first component comprising an analyte-specific MIP saturated with an analyte analog-conjugate comprising the analyte conjugated to biotin, optionally through a spacer; a second component comprising a first biotin binding element saturated with reporter- conjugate comprising a biotin analog having lower affinity to the biotin binding element than biotin conjugated to a reporter, and a third component comprising a second biotin binding element.

4. The device of claim 1 or claim 2 or claim 3 wherein said device is a lateral flow device.

5. The device of claim 1 or claim 2 or claim 3 wherein said device is a flow through device.

6. The device of claim 1 or claim 2 contained in a housing including at least one window aligned with at least one of the results zone, reference zone, and control zone to allow observation of test results on the device.

7. The device of claim 1 or claim 2 wherein the intensity of the signal in the results zone is compared with the intensity of the signal in the reference zone, the device arranged and constructed to provide a positive result when the analyte is present at or above a threshold level, to indicate the concentration of the analyte in the sample.

8. The device of claim 1, wherein the reference zone comprises at least one discrete band of binding element impregnated with a known quantity of the analyte analog:reporter conjugate in the case where the presence or absence of a target analyte in the sample is to be determined, and comprises at least two discrete and non-overlapping bands of binding element impregnated each with increasing quantities of the analyte analog:reporter conjugate and exhibiting a range of color intensities proportional to the amount of the analyte analog:reporter conjugate bound thereto, in the case where the amount of a target analyte in a sample is to be determined, wherein comparison of the intensity of the signal in the results zone with the intensity of one or more bands in the reference zone enables determination of the presence or absence or semi-quantification of the concentration of the analyte in the sample.

9. The device of claim 2, wherein the reference zone comprises at least one discrete band of binding element impregnated with a known quantity of the second binding agent:reporter conjugate in the case where the presence or absence of a target analyte in the sample is to be determined, and comprises at least two discrete and non-overlapping bands of reporter-conjugate binding element impregnated each with increasing quantities of the second binding agentreporter conjugate and exhibiting a range of color intensities proportional to the amount of the second binding agent: reporter conjugate bound thereto, in the case where the amount of a target analyte in a sample is to be determined, wherein comparison of the intensity of the signal in the results zone with the intensity of one or more bands in the reference zone enables determination of the presence or absence or semi-quantification of the concentration of the analyte in the sample.

10. The device of claim lor claim 2 wherein the results zone includes a scale parallel to the immobilized reporter-conjugate binding element calibrated to correspond to the analyte concentration in the liquid sample being analyzed and wherein the presence and concentration of analyte in the liquid is determined by the area covered by the reporter-conjugate along and through the reporter-conjugate binding element in the results zone.

11. The device of claim 1 or claim 2 or claim 3 which comprises more than one solid support, whereby more than one analyte can be detected in a sample.

12. The device of claim 1 or claim 2, wherein said liquid sample is either liquid in its natural state or pre-treated or extracted to be in a liquid state.

13. The device of claim 1 or claim 2, wherein said sample is selected from the group consisting of biological fluids, environment samples, dyes, food stuffs, drugs, toxins, industrial samples and byproducts of industrial production procedures.

14. The device of claim 13, wherein said biological fluid is selected from the group consisting of body fluids, liquid obtained from breath, tissue homogenates, and process fluids.

15. The device of claim 13 wherein said environmental sample is selected from the group consisting of water, oil, liquid waste, liquid extracted from solid waste, soil, plants, and gas dissolved in solution.

16. The device of claim 1 or claim 2 wherein said analyte-specific MIP is fixed to said solid support.

17. The device of claim 2 wherein said releasable first binding agent is selected from the group consisting of biotin, a biotin analog, a biotin derivative, an antigen, Protein A and Protein G, cellulose binding protein, hormones, toxins, lipids, fatty acids, nucleic acids, glycoconjugates, lectins, substrates and ligands.

18. The device of claim 2 wherein said releasable second binding agent is selected from the group consisting of a biotin analog, a biotin derivative, HABA, an antigen, Protein A and Protein G, cellulose binding protein, liposomes, hormones, toxins, lipids, fatty acids, complementary nucleic acids, glycoconjugates, lectins, substrates and ligands or their analogs, provided that said second binding agent has lower affinity to the reporter-conjugate binding element than said first binding agent.

19. The device of claim 1 or claim 2 wherein the detectable reporter is selected from the group consisting of HABA, dyes, fluorescers, fluorescent dyes, radiolabels, magnetic particles, metallic particles, colored particles, fluorescent particles, metal sols, enzyme substrates, enzymes, chemiluminescers, photosensitizers and suspendable particles.

20. The device of claim 2, wherein the first binding area of reporter-conjugate binding element and the second binding area of reporter-conjugate binding element are identical or different and each are selected from the group consisting of avidin, streptavidin, NeutrAvidin, compounds having high specific affinity to biotin, membranes, receptors, immunoglobulins, cellulose, enzymes, lectins, glycoconjugates, complementary nucleic acids and hydrophobic sites having high affinity to their respective binding partners.

21. The device of claim 1 or claim 2, wherein said solid support is selected from the group consisting of a porous material, a porous membrane, a granular material, and an absorbent material.

22. The device of claim 1 or claim 2 wherein said target analyte is any molecule of interest.

23. The device of claim 1 or claim 2, wherein said analyte is selected from the group consisting of a protein, a hormone, an enzyme, a biomarker, metabolites of biomarkers, metabolites of drugs, a natural or synthetic toxin, a steroid, a drug, a drug metabolite, a drug-protein conjugate, a drug metabolite- protein conjugate, a vitamin, a drug of abuse, a chemical or biological warfare agent, a vitamin, antibodies to a drug, antibodies to infectious agents, an environmental pollutant, a lipoprotein, a polysaccharide, an immunoglobulin, a lymphokine, a cytokine, a soluble cancer antigen, a hapten, an oligonucleotide, an oligonucleotide that binds specifically with a protein, a_growth factor, a neurotransmitter, a molecule indicating the safety or quality of a foodstuff, a process chemical, a byproduct of a production process, a pesticide, an insecticide, a herbicide, a fertilizer, a surfactant, an adhesive, and an agent used in the manufacture of food, industrial agents or chemical products.

24. The device of claim 1 or claim 2, wherein said analyte is selected from the group consisting of MMA, homocysteine, troponin sub-units I or T, CKMB, LDH and GOT/SGOT, citrozine, Acetaminophen, Carbamazepine, Chloramphenicol, Digitoxin, Digoxin, Gentamicin, Kanamycin, Lidocaine, Lithium, Methotrexate, Phenobarbital, Propranolol, Quinidine, Theophylline, BNP, FSH, cumadine, vitamin K, aflatoxins, ricin, 2,4-Dichlorophenoxyacetic acid (2-4,D), atrazine, Alachlor, cyanazine, metolachlor, simazine, epinephrine, amphetamine, TSH, free T3 T4, PSA, steroids, estradiol, progesterone, testosterone, estrogen, Arsenic, Cyanide, and Strychnine.

25. The device of claim 1 or claim 2, wherein said molecular imprint polymer comprises a polymer polymerized from monomers cross-linked with cross-linker in the presence of the target analyte and a porogen, said polymer having a capacity for selectively binding the target analyte.

26. The device of claim 1 or claim 2 wherein said molecular imprinted polymer comprises a polymer polymerized from diethylaminoethyl methacrylate monomer cross-linked with ethylene glycol dimethacrylate (EGDMA), in the presence of methylmalonic acid (MMA) and toluene as a porogen, said polymer having a capacity for selectively binding MMA.

27. A method for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample by an assay comprising the steps of:

(a) applying a sample suspected of containing the analyte to the sample application area of the device of claim 1; and

(b) allowing the sample to flow along or through the solid support and contact the MlP-conjugate zone so that, if analyte is present in the sample, analyte binds to the binding sites of the MIP displacing analyte analogrreporter conjugate which flows to the results zone where it is captured by the analyte analog:reporter conjugate binding element, producing a detectable signal that indicates the presence or amount of the analyte in the sample.

28. A method for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample by an assay, said method comprising the steps of:

(a) applying a sample suspected of containing the analyte to the sample application area of the device of claim 2; and

(b) allowing the sample to flow along or through the solid support and contact the MIP-conjugate zone so that, if analyte is present in the sample, analyte binds to the binding sites of the MIP displacing first binding agentanalyte conjugate which flows to said reporter-conjugate binding zone, said first binding area of reporter-conjugate binding element binding said displaced first binding agentanalyte conjugate and thereby displacing second binding agentreporter conjugate which continues along the path of liquid flow to the results zone and is captured by the second binding area of reporter-conjugate binding element, producing a detectable signal that indicates the presence or amount of the analyte in the sample, wherein the amount of second binding agentreporter conjugate captured is proportional to the concentration of said target analyte in the sample.

29. A method for detecting and determining the presence, absence or concentration of a target analyte present in a liquid sample by an assay, said method comprising the steps of:

(a) fixing MIP specific to a target analyte to a solid support;

(b) saturating the binding sites on said analyte-specific MIP with a releasable first binding agentanalyte conjugate to form an MIP-conjugate zone;

(c) immobilizing a first reporter-conjugate binding element to said solid support downstream of said MIP-conjugate zone;

(d) saturating the binding sites of said reporter-conjugate binding element with a detectable, releasable second binding agentreporter conjugate to form an immobilized reporter-conjugate binding zone;

(e) immobilizing a second reporter-conjugate binding element to said solid support downstream of said reporter-conjugate binding zone;

(f) introducing a sample of liquid to be analyzed for the presence of the target analyte onto a sample application area that contacts the solid support to generate a flow of the liquid sample along or through the solid support; (g) allowing an analyte, if present in the sample, to displace the first binding agent:analyte conjugate from the analyte-specific binding sites on the MIP in an amount directly proportional to the amount of analyte present in the sample;

(h) allowing said first binding agent:analyte conjugate to displace the second binding agent:reporter conjugate from the binding sites of the reporter-conjugate binding element in an amount directly proportional to the amount of analyte present in the sample; wherein the affinity of the first binding agentanalyte conjugate to the analyte-specific MIP is lower than the affinity of the analyte to the analyte-specific MIP, and wherein the affinity of the second binding agentreporter conjugate to the reporter-conjugate binding element is lower than the affinity of the first binding agentanalyte conjugate to the reporter-conjugate binding element;

(i) allowing said displaced second binding agent:reporter conjugate to flow freely until it is captured by said second reporter-conjugate binding element; and

(j) detecting the signal displayed by said second reporter-conjugate binding element wherein the concentration of displaced second binding agent:reporter conjugate corresponds to the concentration of the analyte in the sample.

30. The method of claim 27 or claim 28 further including the step of comparing the intensity of the signal in the results zone with the intensity of the signal in a reference zone to determine the concentration of the analyte in the sample.

31. The method of claim 27 or claim 28 wherein the amount of target analyte is determined by means of auxiliary instrumentation or is determined visually as a function of the distribution of said analyte in the results zone without the need for instrumentation.

32. The method of claim 27 or claim 28 wherein the results zone includes a scale calibrated to correspond to the analyte concentration in the liquid being analyzed and wherein the method further comprises the step of determining the concentration of analyte in the sample by determining the area covered by the reporter-conjugate along or through the binding element in the results zone.

33. A method of detecting MMA present in a liquid sample, said method comprising the steps of: a) contacting the sample with the device of claim 1 or claim 2; and b) detecting, in the results zone, the amount of reporter conjugate bound to the reporter-conjugate binding element, wherein the amount of the reporter-conjugate is indicative of the presence or amount of MMA in the sample; c) diagnosing a vitamin B12 deficiency based upon the amount of MMA detected in the sample; and d) optionally, diagnosing methylmalonic aciduria disease based upon the amount of MMA detected in the sample.

34. The method of claim 27 or claim 28 wherein the sample is a solid sample and the method further comprises the preliminary step, before step (a), of applying one or more reagents or diluents to the solid sample to extract or process the sample to elute the analyte and bring it into contact with the device.

35. A kit comprising a. the device of claim 1 or claim 2 or claim 3; b. optionally, one or more reagents or compositions for extracting or processing the sample to elute the analyte; c. optionally, one or more diluents; and d. optionally, instructions for practicing a method of detecting and determining the presence, absence or concentration of a target analyte in a liquid sample.