CN101945990A - Small molecules and protein analysis devices based on molecular imprinted polymers - Google Patents

Small molecules and protein analysis devices based on molecular imprinted polymers Download PDF

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Publication number
CN101945990A
CN101945990A CN2008801268886A CN200880126888A CN101945990A CN 101945990 A CN101945990 A CN 101945990A CN 2008801268886 A CN2008801268886 A CN 2008801268886A CN 200880126888 A CN200880126888 A CN 200880126888A CN 101945990 A CN101945990 A CN 101945990A
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analyte
conjugates
reporter
wedding agent
sample
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拉斐尔·李维
伊多·马葛利特
雅登·德鲁米
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INFIGO DIAGNOSTICS Ltd
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INFIGO DIAGNOSTICS Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2600/00—Assays involving molecular imprinted polymers/polymers created around a molecular template

Abstract

Devices, methods and kits for rapid and simple determination of target molecules, including small molecules, polypeptides, proteins, cells and infectious disease agents in liquid samples that are capable of real-time measurement of these entities in fluid samples that are highly selective, highly sensitive, simple to operate, low cost, and portable. The devices, methods and kits also provide, in at least some embodiments, the use of MIPs in a flow through or lateral flow device.

Description

Small molecules and protein analysis device based on molecularly imprinted polymer
Invention field
The present invention relates to diagnostic field, and relate more particularly to based on using molecularly imprinted polymer to measure circulation (flow through) device or transverse flow (lateral flow) device of analyte level in the liquid sample, with and the method and the test kit that use.
Background of invention
Being used for the level of the analyte of the liquid sample analyte that for example protein is relevant is carried out effective and accurate detection and quantitative methods and device, is to make us interested especially for the use in application miscellaneous.
At present, protein molecule and contain proteinic organism for example bacterium and virus detection and measurement comprise the immunoassay that utilize target-specific antibody.Nowadays use complicated instrument and based on the device of antibody carry out interested various analytes and protein associated molecule in the liquid sample fast, in real time and simple analysis.
Antibody is used to that some fields are for example treated, in the immunoaffinity purification, and especially in the immunoassay.Cause polyclonal antibody by animal is carried out immunity with corresponding antigen, perhaps produce antibody by using fused cell to produce monoclonal antibody with the clone that allows to obtain.
Trial for the optional method of Development and Production antibody or antibody sample compound comprises the recombinant technology that is applied to bacterium or plant.Can produce antibody at most compounds, and the various application of described antibody in being used to from fundamental research to the clinical analysis scope (Kohler and Milstein, 1975, Nature 256,495497; Oellerich .1984, J.Clin.Chem.Clin.Biochem.22,895904 Gosling, 1990.Clin.Chem.36,1,408 1427; Kurstak, 1986, in Enzyme Immunodiagnosis (enzyme immunodiagnosis), Kurstak, editor, 5-11 page or leaf, Academic Press, London).
The immunological technique of the reporter of applying marking has been widely used in biology and medical research and the clinical diagnosis.Although known reporter and step have a lot (Oellerich, 1984, Clin.Chem.Clin.Biochem.22,895-904; Gosling, 1990, Clin.Chem.36,1408-1427), but all present known immune diagnostic techniques all utilize the significant avidity and the specificity of antibody.
Yet antibody is unsettled biomolecules, and it needs careful operation and storage.Their production is time-consuming process (Kurstak, 1986, in Enzyme Immunodiagnosis (enzyme immunodiagnosis), Kurstak, editor, 5-11 page or leaf, Academic Press, London), the step that comprises effort for example haptens to the coupling of carrier proteins, the immunization of animal, the bloodletting of animal and the separation and the purifying of immunoglobulin (Ig).
Abiotic antibody analog (artificial antibody) is the useful surrogate of natural antibody.The use of this system can reduce the needs to animal-origin.In addition, can be at being considered to usually to be difficult to or can not for example immunosuppressor, small peptide or other small molecules obtain antibody analog at its molecule that produces antibody.This abiotic system for preparing by chemical process also will be more stable than natural antibody, and repeatability is criticized in pursuing that its permission repeated use, high temperature move down, sterilize easily and increases.
Use for example synthetic polymkeric substance and the technology that is similar to immunoassay of non-antibody is known of high selectivity reagent stable and preparation easily therein.This method utilize molecularly imprinted polymer (molecular imprinted polymer, MIP).Molecularly imprinted polymer is by function monomer being centered on the polymkeric substance that template or " marking (print) " molecule aggregation prepare, then by extracting or additive method is removed this template or " marking " molecule from polymkeric substance, so that polymkeric substance optionally absorbs template or marking molecule being exposed to the imprint dividing period of the day from 11 p.m. to 1 a.m again.People's such as Mosbach United States Patent (USP) the 5th, 821,311,5,872,198 and 5,959, the symmetric pearl of describing some MIP polymkeric substance, polymerization procedure for No. 050 and producing as chromatographic media by the suspension polymerization of function monomer.
The method of molecular imprinting has attracted many attentions (people 2006 such as Alexander, J.Mol.Recognit. in recent years; 19:106-180).Molecular imprinting stems from notion (people such as Mosbach K., Bio/Technology, 1996,14, the 163-170 that acts on the recognition site customized of generation in the polymkeric substance by template polymerization; People such as Ansell R.J., Curr.Opin.Biotechnol., 1996,7,89-94; Wulff G.Angew.Chem.Int.Ed.Engl, 1995,34,1812-32; People such as Vidyasankar S., Curr.Opin.Biotechnol., 1995,6,218-224; With Shea K.J, Trends In Polymer Science, 1994,2,166-173).Molecularly imprinted polymer has shown surprising evident characteristics, its be used to various fields for example medicine separate (people such as Fischer L., J.Am.Chem.Soc, 1991,113,9358-9360; People such as Kempe M, J.Chromatogr., 1994,664,276-279; People such as Nilsson K., J.Chromatogr., 1994,680,57-61), receptor mimics (people such as Ramstrom O., Tetrahedron:Asymmetry, 1994,5,649-656; People such as Ramstrom O., J.Mol.Recogn., 1996,9,691-696; People such as Andersson L.I., Proc.Natl.Acad.Sci., 1995,92,4788-4792; Andersson L.I., Anal.Chem., 1996,68,111-117), bionical inductor block (people such as Kriz D., Anal.Chem., 1995,67,2142-2144), antibody analog (people such as Vlatakis G., Nature, 1993,361,645-647), auxiliary synthetic (people such as Bystrom S.E., J.Am.Chem.Soc., 1993 of template, 115,2081-2083) and catalysis (people such as Muller R., Makromol.Chem., 1993,14,637-641; BeachJ.V. wait the people, J.Am.Chem.Soc., 1994, volume 116,379-380).
The whole bag of tricks that is used for protein and other macromole are carried out the marking has been described.For example, the ionic molecule image (United States Patent (USP) the 5th, 756, No. 717) that has produced polypeptide by wanting the complete polypeptide chain of bonded to mix on substrate substance and Molecular Graphs picture.The molecular imprinting (United States Patent (USP) the 5th, 814, No. 223) by in the presence of every kind of intact proteins, acrylamide polymerization being prepared cytochrome c, oxyphorase and myohaemoglobin respectively.The marking of h-Mb in the protein mixture that contains the whale myohaemoglobin optionally in conjunction with h-Mb (United States Patent (USP) the 5th, 814, No. 223).
In No. the 6582971st, the United States Patent (USP) method of by monomeric interfacial polymerization the mcroorganism molecule being carried out the marking under the situation that has marking molecule and host's polymkeric substance at the interface between the organic solvent and the aqueous solution has been described.Described the marking composition of the substrate substance that comprises the marking that defines template molecule in No. the 6979573rd, the United States Patent (USP), wherein said template molecule is usually corresponding to an interested macromolecular part.
Show, MIP can be produced and the target tobacco mosaic virus (TMV) (people such as Linden, 2006, Biomaterials 27,4165-4168).
The molecular imprinting method can comprise " the epi-position marking " (Rachkov and Minoura, 2000, J.Chromatogr A.; 889,111-118), wherein on protein surface, identify crucial epi-position and prepare MIP as target with the linear peptides of representing this epi-position.
Numerous analytical equipments and the method that is used to detect various analytes based on molecular imprinting summarized by Ye and Haupt (Anal.Bioanal.Chem.2004,378,1887-1897).Main challenge is to obtain tangible signal from polymkeric substance-analyte binding events.Proposed various means, yet wherein most of means comprise complicated Method and kit for.Some examples based on the inductor block of MIP are as follows:
People such as Yan (United States Patent (USP) the 5th, 587, No. 273) describe such inductor block, and it utilizes the molecular imprinting film and measures the electric capacity of this film or light characteristic or film carried out spectroscopic analysis after step of exposure.Huang (United States Patent (USP) the 6th, 680, No. 210) disclose be used for to macromole detect, analysis and quantitative device based on MIP.Then its analysis is detected by after combination, analyte molecule being dissociated from polymkeric substance.
People such as Williams (United States Patent (USP) 6,807,842) disclose the existence that is used for the check and analysis thing and the molecular recognition inductor block system of concentration, it comprises the resistance-type inductor block with semiconductive polymer film, and described semiconductive polymer film expands when being exposed to analyte.
People such as Kroeger (English Patent the 2nd, 337, No. 332) disclose the electrode that has with the surface of the synthetic polymer modification of the marking, the synthetic polymer of the wherein said marking discerns specifically, in conjunction with and be concentrated in the analyte of being close to nearly electrode surface place.Directly bonded analyte self or electrochemical activity derivative pre-incubated with electrode or that be added in the sample (competition/replacement mensuration) are carried out quantitatively at electrode surface by electrochemistry (for example by differential pulse or square wave voltammetry).
Penelle (United States Patent (USP) the 6th, 890, No. 486) discloses the inductor block based on Perchlorobenzene MIP with MIP technology and quartz crystal microbalance (QCM) associating.
People such as Green (United States Patent (USP) the 6th, 638, No. 498) disclose to utilize has that for example DCA and CDCA have the device of the MIP of specific binding capacity to specific bile acide and/or salt.Detect by replacing radiolabeled CDCA with the CDCA in the sample.In to above-mentioned modification, the fluorescent derivative of cholic acid is used as the mensuration material.
The open hydrocortisone fibre optics inductor block of people such as Lawrence (United States Patent (USP) the 6th, 833, No. 274), it uses the polymkeric substance of the hydrocortisone marking and the replacement of hydrocortisone-fluorescent chromophore conjugates.
People such as Schwartz (United States Patent (USP) the 6th, 967, No. 103) disclose explosion detector (explosive detector), and it utilizes a series of fiber optic cables that have been coated with the bifurcated of MIP.The single inductor block fibrous bundle that respectively carries gauged air-flow is used to fiber is exposed to target molecule.Detector excites the excitation light source of every fiber special use, and reads and handle the gained intensity of fluorescence simultaneously by the detector that contains strainer and photorectifier, and described intensity is indicative for the concentration of target molecule.
In some laboratories, show, with the use of the MIP of the replacement of analyte-mark conjugates associating be feasible (people such as Vlatakis G., Nature, 1993,361,645-647, people such as Levi, 1997, Anal.Chem.69.2017-2021; People such as Nathaniel T., J.Am.Chem.Soc.2005,127,5695-5700; People such as Nicholls C., Biosens.Bioelec., 2006,21,1171-1177).Analyte with the competition of analyte-mark conjugates also with MIP as inducing method (people such as Piletsky S.A., Analytical Letters, 1997,30,445-455; People such as Surugiu I., Analyst, 2000,125,13-16).
PCT application WO 07/002237 has described the method for analyzing liquid sample, and it comprises sample is contacted with MIP.
United States Patent (USP) the 6th, 890 is described quartz crystal microbalance inductor block and the use together of MIP technology No. 486.
The invention summary
The use of the unexposed MIP of background technology in circulation device or lateral flow device.In addition, the background technology method comprises heavy specimen preparation, and requires to use complicated assistant analysis device by the personnel through training.
Therefore, exist for use MIP diagnosis small molecules, macromole, cell and whole organism fast and the needs of extensively admitting of simple assembly and test kit, and to having of they will be highly favourable, it lacks at least some restrictions of prior art.
The target molecule that the present invention is provided in its various embodiments in the liquid sample comprises vectorial quick and simple device, method and the test kit of determining of small molecules, polypeptide, protein, cell and transmissible disease, described device, method and test kit can be measured these entities in the liquid sample in real time, and be high selectivity, extremely sensitive, simple to operate, low cost with portable.Described device, method and test kit also provide MIP use in circulation or lateral flow device at least some embodiments.
Described device, method and test kit are fit to be used by deconditioned personnel, and do not need uncommon and instrument complicated operation.When test kit of the present invention randomly needs supplementary instrument, it be preferably portable and hand-held, relatively cheap with use simple.
It is vectorial fast and use simple determinator, method and test kit to the invention provides the target polypeptide, protein, cell and the transmissible disease that are used for determining liquid sample, and it is designed to be used for by family, clinic, doctor's office, the hospital bed, factory or field.This determinator obtains bigger susceptibility and specificity is given a discount than traditional quick measurements determination, the minimizing that causes the stronger and/or more stable visual signalling that produces than conventional apparatus, easier result explanation and uncertain result to take place.In a small amount of time, this device can be used to detect the target analyte in various biologies, environment and the production piece and not need the complicated sample preparation process, and therefore be fit to by in addition field conditions in deconditioned personnel use.
The present invention combines the advantage of lateral flow device and circulation device (quick, relatively cheap and use simply) with brand-new two replacements/competitive methods with the advantage (specificity, controlled generation and highly stable are arranged) of diagnostic MIP.
Be contemplated that device of the present invention will provide " in real time " to measure at least some embodiments, be i.e. detection time relatively fast, be preferably about 15 minutes, more preferably for being less than 15 minutes.Alternatively, depend on used detectable reporter, can use that commercial that get for example reader, scanner wait explanation results with using simple supplementary unit.
Based on the combination of MIP to analyte, the existence that the molecularly imprinted polymer (MIP) of use target-specific comes test sample target test thing.
According to embodiments more of the present invention, provide the method for at least a analyte in the tracer liquid sample.Described method comprises: provide to comprise the molecularly imprinted polymer with analyte specific binding site that is fixed in solid support, and liquid sample is applied to the diagnostic device in the sample application zone of described device, wherein said liquid sample can along along or the flowpaths that passes solid support move; Liquid sample is applied to sample application zone and allow this sample along or pass solid support and flow, and contact with molecularly imprinted polymer; And detect combining of described analyte and described molecularly imprinted polymer.
According to embodiments more of the present invention, the diagnostic device of at least a analyte in the tracer liquid sample is provided, described device comprises: be fixed in the molecularly imprinted polymer with analyte specific binding site of solid support, wherein said liquid sample can move along the flowpaths along solid support; Be used for that liquid sample is applied to described device and with its sample application zone that contacts with molecularly imprinted polymer; And the bonded surveyed area that is used to detect described analyte and described molecularly imprinted polymer.
Some embodiments of the method according to this invention or device, described device comprises lateral flow device, circulation device or made up the device of transverse flow and flow-through element, wherein liquid sample from the sample application zone along or the solid support assembly that passes device flow along the flowpaths of device.
According to some embodiments, analyte is replaced the analyte analog that is bonded to molecularly imprinted polymer releasedly: reporter conjugates, wherein analyte analog: the replacement of reporter conjugates shows the existence of analyte in the sample.
According to some embodiments, releasable analyte analog: the reporter conjugates is incorporated in to molecularly imprinted polymer, and wherein said analyte equals analyte analog at least to the avidity of the binding site of molecularly imprinted polymer: the reporter conjugates is to the avidity of the analyte specific binding site of molecularly imprinted polymer.When with molecularly imprinted polymer when analyte in the liquid sample contacts, the combined and analyte analog of analyte: the reporter conjugates is replaced.This device randomly also comprises results area, this results area is included in the analyte analog that is fixed in solid support in the sample flow approach: reporter conjugates binding member, and also reporter-conjugates binding member can be in conjunction with the analyte analog of replacing: the reporter conjugates.Analyte analog: the concentration of analyte is randomly proportional in the replacement of reporter conjugates and the liquid sample, and make the bonded of analyte and molecularly imprinted polymer detect the analyte that comprises replacement: reporter conjugates and analyte analog: the bonded of reporter conjugates binding member detects.
According to some embodiments, analyte is replaced first wedding agent that is bonded to described molecularly imprinted polymer releasedly: the analyte conjugates, first wedding agent of Ti Huaning wherein: the analyte conjugates is replaced second wedding agent that is bonded to solid support releasedly: reporter conjugates, wherein second wedding agent: the replacement of reporter conjugates shows the existence of analyte in the sample.
According to some embodiments, releasable first wedding agent: the analyte conjugates is incorporated in to molecularly imprinted polymer, and wherein said analyte equals first wedding agent at least to the avidity of the binding site of molecularly imprinted polymer: the analyte conjugates is to the avidity of the analyte specific binding site of molecularly imprinted polymer.When with molecularly imprinted polymer when analyte in the liquid sample contacts, combined and first wedding agent of analyte: the analyte conjugates is replaced.This device randomly also comprises reporter-conjugates calmodulin binding domain CaM, described reporter-conjugates calmodulin binding domain CaM is included in the reporter-conjugates binding member that is fixed in solid support in the liquid sample flowpaths, and described reporter-conjugates binding member has for combination detectable, releasable second wedding agent thereon: the binding site of reporter conjugates.Randomly and preferably, first wedding agent: the analyte conjugates equals second wedding agent at least for the avidity of the analyte specific binding site of reporter conjugates binding member: the reporter conjugates is to reporter: the avidity of the binding site of conjugates binding member.First wedding agent: the analyte conjugates in conjunction with replacing second wedding agent: reporter conjugates, and second wedding agent: the concentration of analyte is randomly and preferably proportional in the replacement of reporter conjugates and the liquid sample.This device randomly also is included in second wedding agent that is fixed in solid support in the sample flow approach: reporter conjugates binding member, described reporter conjugates binding member can be in conjunction with second wedding agents of replacing: the reporter conjugates.Bonded detection for analyte and molecularly imprinted polymer randomly comprises for second wedding agent: reporter conjugates and analyte analog: the bonded of reporter conjugates binding member detects.
According to some embodiments, method of the present invention or device comprise analyte analog: the reporter conjugates, wherein said analyte and analyte analog: the analyte specific binding site of reporter conjugates competition molecularly imprinted polymer, wherein unconjugated analyte analog: the existing detected and show the existence of analyte in the sample of reporter conjugates.
According to some embodiments, method of the present invention or device comprise analyte analog: the reporter conjugates, wherein said analyte equals analyte analog at least to the avidity of the analyte specific binding site of molecularly imprinted polymer: the reporter conjugates is to the avidity of the analyte specific binding site of molecularly imprinted polymer.When with molecularly imprinted polymer when analyte in the liquid sample contacts, analyte and analyte analog: the binding site of reporter conjugates competition molecularly imprinted polymer.This device preferably also comprises results area, this results area comprises the analyte analog that is incorporated into solid support: reporter conjugates binding member, reporter conjugates binding member can be in conjunction with unconjugated analyte analog: the reporter conjugates also provides detectable signal, the concentration of analyte in the described detectable signal indicating liquid sample.
According to some embodiments, method of the present invention or device comprise first wedding agent: analyte conjugates, second wedding agent: the reporter conjugates and second wedding agent: acceptor conjugates binding member.The analyte and first wedding agent: the analyte specific binding site of analyte conjugates competition molecularly imprinted polymer makes described first wedding agent of at least a portion in the presence of analyte: the analyte conjugates is unconjugated.Unconjugated then first wedding agent: the analyte conjugates and second wedding agent: reporter conjugates competition is for second wedding agent: the combining of acceptor conjugates binding member.
According to some embodiments, method of the present invention or device comprise first wedding agent: the analyte conjugates, wherein said analyte equals first wedding agent at least to the avidity of the analyte specific binding site of molecularly imprinted polymer: the analyte conjugates is to the avidity of the analyte specific binding site of molecularly imprinted polymer, wherein when with described molecularly imprinted polymer when analyte in the liquid sample contacts, the analyte and first wedding agent: the analyte specific binding site of analyte conjugates analyte competition molecularly imprinted polymer.Unconjugated first wedding agent: the analyte conjugates flows in the liquid sample flow approach.This device randomly also comprises: second wedding agent: the reporter application region, and it comprises second wedding agent of drying regime: the reporter conjugates; The reporter conjugates calmodulin binding domain CaM in downstream, it is included in the reporter-conjugates binding member that is fixed in solid support in the liquid sample flowpaths.First wedding agent: the analyte conjugates equals second wedding agent at least to the avidity of reporter-conjugates binding member: the reporter conjugates is to the avidity of binding site.When with exsiccant second wedding agent: reporter conjugates and unconjugated first wedding agent: when the analyte conjugates contacts, second wedding agent: reporter conjugates and unconjugated first wedding agent: the competition of analyte conjugates combines with reporter-conjugates binding member, and unconjugated second wedding agent: the reporter conjugates is flow further downstream in the liquid sample flowpaths.This device randomly also comprises: comprise and solid support bonded second wedding agent: the results area of reporter conjugates binding member, can be in conjunction with unconjugated second wedding agent: the reporter conjugates also provides the reporter-conjugates binding member of detectable signal, the concentration of analyte in the wherein said detectable signal indicating liquid sample.
According to some embodiments, method of the present invention or device also comprise first wedding agent: analyte analog, wherein unconjugated first wedding agent: analyte analog by with analyte to the competition of the binding site of molecularly imprinted polymer and by at least a produce of described analyte from the replacement of molecularly imprinted polymer.This device randomly also comprises second wedding agent: reporter conjugates binding member, wherein unconjugated second wedding agent: the reporter conjugates by with first wedding agent: the competition of analyte conjugates and by first wedding agent: at least a generation in the replacement of analyte conjugates, wherein unconjugated second wedding agent: the existence of reporter conjugates shows the existence of analyte in the sample.
According to some embodiments, first wedding agent: analyte analog is provided in the sample application zone, perhaps is incorporated in to molecularly imprinted polymer.Reporter conjugates binding member randomly is fixed on the solid support in the liquid sample flow approach.Second wedding agent: the reporter conjugates is provided in reporter conjugates application region, perhaps is bonded to reporter conjugates binding member releasedly.This device preferably also comprises results area, it comprises second wedding agent that is bonded to solid support: reporter conjugates binding member, described reporter-conjugates binding member can be in conjunction with unconjugated second wedding agent: the reporter conjugates also provides detectable signal, the concentration of analyte in the described detectable signal indicating liquid sample.
According to some embodiments, at least a reporter conjugates or wedding agent conjugates comprise vitamin H, and binding member is selected from the group of being made up of avidin, NeutrAvidin and Extravidin.
According to some embodiments, reporter-conjugates-binding member flatly is fixed on the sample fluid flow approach on the solid support, and unconjugated reporter conjugates makes the binding site of reporter-conjugates binding member saturated.The result is advancing post by video picture randomly, its have with sample in the proportional length of amount of analyte.
According to some embodiments, this device also comprises at least one in following: positive control area; Reference zone; The absorption region in results area downstream on the liquid sample flowpaths, described absorption region comprises the absorbent material that can absorb liquid sample; And comprise with results area, reference zone and control zone at least one aligns with the shell (housing) of at least one window of allowing the test result on the observing device.
According to some embodiments, strength of signal in the results area and the strength of signal in the reference zone are compared, described device is arranged and is configured to provide positive findings at analyte when threshold level exists to be equal to or higher than, to show the concentration of analyte in the sample.
According to some embodiments, this device also comprises reference zone, it comprises at least one discrete bands of binding member, described binding member comprises the analyte analog of known quantity: the reporter conjugates, wherein the existence of sample target test thing or do not exist is to determine by strength of signal in the reference zone relatively and the strength of signal in the results area.
According to some embodiments, this device lacks reference zone, and results area also comprises and fixed reporter-parallel scale of conjugates binding member, described scale is calibrated with the analyte concentration in the analyzed liquid sample of correspondence, so as by by along with pass reporter in the results area-area that reporter-conjugates covered of conjugates binding member and determine the existence and the concentration of analyte in the liquid.
According to some embodiments, sample is selected from the group of being made up of the byproduct of biological liquid, environmental sample, food, toxin, production piece and industrial production step.
According to some embodiments, analyte is selected from by cell, organism, small molecules, protein, hormone, enzyme, biomarker, the meta-bolites of biomarker, the meta-bolites of medicine, medicine, drug metabolite medicine-protein-coupled thing, drug metabolite-protein-coupled thing, VITAMIN, Drug abuse, natural or synthetic toxin, chemistry or biological warfare agent, the antibody of medicine, the antibody of infectious agent, environmental pollutant, immunoglobulin (Ig), lymphokine, cytokine, the solubility cancer antigen, somatomedin, neurotransmitter, the molecule that shows food safety or quality, operating chemical product (process chemical), the byproduct of production process, sterilant, insecticide, weedicide, fertilizer, tensio-active agent, tackiness agent and be used to make food, the group that the agent of industrial agent or chemical products is formed.
According to some embodiments, analyte is selected from by MMA, homocysteine, troponin subunit I or T, CKMB, LDH and GOT/SGOT, citrozine, acetaminophen, Carbamzepine, paraxin, digitoxin, digoxin, gentamicin, kantlex, lignocaine, lithium, methotrexate, phenylethyl barbituric acid, Proprasylyte, Quinidine, theophylline, BNP, FSH, cumadine, vitamin K, aflatoxin, Ricin, 2, the 4-dichlorphenoxyacetic acid (2-4, D), atrazine, alachlor, bladex, metolachlor, simazine, suprarenin, Amphetamine, TSH, free T3 T4, PSA, steroid, estradiol, progesterone, testosterone, oestrogenic hormon, arsenic, prussiate, Strychnine, avian influenza virus, HIV virus, hepatitis virus, poliovirus, cytomegalovirus, dengue virus, Ebola virus, simplexvirus, Measles virus, EBV, rabies virus, respiratory syncytial virus, rotavirus, SARS virus, west Nile virus, yellow fever virus, campylobacter, chlamydiaceae, cholera, genus clostridium, diphtheria, intestinal bacteria, eisseria, Helicobacterium, hemophilus, Mycobacterium, Staphylococcus, Whooping cough, salmonella, Shigella, streptococcus, treponema, tetanus, Yersinia (Yershinia), staphyloentero-toxin, staphylococcal enterotoxin B, Ricin, Toxins, botulin, mycotoxin, tetanus toxin, Toxins,exo-, cholera, the trichothecene mycotoxin, transmissible disease vehicle there are specific antibody and the autoantibody relevant with autoimmune disorders, cancer cells, the cell of virus infection, virus, the group that bacterial cell and fungal cell are formed.
According to some embodiments, the group that releasable first wedding agent is selected from is conjugated protein by vitamin H, vitamin H analogue, biotin derivative, antigen, albumin A and Protein G, Mierocrystalline cellulose, hormone, toxin, lipid, lipid acid, nucleic acid, sugared conjugates, lectin, substrate and part are formed.
According to some embodiments, the group that releasable second wedding agent is selected from is conjugated protein by vitamin H, vitamin H analogue, biotin derivative, HABA, DTB, antigen, albumin A and Protein G, Mierocrystalline cellulose, liposome, hormone, toxin, lipid, lipid acid, nucleic acid, sugared conjugates, lectin, substrate and part or their analogue are formed.
According to some embodiments, but reporter is selected from the group of being made up of HABA, dyestuff, fluorescent agent, fluorescence dye, radio-labeling, magnetic-particle, metallic particles, semiconductor grain, quantum dot, coloured particle, fluorescent grain, metal-salt, enzyme substrates, enzyme, chemoluminescence agent, sensitizer and suspended particle (suspendable particle).
According to some embodiments, first wedding agent is identical or different with second wedding agent, and be selected from separately by the avidin, streptavidin, NeutrAvidin, the ExtrAvidin compound that have the high specific avidity of vitamin H, film, acceptor, immunoglobulin (Ig), Mierocrystalline cellulose, enzyme, lectin, the sugar conjugates, complementary nucleic acid and have the group that the hydrophobic site of the high-affinity of they corresponding binding partners is formed.
According to some embodiments, solid support is selected from the group of being made up of porous material, porous-film, granulate material and absorbent material.
According to some embodiments, molecularly imprinted polymer comprises the polymkeric substance that is selected from by the following group of forming: in the presence of target analyte or target analyte-conjugates and pore former (porogen) with linking agent with one or more are monomer crosslinked and the polymkeric substance that polymerization obtains, wherein said polymkeric substance has the ability of selective binding target analyte and its conjugates; The polymkeric substance that polymerization obtains in the presence of MMA, wherein said polymkeric substance has the ability of selective binding MMA; The synthetic peptide that contains amino acid LSCKCGKCNTDY or represent TSH the β subunit epi-position peptide in the presence of polymerization obtains in pore former polymkeric substance, described polymkeric substance has the ability of selective binding TSH.
According to some embodiments, the method of the TSH that exists in the tracer liquid sample is provided, described method comprises the steps: sample is contacted with device of the present invention, in results area, detect the amount of the reporter conjugates be bonded to reporter-conjugates binding member, the amount of wherein said reporter-conjugates for the existence of TSH in the sample or amount be have tell-tale; And diagnosis hypothyroidism or hyperthyroidism illness.
According to some embodiments, test kit is provided, it comprises device of the present invention; Randomly, be used to extract or processed sample with one or more reagent or the composition of elution analysis thing; Randomly, one or more thinners; And randomly, be used to put into practice and detect and the existence of definite liquid sample target test thing, do not exist or the specification sheets of the method for concentration.
According to some embodiments, the detection of analyte comprises uses the auxiliary detection apparatus, and wherein said detector means is selected from the group of being made up of fluorescence inductor block, Magnetic Induction device, chemoluminescence inductor block, densometer, image dissector and radioactivity inductor block.
According to some embodiments, sample is pretreated to modify the characteristic of sample.
According to some embodiments, pre-treatment is selected from by dilution, filters, distillation, concentrate, deactivation and the interpolation of reagent or the group that its combination is formed of interfering component.
According to some embodiments, this device comprises at least one the one or more porous pad that places sample application zone, results area, reference zone, control zone or described absorption region.Randomly, the pretreated flow velocity that passes device with promotion or control sample of this pad.
The present invention is aforementioned will to become obviously from the following description with other features and advantage, and the carrying out of described description is with reference to the figure and the claim of enclosing.
The accompanying drawing summary
This paper has only described to illustrative the present invention with reference to figure that encloses and following detailed description, should understand, shown detail file only are used for for example and illustrative discussion, and it is presented the description the most useful and that understand easily that is considered to embodiment of the present invention to provide.Do not attempt the understanding more basic than the present invention required show CONSTRUCTED SPECIFICATION of the present invention in more detail, the description of accompanying drawing makes those skilled in the art understand how some forms of the present invention can realize in practice.In the accompanying drawings:
Figure 1A has competition or single the replacement and the side-view of first embodiment of the lateral flow device of vertical imaging as described in example 1 above.
Figure 1B is the side-view with lateral flow device of competition or single replacement and horizontal imaging as described in example 2 above.
Fig. 2 A is the side-view with lateral flow device of competition/pair replacement and vertical imaging as described in example 3 above.
Fig. 2 B is the side-view with lateral flow device of competition/pair replacement and horizontal imaging as described in example 4 above.
Fig. 3 A is the side-view with circulation device of competition or single replacement and vertical imaging as described in example 5 above.
Fig. 3 B is the side-view with circulation device of competition or single replacement and horizontal imaging as described in example 6 above.
Fig. 4 A is the side-view with circulation device of competition/pair replacement and vertical imaging as described in example 7 above.
Fig. 4 B is the side-view with circulation device of competition/pair replacement and horizontal imaging as described in example 8 above.And
Fig. 5 is the side-view of the transverse flow described in the embodiment 9.
The description of embodiment preferred
The invention provides and be used for determining the existence of at least a analyte of liquid sample or do not exist or diagnostic method, device and the test kit of concentration.
With reference to figure, embodiment and the description of enclosing, diagnostic method of the present invention, device and test kit can be better understood.Expection the present invention be not limited to it below description or accompanying drawing in listed or by the application in the details of embodiment institute illustration.Can put into practice the present invention in various other modes, and it can be put into practice with other embodiments.And, expect that wording used herein and term are not to be regarded as restriction for purpose of description.
Before describing ad hoc approach of the present invention and device, with the general feature of describing at least some embodiments of the present invention.
According to some embodiments, the diagnostic device of at least a analyte in the tracer liquid sample is provided, described device comprises in conjunction with at least one solid support with molecularly imprinted polymer (MIP) of analyte specific binding site; The sample application zone; And surveyed area.
MIP is bonded to solid support by physical method or the chemical bonding (immobilization role) by object and upholder.
Liquid sample can along along or the flowpaths that passes solid support move, for example by capillary action or flowing by any kind.
Liquid sample is applied to the sample application zone, and it is contacted with the molecularly imprinted polymer in downstream, sample application zone on the liquid sample flowpaths.
Determine combining of analyte and molecularly imprinted polymer in surveyed area, described surveyed area is preferably located in the downstream of molecularly imprinted polymer position.Preferably, surveyed area provides detectable signal, and it shows the existence of analyte in the liquid sample.Yet, it should be noted that this detection randomly comprises the direct detection of analyte in the sample, and (or additionally) alternatively, this detection randomly comprises the indirect detection of analyte in the sample, for example according to competition and/or replace or its combination.
According to some embodiments, the method of at least a analyte in the tracer liquid sample is provided, described method comprises: be provided for the diagnostic device of at least a analyte in the tracer liquid sample, described device comprises at least one solid support, sample application zone and the surveyed area that combines the molecularly imprinted polymer with analyte specific binding site; Liquid sample is applied to the sample application zone; And the check and analysis thing combines with molecularly imprinted polymer.
As used herein, term " analyte " is meant molecule, group of molecules that will detect or measure or the compound with natural or synthetic source, perhaps its analogue or derivative, and it can be specifically in conjunction with at least one binding partners (for example MIP).Can use analogue or derivative, when analogue or derivative participation mensuration, mode that can be substantially the same with analyte self is used as them in conjunction with a right member.
Can use almost the mensuration of any analyte to put into practice method of the present invention.The analyte size is different, and scope can be from small molecules, have and be less than 30 amino acid whose polypeptide analysis things to whole organism such as bacterium.
Target analyte can be interested any molecule, polypeptide, protein or transmissible disease vehicle.Can use the methods and apparatus disclosed easily detect and/or quantitatively to any known analyte of the analyte specificity MIP that can be fit at its preparation.Can under the background of the development﹠ testing of basic scientific research, the medical practice that comprises animal doctor and dentist, forensic analysis, environment protection and monitoring and research, industry or chemical manufacturing and medicine, food and cosmetic product, use method and apparatus as herein described.
Detected analyte can include but not limited to be selected from the analyte of the group of being made up of antibody, immunoglobulin (Ig), lymphokine, cytokine, solubility cancer antigen, somatomedin and the transmissible disease vehicle of the antibody of biomarker, sterilant, Drug abuse, albumen, hormone, enzyme, biomarker, natural or synthetic toxin, chemical warfare agent, biological warfare agent, medicine, infectious agent.
As an example but limit never in any form, analyte can comprise the biomarker that is selected from the group of being made up of Methylpropanedioic acid (MMA), homocysteine, creatinine, TSH, BNP, FSH, troponin, CK, CKMB, AST, GOT, LDH, myohaemoglobin, PSA, AST, ALT, ALKP, GGT and bilirubin (human and animal the two).Analyte can comprise and being selected from by avian influenza virus, HIV virus, hepatitis virus, poliovirus, cytomegalovirus, dengue virus, Ebola virus, simplexvirus, measles, EBV, rabies virus, respiratory syncytial virus, rotavirus, SARS virus, west Nile virus, yellow fever virus, campylobacter, chlamydiaceae, cholera, genus clostridium, diphtheria, intestinal bacteria, eisseria, Helicobacterium, hemophilus, Mycobacterium, Staphylococcus, Whooping cough, salmonella, Shigella, streptococcus, treponema, the transmissible disease vehicle of the group that tetanus and Yersinia are formed.Analyte can comprise the toxin that is selected from the group of being made up of staphyloentero-toxin, staphylococcal enterotoxin B, Ricin, Toxins, botulin, mycotoxin, tetanus toxin, Toxins,exo-, cholera and trichothecene mycotoxin.The group that the optional freedom of analyte is made up of specific antibody or the autoantibody relevant with autoimmune disorders transmissible disease vehicle.
According to some embodiments, liquid sample of the present invention randomly comprises any liquid that can contain specific target analyte.This liquid comprises the byproduct of biological liquid, environmental sample, food, medicine, toxin, production piece and industrial production step.Biological liquid randomly and preferably is selected from the group of being made up of liquid, tissue homogenate and the process liquid of body fluid, breathing gained.
Liquid sample can randomly comprise and being selected from by liquid for example water, oil, waste liquid or from the liquid of the solids extract environmental sample as the group formed from the liquid of solid waste, soil and plant extract.
Can or after pre-treatment is with the characteristic of modifying it, randomly use sample as directly obtaining from originating.Pre-treatment for example can comprise from blood separation blood plasma, dilution mucus, dilute sample or like that.Treatment process can comprise filtration, distillation, concentrates, the deactivation of interfering component and the adding of reagent.For example, can be with sample centrifugal or filter to remove particulate matter, maybe it can be dissolved in or replenish damping fluid or tensio-active agent so that suitable medium to be provided so that allow more effective detection of analyte.The damping fluid that is fit to comprises in those any known to the skilled, 1-1 for example, Tris solution (the TRIZMA of 000mM, Sigma Chemical Co., St.Louis, Mo.) or 1-1, tensio-active agent polysorbate20 (the commercial tween that also is known as of 000mM TRIS (2-amino-2-(hydroxyl-methyl)-1, ammediol) or 0.05-0.5%
Figure BPA00001204794800171
20).Other damping fluid comprises phosphate buffered saline (PBS) (PBS), citrate buffer or bicarbonate buffer.
The present invention also can randomly be used for detecting the analyte that is included at first in the solid phase sample.Before analysis, be used for extracting or processed sample extracts these analytes simply with the suitable reagent of elution analysis thing or composition and suspends or be dissolved in the liquid.Leaching process can be with simple as shaking solid sample in top listed those at thinner or damping fluid, and it can be applied to solid support then.Randomly, for example hand operated press, mortar and pestle, homogenizer, juice extractor, mixing tank or food-processor can be used to pre-treatment or extract sample target analyte is become the liquid form that is adapted at testing in the device instrument.Solid sample can comprise biological tissue's (for example obtaining), soil or leaf in carrying out examination of living tissue.
According to some embodiments, can prepare the specific molecularly imprinted polymer of analyte (MIP) according to any technology well known by persons skilled in the art.These methods comprise the covalency marking (Wullf G, 1982, Pure ﹠amp; Appl.Chem., 54,2093-2102), whereby monomer covalently is connected to analyte and uses linking agent its polymerization.Next, analyte is cut down from polymkeric substance, stay the specific binding cavity of analyte.Alternatively, can use for example by Mosbach (United States Patent (USP) the 5th, 110, No. 833) disclosed non-covalent marking method, monomer interacts by noncovalent force and target molecule and connects to form the target-specific binding site after removing target molecule by linking agent then whereby.Can use the mutation of these methods to make up: thin molecular imprinting film and film (people 1998Chem.Mater. such as Hong, 10,1029-1033); The marking on the solid support surface (people such as Blanco-L ó pez, 2004, Anal.Bioanal.Chem., 378,1922-1928; People such as Sulitzky C., 2002Macromolecules, 35,79-91); Microsphere (people such as Ye, 2000, Macromolecules, 33,8239-8245) and or even protein and complete organism (people 2006 Adv.Funct.Mater. such as Hayden, 16,1269-1278).
Preferably, molecularly imprinted polymer is included under the existence of target analyte and pore former with linking agent monomer crosslinked and polymeric polymkeric substance, and described polymkeric substance has the ability of selective binding target analyte.Protein imprinted methods involving is so-called " the epi-position marking " (Rachkov and Minoura, 2000, J.Chromatogr A.; 889,111-118), wherein Guan Jian epi-position is identified on protein surface and MIP is with will representing the wire peptide of this epi-position to prepare as target.
Can prepare MIP at any conjugates of analyte or analyte and other molecules, if MIP specifically bound analyte and it conjugates the two.
Some embodiments according to device of the present invention, in MIP-conjugates zone, provide MIP, described zone is to be fixed on the zone that has comprising on the solid support on the device of the analyte specific molecular imprinted polymer (" MIP ") of the specificity binding affinity of tested analyte directly or indirectly, and wherein said specificity binding affinity to tested analyte is meant to whole analyte, to the discrete portions of analyte or to the specificity binding affinity of the peptide of representing defined epitope on the analyte.
According to some embodiments, use releasable analyte analog: reporter conjugates or releasable first wedding agent: the molecule of analyte conjugates makes the analyte specific binding site of MIP saturated.
As used herein, term " analyte is specific " is meant determines the association reaction that analyte exists in the presence of heterogeneous molecule colony.
As used herein, term " analyte analog " is meant the analyte of modification or any other molecule or its combination, and the form of the analyte of potential discovery has structural similarity in itself and the sample, and it can be in conjunction with at least a analyte binding partners.In certain embodiments of the invention, analyte analog is detectable analyte analog-reporter conjugates.For example, analyte analog-conjugates can comprise and be coupled to vitamin H or any other reporter form right half of reporter to binding member analyte that wherein vitamin H is the example of this element.Analyte analog can be MMA, target protein or the peptide of representing discrete epi-position on the tested analyte.
As used herein, term " reporter " is meant can be by any molecule, particle or the composition that can be connected to analyte, analyte analog or binding partners of spectrum, photochemistry, biological chemistry, immunochemistry, electricity, optics, radioassay or chemical means detection.According to the expectation attribute of analytical equipment, reporter is selected from the molecule and the particle that can get in a large number.Some examples that are not considered to this reporter that limits by any way comprise: HABA, dyestuff, fluorescence dye, radio-labeling, magnetic-particle, metallic particles, coloured particle (for example gold and latex colloidal sol) but, fluorescent grain, metal-sol, enzyme substrates and enzyme, chemiluminescent molecule, sensitizer, semiconductor grain, quantum dot and suspended particle.The coupling of compound (for example analyte) and reporter can be passed through covalent linkage, ionic linkage, hydrogen bond, inner complex formation, adsorption process, hydrophobic interaction, aqueous favoring mutual effect, electrostatic interaction and the like or these keys and interactional any combination, and/or can comprise linking group.
But detectable reporter randomly is selected from the group of being made up of HABA, dyestuff, fluorescent agent, fluorescence dye, radio-labeling, magnetic-particle, metallic particles, coloured particle, metal-sol, enzyme substrates, enzyme, chemoluminescence agent, sensitizer suspended particle, polymer beads, semiconductor grain, quantum dot.
Clearly, detectable reporter can be the visible material in the previous list, for example painted latex pearl, and perhaps it can participate in the reaction that produces colored product.When detecting by an unaided eye, reaction product may be a visible, and perhaps for example when being exposed to special light source for example during UV-light, it may be tangible.Though expectation will be the main path that obtains test result to the observation of results area (directly or indirectly), but also consider additive method within the scope of the invention, for example when analyte when detected fluorescent substance associates by being exposed to scanner subsequently.
The concentration of analyte in the sample, no matter be qualitatively or quantitative, indicated by the amount of detectable reporter, described reporter associates with results area subsequently.The detectable signal that shows analyte concentration produces in results area.
As used herein, term " analyte analog: reporter conjugates " is meant and is used to detect micromolecular conjugates, and be used to produce the specific MIP of analyte when " the epi-position marking " method.Synthetic peptide or can represent other synthetic of group of individual molecule or this molecule or the molecule of natural acquisition to represent analyte specificity epitope or its analogue, and be coupled to reporter molecule, it can for example be selected from the group of the molecule of discussing below, for example gold sol.Analyte analog: the reporter conjugates has the high binding affinity to analyte specificity MIP, but it is lower than the avidity of the analyte of unmodified.The molecule of coupling even keep association with the binding cavity of analyte specificity MIP under dampness is unless they randomly and are preferably replaced with the target analyte in the tested liquid sample of the mode of dose-dependently.When being replaced from polymkeric substance, analyte analog: the reporter conjugates moves freely in the solid support of humidity.
As used herein, term " analyte analog: reporter: binding partners conjugates " is meant the conjugates that uses when the group of whole polypeptides or other molecules or molecule is carried out the marking.Target analyte or its analogue are coupled to from the reporter of the group of the molecule of discussing below and particulate group gold sol for example, and are coupled to the binding partners biological example element from the group of discussing below.Analyte analog: reporter L binding partners conjugates has the high binding affinity to analyte specificity MIP, but it is lower than the avidity of the analyte of unmodified.The molecule of coupling even keep association with the binding cavity of analyte specificity MIP under dampness is unless they are replaced with the target analyte in the tested liquid sample of the mode of dose-dependently.When being replaced from polymkeric substance, analyte analog: reporter: the binding partners conjugates moves freely in the solid support of humidity.Under polypeptide and proteinic situation, analyte analog: reporter: the binding partners conjugates is coupled to reporter for example gold sol and wedding agent biological example element simultaneously, and it can be used to catch the analyte analog of replacement: reporter: the binding partners conjugates.
As used herein, term " binding affinity " is meant a molecule and another bonded intensity.If specific molecule is bonded to or another specific molecule that associates specifically, say this two binding affinities that molecular display is mutual so.The association constant of binding affinity and a pair of molecule is relevant with the constant that dissociates, but the measurement of these constants or definite be not critical for the present invention.On the contrary, be used for describing the avidity observed apparent avidity (except as otherwise noted) in positive research normally of the molecular interaction of described method and apparatus herein, it can be used to the relative intensity of molecule of comparison (for example MIP or other specific binding partners) in conjunction with two other molecule (for example analyte and analyte-reporter conjugates).The notion of binding affinity, association constant and the constant that dissociates is known.
According to some embodiments, the analyte specific binding site of MIP is not occupied, and can assay or in conjunction with the competitor combination.
According to some embodiments, MIP is that TSH is specific.Randomly, this MIP is included in corresponding to the epi-position on the TSH β subunit for example in the presence of the synthetic peptide of the amino-acid residue (LSCKCGKCNTDY) of TSH β subunit 9 5-112 position, by as monomeric methacrylic acid (MAA), as the Ethylene glycol dimethacrylate (EGDMA) of linking agent, as starting 2 of the factor, 2P-azo two (2, the different heptonitrile of 4-) (ABDV) and the polymkeric substance that generates as 3% water institute polymerization in the acetonitrile of pore former.Can use standard step on the peptide synthesizer of routine, randomly to prepare synthetic peptide.
According to some embodiments of apparatus of the present invention, solid support can comprise and be used for flow-through appt industry, and has and promote liquid by any porous material of capillary force along device progressive vesicular structure.Preferably, solid support is selected from the group of being made up of porous material, porous-film, granulate material material and absorbent material.These materials also have good protein-bonded ability usually.The example of restriction never in any form of this material comprises: nitrocellulose (high protein binding) cellulose acetate (low protein binding) glass fibre membrane (non-protein binding), the film that nylon, poly(vinylidene fluoride) (PVDF), polyethersulfone (PES) are made or can by or will be by the company of special manufacturing diagnostic film other films of this character of being developed of Millipore company, Pall company and Whatman for example, and brand-new microfluid system disclosed system in No. the 20050042766th, the U.S. Patent application for example.
This device can randomly comprise above a kind of solid support, can detect in sample whereby above a kind of analyte.
According to some embodiments of apparatus of the present invention, the sample application zone comprise suction, porous or fibrous material are made can rapid absorption liquid absorption pad.Its porousness may be unidirectional (being that hole or fiber fully or mainly are parallel to the extension of mobile axis) or multidirectional (being structure omnidirectional, that be similar to sponge).Can use the porous plastic material, for example polypropylene, polyethylene (preferably having very high molecular weight), poly(vinylidene fluoride), vinyl acetate ethylene, vinyl cyanide and tetrafluoroethylene.Pre-treatment with tensio-active agent can randomly be used for reducing any inherent hydrophobicity and be improved the ability that absorbs and transport moist sample fast and effectively in the mill.This material also can be made by paper or other cellulosic materials.The opening in the cover of this material coupling device (casing) and the solid support of device.
With the pad of serum from separation of whole blood, for example United States Patent (USP) the 5th, 660, described in No. 798, can in test sera, use by the device of analyte.The sample that is applied to the sample application zone is absorbed by absorption pad, contacts by capillary force unrestricted flow and with solid support.Depend on type of device, the sample volume of access to plant can be at random or be subjected to intelligent use pad control, described intelligent use pad allows accurate predetermined sample volume access to plant (referring to No. the 6th, 008,056, United States Patent (USP) for example).
According to some embodiments, device of the present invention also comprises cover.Cover remains on suitable configuration with solid support so that guarantee the correct function of whole device.
Cover randomly comprises window, the preferably window between 1 and 4.More preferably, this cover comprises results window, and its effect is to allow observations.Further preferably, this cover comprises the contrast window, and it allow to observe fully carrying out that control reaction for example confirms to test.Also randomly, this cover is included in the 3rd window or opening in this cover, and described window or opening allow at sample window by directly device being placed sample (allowing the contact of sample at openning) or by with dropper or similar device sample application being applied to the sample application zone in sample window with liquid sample.In addition, this cover can comprise randomly that reference window is with intensity as a result in the permission comparative result zone and the bar band strength in the reference zone.
Randomly, solid support also comprises reference zone.As used herein, reference zone is that the analyte analog-reporter binding member of wherein several row is fixed on the zone on the solid support, it has the analyte analog-reporter conjugates of the known quantity that is bonded to them, and to produce distinctive signal with the proportional intensity of the amount of bonded reporter.
This device can randomly also comprise the positive control area that contains the instrument that produces positive control, and it confirms analyte analog: thus the reporter conjugates suitably flow and its to determine to test with combining of results area be work.
Randomly, device of the present invention can comprise one or more absorption pads, and it is placed to promote that analyte flows through results area, reference zone and/or control zone.
In addition, upholder can randomly comprise the absorption region of the pad that contains absorbent material, and described pad is connected by liquid with solid support when pad and solid support are to wet, and described spacer has enough porousness and ability to absorb excessive liquid.
According to some embodiments, device of the present invention can comprise that expectation comprises the lateral flow device of the device of (dipstick) form that has dipstick, perhaps circulation device, perhaps its combination.
According to some embodiments, detection system can be the direct monitoring by the reporter of single replacement or competition and coupling, perhaps replaces or competitive method following will being further explained in detail by two.Yet,, can randomly circulate or transverse flow for any following embodiment.
The single replacement
Some embodiments according to the present invention provide the existence that is used for determining being present in the target analyte of liquid sample, have not existed or single diagnostic device of replacing of concentration.This device comprises the analyte analog that is bonded to the molecularly imprinted polymer on the solid support releasedly: the reporter conjugates; Be used for sample application in device and with it and the contacted sample application of solid support zone; And surveyed area.Analyte has the analyte analog of being equal to or greater than: the avidity to the MIP binding site of reporter conjugates, so that the contact between analyte and the MIP causes analyte analog: reporter is replaced.
Aspect further one, the present invention relates to measure the existence that detects and determine to be present in the target analyte in the liquid sample by a kind of, do not exist or the method for concentration, described mensuration comprises the steps: suspection is contained the sample application of analyte in single sample application zone of replacing diagnostic device of the present invention, and allow sample along or pass that solid support flows and contact MIP-conjugates zone, analyte is bonded to the binding site of MIP so that if analyte is present in the sample, replacement analysis thing analogue: reporter conjugates, described analyte analog: the reporter conjugates flow to results area and at this assay analogue: reporter conjugates binding member is caught, produce detectable signal, the existence or the amount of analyte in the described detectable signal indicating sample.
Surveyed area preferably includes and contains the analyte analog that is fixed in solid support: the results area of reporter conjugates binding member, wherein said reporter conjugates binding member can be in conjunction with the analyte analog of replacing: the reporter conjugates.
As used herein, term " analyte analog: reporter conjugates binding member " is to contain on the finger device analyte analog: reporter conjugates molecule has the zone of the element of high-affinity.These elements can randomly include but not limited to vitamin H binding member (for example avidin, streptavidin, NeutrAvidin and ExtrAvidin), reporter specific antibody, enzyme, substrate and especially at analyte analog: the MIP of reporter conjugates preparation.
Single replacement method comprises the liquid sample introducing sample application zone of suspection being contained test analyte, and allows sample to move along upholder.If analyte is present in the sample, it is bonded to the analyte specific binding site of MIP, and replacement analysis thing analogue: the reporter conjugates.The reporter conjugates that is replaced along or pass solid support and move to results area, it is bonded to reporter conjugates binding member at this, and detectable signal is provided.The existence of detectable signal and/or intensity preferably at least with sample in analyte amount quantitatively or relevant qualitatively, though can randomly obtain " be/not " answer of binary; Randomly this also is possible for following other embodiments.
Two replacements
On the other hand, the present invention relates to be used for directly to detect and the existence of definite liquid sample target test thing, do not exist or two diagnostic devices of replacing of concentration.This device comprises in conjunction with the solid support of MIP and is bonded to releasable first wedding agent of MIP: the analyte conjugates; The sample application zone; And at the reporter in downstream, sample application zone: the conjugates calmodulin binding domain CaM.
The two replacement methods of design promote signal to form and explain, and it can be applied to having other instruments of similar quality.This method finds the special applications of perfect biotin-avidin system, and it provides very strong binding affinity with numerous analogues that can be used and derivative.When handling " the epi-position marking " method, when wedding agent: the analyte conjugates is at its preparation specificity MIP and when being coupled to the synthetic peptide of the epi-position on the representative target analyte of wedding agent biological example element, and two replacement methods are highly to be suitable for.
Common single replacement method that two replacement methods and reporter directly are bonded to target molecule is compared has some advantages.Vitamin H is relatively little molecule, and its is added the interaction that target molecule should disturb it and analyte specificity MIP not manyly.The chemical feature of vitamin H is established very much, and many commercial reagents can be modified target analyte.Vitamin H is very fast and strong with combining of various vitamin H binding members, and it is contributed for the overall performance of device.Because reporter directly is not bonded to analyte, there is big handiness aspect the ability of biotin derivative so select and it is coupled at reporter.Incompetent or the difficulty that reporter molecule is bonded to some analyte may be a limiting factor of using the replacement method in these cases.In addition, it allows to increase the sensitivity of device, because the whole bag of tricks that can use signal well known by persons skilled in the art to amplify owing to the separation between sensing element and the results area.
Another advantage is the device for various analytes, and the present invention uses identical signal formation method, and producing is similar mostly, and only need develop specific MIP and the analyte that is coupled to vitamin H at every kind of independent product.This makes the listing of product innovation shorten the time and reduces exploitation and production cost.
If complete albumen is carried out the marking, wedding agent so: the analyte conjugates can be randomly by coupling the two target protein of wedding agent and reporter molecule formed.In this case, conjugates can be loaded with enough reporter molecules so that may not need amplification.
As used herein, term " wedding agent: analyte conjugates " is meant directly or is coupled to by introns the target analyte of wedding agent (being preferably vitamin H or biotin derivative).Alternatively, wedding agent: the analyte conjugates can randomly be the peptide corresponding to the epi-position on the analyte that is coupled to wedding agent.Wedding agent has the specific avidity to its corresponding binding partners.When wedding agent was vitamin H and its derivative, corresponding binding partners or agent were avidin, streptavidin, NeutrAvidin and similar vitamin H wedding agent.
First wedding agent: the analyte conjugates has high binding affinity to analyte specificity MIP, but it is lower than the avidity of the analyte of unmodified.Even under dampness, first wedding agent: the maintenance of analyte conjugates molecule is connected with analyte specificity MIP binding site, unless they are replaced with the target analyte in the tested liquid sample of dose-dependent mode.When being replaced from polymkeric substance, first wedding agent: the analyte conjugates moves freely in the solid support of humidity.
Two alternatives also comprise results area, and it is included in second wedding agent that is bonded to solid support on the sample flow approach: reporter conjugates binding member.Reporter-conjugates binding member has and has connected detectable, releasable second wedding agent: the binding site of reporter conjugates.
As used herein, term " binding member " is meant can be with enough avidity specifically in conjunction with its molecular structure of corresponding binding partners.The particular sequence of this element and boundaries for certain are not crucial, as long as it represents in conjunction with active.Binding characteristic comprises a series of avidity, affinity (avidity) and specificity and combination thereof necessarily, as long as it represents in conjunction with active.
Wedding agent: the reporter conjugates comprises as above for analyte analog: the described any reporter entity of reporter conjugates, its be coupled to can with wedding agent: the analyte conjugates is bonded to the wedding agent of identical binding member specifically.
Second wedding agent has the high affinity to this binding member, but it is lower than first wedding agent that is used for this device: the avidity of analyte conjugates.So first wedding agent: the contact between analyte conjugates and the binding member causes second wedding agent: the replacement of reporter conjugates.Second wedding agent: the concentration of analyte is proportional in the replacement of reporter conjugates and the liquid sample, so that the existence of detectable signal and/or intensity are relevant with the amount of analyte in the sample.
When the biological example element is used as wedding agent in device: during wedding agent in the analyte conjugates, have than vitamin H low the vitamin H of the avidity of vitamin H binding member (for example avidin, streptavidin, NeutrAvidin and ExtrAvidin) or the derivative of other molecules (for example HABA and DTB) are used to make up wedding agent: the reporter conjugates.
As used herein, term " binding partners " or " wedding agent " are meant any molecule or the composition of the ad hoc structure aspect that can discern and be bonded to another molecule or composition.The example of this binding partners and corresponding molecule or composition comprises vitamin H/avidin, antigen/antibody, haptens/antibody, hormone/acceptor, nucleic acid chains/complementary nucleic acid chain, substrate/enzyme, inhibitor/enzyme, albumin A (or G)/immunoglobulin (Ig), carbohydrate/lectin, virus/cell receptor and apoprotein/lipid.
The group that releasable first wedding agent is selected from is conjugated protein by vitamin H, vitamin H analogue, biotin derivative, antigen, albumin A and Protein G, Mierocrystalline cellulose, hormone, toxin, lipid, lipid acid, complementary nucleic acid sequence, sugared conjugates, lectin, substrate and part are formed.
The group that the optional free vitamin H analogue of releasable second wedding agent, HABA, DTB, antigen, albumin A and Protein G, Mierocrystalline cellulose are conjugated protein, liposome, hormone, toxin, lipid, lipid acid, complementary nucleic acid, sugared conjugates, lectin, substrate and part or their analogue are formed, condition is that described second wedding agent has the avidity to reporter-conjugates binding member lower than described first wedding agent.The vitamin H analogue is in Advances in Protein Chemistry (protein chemistry progress), Anfinsen, and Edsall and Richards edit, and Academic Press (1975) is identified in the 104-111 page or leaf.
Single competition
The present invention also provides a kind of diagnostic device, and wherein the sample application zone comprises analyte: the analogue conjugates, and also the binding site of MIP is not occupied.Also can for example provide conjugates in the pad, so that conjugates is in case by the sample hydration unrestricted flow that just becomes.In this case, analyte analog: the analyte specific binding site of the analyte competition MIP in reporter conjugates and the sample.Then, unconjugated conjugates moves and is as above described detected for the conjugates of replacing.
More further aspect, the present invention relates to detect and determine to be present in the existence of the target analyte in the liquid sample, do not exist or the method for concentration by a kind of mensuration, described method comprises the steps: suspection is contained the sample application zone of the sample application of analyte in state of conflict diagnostic device of the present invention, and allow sample along or pass that solid support flows and contact MIP-conjugates zone, so that if analyte is present in the sample, then analyte and analyte analog: the binding site of reporter conjugates competition MIP.Unconjugated analyte analog: the reporter conjugates flow to results area, therein its assay analogue: reporter conjugates binding member is caught, and produces detectable signal, the existence or the amount of analyte in the described detectable signal indicating sample.
Two competitions
The present invention further provides a kind of diagnostic device, it comprises first wedding agent: analyte conjugates, second wedding agent: the reporter conjugates and second wedding agent: reporter conjugates binding member.Randomly in the sample application zone, provide first wedding agent: analyte conjugates, and the analyte specific binding site of itself and analyte competition MIP.Unconjugated first wedding agent: analyte conjugates flow further downstream, and with second wedding agent: the reporter conjugates competition and second wedding agent: the combining of reporter conjugates binding member.
Again aspect another, the present invention relates to detect and determine to be present in the existence of the target analyte in the liquid sample, do not exist or the method for concentration by a kind of mensuration, described method comprises the steps:
(a) suspection is contained the sample application of analyte in two sample application zones of replacing diagnostic device of the present invention; And
(b) allow sample along or pass that solid support flows and contact MIP-conjugates zone, so that if analyte is present in the sample, then analyte combines with the binding site of MIP, replace first wedding agent: the analyte conjugates, described first wedding agent: the analyte conjugates flow to reporter-conjugates calmodulin binding domain CaM, first calmodulin binding domain CaM of reporter-conjugates binding member is in conjunction with first wedding agent of replacing: thus analyte conjugates and replace second wedding agent: the reporter conjugates, described second wedding agent: the reporter conjugates continues along the approach of liquid-flow, second calmodulin binding domain CaM to results area and the son that is in the news-conjugates binding member is caught, produce detectable signal, the existence and/or the amount of analyte in the described detectable signal indicating sample, second wedding agent that wherein is captured: the concentration of target analyte is proportional described in the amount of reporter conjugates and the sample.
In yet another aspect, the present invention relates to comprise the device of solid support, comprise: first assembly, it comprises the sample application pad that has flooded analyte analog-conjugates, described analyte analog-conjugates comprises randomly the analyte (randomly, analyte: the analogue conjugates can be submerged in the special agent pad between sample pad and the MIP) that is coupled to vitamin H by introns; Second assembly, it comprises analyte specificity MIP; The 3rd assembly, it contains the first saturated vitamin H binding member of useful reporter-conjugates, and described reporter-conjugates comprises and is coupled to having of reporter the vitamin H analogue to the avidity of vitamin H binding member lower than vitamin H (desthiobiotin-DTB) for example; And the 4th assembly, it comprises the second vitamin H binding member.
Again aspect another, the present invention relates to comprise the device of solid support, comprise: first assembly, it comprise with analyte analog-conjugates saturated analyte specificity MIP, described analyte analog-conjugates comprises the analyte that randomly is coupled to vitamin H by introns; Second assembly, it comprises with the first saturated vitamin H binding member of reporter-conjugates, described reporter-conjugates comprises and is coupled to having of reporter the vitamin H analogue to the avidity of vitamin H binding member lower than vitamin H; And the 3rd assembly, it comprises the second vitamin H binding member.
Competition adds replacement
The present invention also can randomly provide diagnostic device and the method that comprises competition and replacement method with any order and combination.
For example, this method can comprise: first step wherein is bonded to first wedding agent of MIP: analyte conjugates assay is replaced; And second step, the conjugates that wherein is replaced and second wedding agent: the binding site of reporter conjugates competition reporter conjugates binding member.
Alternatively, first step can comprise first wedding agent: between analyte conjugates and the analyte to the competition of MIP binding site; And second step can comprise second wedding agent from reporter conjugates binding member: the reporter conjugates is by the replacement of unconjugated analyte conjugates.
More further aspect, this method comprises in the comparative result zone that strength of signal in the strength of signal and reference zone determines the step of the concentration of analyte in the sample.
Still further aspect, results area comprises the scale with respect to analyte concentration calibration in the liquid sample to be analyzed that is parallel to fixed reporter-conjugates binding member, and the existence of analyte and concentration are by by along determining with the area that reporter-conjugates covered that passes reporter in the results area-conjugates binding member in the wherein said liquid.
Again aspect another, the present invention relates to detect the method that is present in the TSH in the liquid sample, described method comprises the steps:
A) sample is contacted with competition according to the present invention, list or two alternative, and
B) detect the amount of the reporter conjugates be bonded to reporter-conjugates binding member in results area, the amount of wherein said reporter-conjugates is indicative for existence or the amount of TSH in the sample;
C) diagnose thyroid activity.
The method that detects TSH can be used to identify under-active Tiroidina (hypothyroidism), it can cause for example weight increase, fatigue, xerosis cutis, constipation of symptom, feel too cold or the menstrual period frequent; Perhaps be used to identify overactive Tiroidina (hyperthyroidism), it can cause, and symptom is for example lost weight, heart rate quickening, nervousness, suffer from diarrhoea, feel the too hot or erratic menstrual period.
On the other hand, device of the present invention can be used to extract or processed sample with the elution analysis thing and detect any or all thinner of given analyte or reagent is packaged advances diagnostic kit.
Again other aspect, diagnostic kit also comprises wrapping material and the relevant specification sheets that carries out quantitative analysis on the liquid sample of at least a type.
The sensitivity of specific mensuration is relative affinity and all ingredients concentration, the function of the intensity of time of contacting with each other of particular agent and analyte and the signal that produces by the report system therein.Those skilled in the art are familiar with being used to optimize and characterize the sensitivity of the tradition mensuration that is used for lateral flow device and circulation device and the method for dose response feature.
Another important embodiment of the present invention is visual monitoring and the method for explaining signal in the results window.In a variant of being called of Monitoring systems " vertical developing method ", reporter-conjugates binding member (vitamin H in conjunction with or other reporters in binding member or the analyte-reporter binding member any) placed perpendicular to the sample flow approach so that catch and concentrate free-pouring reporter conjugates molecule as much as possible with order at random.The amount (it is the direct representative of the amount of analyte in the sample) that is bonded to the reporter conjugates of binding member is big more, and the visual signalling of acquisition is strong more.The results window next door is a reference window, and its some indicatrix by the binding member of the reporter conjugates that has flooded the accurate amount that increases is separately formed.These lines will represent a series of colour intensities, and its known quantity with the reporter conjugates that is bonded to them is proportional.In the results window in strength of signal and the adjacent reference window line strength make that relatively the good estimation of the amount of analyte becomes possibility in the sample.
In another variant that is called " horizontal developing method " of Monitoring systems, the boundary band of reporter-conjugates binding member is clearly accurately flatly placed the sample circulating pathway.Should be to allow liquid only to flow through the zone of the son that is in the news-conjugates binding member covering and to take measures to avoid sample to operate the device of this position around zone that binding member was covered or the mode that flows through from its below.This can for example physically cut porous material by the mode of staying in the flowpaths with the zone of having only binding member to cover, perhaps by for example using by photolithography (photolithography) to methods such as the channel forming that flows (people such as Martinez, 2007 Angwe.Chem.Int.119 1340-1342) realize.The reporter molecule of all couplings that this assurance is replaced in response to the existence of analyte in the sample arrives the fixed binding member.The reporter conjugates molecule that is replaced that arrives binding member moves by wicking action, and the combined element of any reporter-conjugates molecule is caught.Yet, because the receptivity of binding member is limited, and because the binding site of binding member has been full of reporter-conjugates molecule, therefore be with forward binding member molecular change to get saturated and also reporter-conjugates molecule must along band further migration downwards to find the free binding site.The concentration of analyte is high more in the sample, and reporter-conjugates is big more with the area of the binding member of covering.To be made that by the pre-calibration of the area that reporter-conjugates covered of known quantity it introduces the scale that allows the user to determine the calibration of the amount of analyte in the sample by the next door of fixed binding member in device according to along the distance that reporter-conjugates covered of binding member band the accurate mensuration of analyte concentration being become possibility.
This device can comprise above a kind of solid support, can detect in sample whereby above a kind of analyte.
Second calmodulin binding domain CaM of first calmodulin binding domain CaM of reporter-conjugates binding member and reporter-conjugates binding member can be identical or different, and be selected from separately by the avidin, streptavidin, NeutrAvidin, the ExtrAvidin compound that have the high specific avidity of vitamin H, film, acceptor, immunoglobulin (Ig), Mierocrystalline cellulose, enzyme, lectin, the sugar conjugates, complementary nucleic acid and have the group that the hydrophobic site of the high-affinity of they corresponding binding partners is formed.
The detailed description of drawings and Examples
The following examples are illustrative for the present invention, but not are intended to be limited by any way.Those skilled in the art will find can reformed various non-key parameters.For clear, various assemblies are flatly presented in the lateral flow device, but can make as required one on another or the stack.Fig. 1-2 shows four kinds of different embodiments of lateral flow device of the present invention, Fig. 3-4 shows four kinds of different embodiments of circulation device of the present invention, and Fig. 5 shows that with the embodiment of the device of transverse flow and flow-through element merging, each embodiment will be described in respectively among the embodiment 1-9.
Embodiment 1: the embodiment of Figure 1A
The structure and the operation of the lateral flow device that has competition or single replacement and vertical video picture are described below.
Figure 1A is used for detecting and the existence of the target analyte that definite liquid sample exists, does not exist or the side-view of the lateral flow device of concentration.Analytical test set comprises and containing as can be from the hollow solid cover 10 of the solid support 12 of the porous calibration tape form of the carrier of transmit fluid sample wherein, can be by the solid support mobile example of wicking action in the liquid-flow approach.Solid support 12 has the definite zone that comprises the sample application zone, and wherein said sample application zone comprises and being used for sample application in device and sample application pad 14 that it is contacted with solid support 12.Sample application pad 14 is arranged near cover 10 opening or the window 16 to be used for the using liquid sample.When using type of competition, with the releasable analyte analog of sample application pad 14 impregnation drying states: the reporter conjugates, can add special-purpose pad alternatively.
If target analyte is present in the liquid sample, then will be transported to the MIP-conjugates zone in downstream, sample application zone from the sample application zone, it is included in the analyte specificity MIP 18 that is fixed in solid support 12 on the sample circulating pathway.MIP has the releasable analyte analog with drying regime: the analyte specific binding site that the reporter conjugates is saturated, it comprises the target analyte that is coupled to binding partners and reporter molecule.Analyte to the avidity of the binding site of analyte specificity MIP greater than analyte analog: the reporter conjugates is to the avidity of the binding site of analyte specificity MIP.When containing the liquid sample contact analysis thing specificity MIP of analyte, the analyte in the sample is bonded to the analyte specificity chamber of MIP and thereby to replace the analyte analog-reporter conjugates that occupies these chambeies with the direct proportional amount of the concentration of specific analyte, cause the analyte analog of replacing: the reporter conjugates is flow further downstream in the liquid-flow approach.Under the situation of competition, the analyte analog of liquid sample dissolving dipping: mix with analyte in the sample reporter conjugates and it.When liquid arrives analyte specificity MIP, two kinds of molecule competition analysis thing specific binding sites.With the proportional quantitative analysis thing of the concentration of specific analyte analogue: the reporter conjugates not combined and in the liquid-flow approach flow further downstream.
Further be analyte analog downstream on the solid support 12: reporter conjugates binding member, it is to be bonded to the analyte analog that is fixed in solid support 12 on the flowpaths in analyte specificity MIP 18 sample downstreams: the binding partners of the binding member of reporter conjugates 20.Opening in the cover 10 is positioned at analyte analog: reporter conjugates binding partners 20 tops, it comprises the results window 22 of device.When the liquid sample that contains analyte flows to the flowpaths zone, reporter conjugates binding partners 20 is bonded to unconjugated analyte analog: reporter conjugates or replace analyte analog from the replacement of analyte specificity MIP 18: the reporter conjugates, and detectable signal is provided in results window 22, it shows the existence or the concentration of analyte in the sample.
Further be analyte analog downstream on solid support 12: three of reporter conjugates binding partners discrete with nonoverlapping indication band, its each all extend along the length of band, wherein 24a represents lower concentration with reference to band, and 24c represented that high density is with reference to band to concentration with reference to band during 24b represented.These three indication bands are formed in the reference zone that is used for establishing reference point in the existence of determining analyte in the sample or the sxemiquantitative.Corresponding reference window 26 appears at reference in the cover 10 above band 24a, 24b and the 24c.Under the existence that will determine sample target test thing or the situation of not depositing, reference zone can comprise the analyte analog with known quantity: at least one discrete bands of the binding member of reporter conjugates dipping.Under the situation of the amount that will determine sample target test thing, reference zone can comprise the analyte analog of each personal increasing amount: the indication band at least two intervals of the binding member of reporter conjugates dipping, and represent a series of and the analyte analog that is incorporated into it: the proportional colour intensity of the amount of reporter conjugates.The user comes explanation results by the intensity of one or more bands in strength of signal in the visual comparison results window 22 and the reference window 26 and determines the existence of analyte in the sample, do not exist or the sxemiquantitative of concentration.
Downstream with reference to band 24a, 24b and 24c is to use analyte analog: the contrast pad 32 of reporter conjugates dipping, but this conjugates is static when solid support 12 unrestricted flow that becomes during by the liquid wets of sample under drying regime.It further is the another kind of analyte analog that is anchored on the solid support 12 downstream: reporter conjugates binding partners 34.At analogue: there is the corresponding contrast window 30 that is used for observations in the cover of reporter conjugates binding partners 34 tops.Filled up 32 analyte analog that discharge by the liquid of sample from contrast: the reporter conjugates arrives binding partners 34 and becomes and is fixed in solid support 12, produces visual signal in contrast window 30.Contrast pad and analyte analog: reporter conjugates binding member 34 is configured for producing the control zone of positive control jointly, described positive control is confirmed analyte analog: the reporter conjugates suitably flow and to the combination of results area, thereby determine that test suitably works.
Far-end at device is the absorption pad 36 of absorbent material, and when pad 36 and solid support 12 are when wetting, described absorption pad 36 and solid support 12 communicate by liquid.Described spacer has enough porousness and ability to absorb excess liquid, and guarantees the continuous flow of through-going device.
Embodiment 2: the embodiment of Figure 1B
The structure and the operation of the lateral flow device that has competition or single replacement and the horizontal video picture of test result are described below.
Described lateral flow device identical with described in the top embodiment 1 is with the similar part of similar numeral indication, except the modification of reference number and additional letter " a " expression.Modify as follows: do not have reference zone in the device of Figure 1B, therefore, cover 38 has less window or opening, and has the results window 42 of expansion.Only according to analyte analog: the distance that the reporter conjugates is covered reads the result from results window 42.By the fixed analyte analog: it is regional extended and have a scale 46 that is parallel to its and places that reporter conjugates binding member 44 is covered.Scale 46 and by the fixed analyte analog: the zone that reporter conjugates binding member 44 is covered by common calibration with corresponding to the analyte concentration in the analyzed liquid sample.By by along with pass analyte analog: the analyte analog of reporter-conjugates binding member 44: the area that reporter-conjugates covered is determined the existence and the concentration of analyte in the liquid in results window 42.In contrast window 30a, observe the result of control signal.
Embodiment 3: the embodiment of Fig. 2 A
The structure and the operation that have the competition/pair replacement and the lateral flow device of the vertical video picture of test result are described below.
Fig. 2 A is used for detecting and the existence of the target analyte that definite liquid sample exists, does not exist or the side-view of the lateral flow device of concentration.Device comprises that contain can be from the hollow solid cover 10b of the solid porous upholder 12b of the calibration tape form of transmit fluid sample wherein, the sample that can move along the solid support in the liquid-flow approach by wicking action.Solid support 12b has the definite zone that comprises the sample application zone, and wherein said sample application zone comprises and being used for sample application in device and sample application pad 14b that it is contacted with solid support 12b.It is neighbouring to be used for the using liquid sample that sample application pad 14b is arranged in the opening or the window 16b that cover 10b.When using type of competition, with the releasable analyte analog of sample application pad 14b impregnation drying state: the reporter conjugates, can add special-purpose pad alternatively.
If target analyte is present in the liquid sample, then will by from sample application pad 14b along with the MIP-conjugates zone carrying of passing 14b downstream, sample application zone, described MIP-conjugates zone is included in the analyte specificity MIP 50 that is fixed in solid support 12b on the sample circulating pathway.MIP 50 has with releasable first wedding agent as target drying regime, that be coupled to binding member: the analyte conjugates saturated analyte specific binding site.Analyte to the avidity of the binding site of analyte specificity MIP 50 significantly greater than first wedding agent: the analyte conjugates is to the avidity of the binding site of analyte specificity MIP 50, so that cause first wedding agent in the presence of analyte: the analyte conjugates is from the replacement of the analyte specific binding site of MIP 50.When containing the liquid sample contact analysis thing specificity MIP 50 of analyte, thereby the analyte in the sample is bonded to the analyte specificity chamber of MIP also to replace first wedding agent that occupies these chambeies with the direct proportional amount of the concentration of specific analyte: the analyte conjugates, cause first wedding agent of replacing: the analyte conjugates is flow further downstream in the liquid-flow approach.Under the situation of competition, the analyte analog of liquid sample dissolving dipping: mix with analyte in the sample reporter conjugates and it.When liquid arrives analyte specificity MIP, two kinds of molecule competition analysis thing specific binding sites.With the proportional quantitative analysis thing of the concentration of specific analyte analogue: the reporter conjugates not combined and in the liquid-flow approach flow further downstream.
Further be reporter-conjugates calmodulin binding domain CaM downstream on solid support 12b, it is included in first calmodulin binding domain CaM that is fixed in reporter-conjugates binding member 52 of solid support 12b on the sample flow approach in analyte specificity MIP 50 downstreams, and this calmodulin binding domain CaM comprises detectable, releasable second wedding agent of binding site with drying regime: the reporter that the reporter conjugates is saturated-conjugates binding member 52.First wedding agent: the analyte conjugates to the avidity of the binding site of reporter-conjugates binding member 52 greater than second wedding agent: the reporter conjugates is to the avidity of the binding site of reporter-conjugates binding member 52.First calmodulin binding domain CaM of reporter-conjugates binding member 52 with sample in the direct proportional amount of concentration of specific analyte combine first wedding agent: the analyte conjugates is also replaced second wedding agent: the reporter conjugates, cause second wedding agent of replacing: the reporter conjugates continues migrated downstream in the liquid-flow approach.Alternatively, can use competition and can be: reporter conjugates drying with second wedding agent on pad, by sample liquids hydration again and with first wedding agent: the analyte conjugates is competed the binding site in first calmodulin binding domain CaM, in this case, do not need first wedding agent: analyte is to the bigger avidity of the binding site of reporter-conjugates binding member 52.Excessive unconjugated second wedding agent: the reporter conjugates will continue migrated downstream in the liquid-flow approach.
On the solid support 12b further be second calmodulin binding domain CaM that is fixed in reporter-conjugates binding member 54 of solid support 12b on the sample flow approach in the first calmodulin binding domain CaM downstream of reporter-conjugates binding member 52 downstream.Opening among the cover 10b is positioned at second calmodulin binding domain CaM top of reporter-conjugates binding member 54, comprises the results window 22b of device.When the liquid sample that contains analyte flows in the flowpaths zone, second calmodulin binding domain CaM of reporter-conjugates binding member 54 is bonded to second wedding agent of replacing from first calmodulin binding domain CaM of reporter-conjugates binding member 52: the reporter conjugates, and the detectable existence of analyte in the sample or the signal of concentration of showing is provided.
As described in about the device among the embodiment 1A, what further be positioned at solid support 12b downstream is second wedding agent: three of reporter conjugates binding member discrete with nonoverlapping indication band, wherein 56a represents lower concentration with reference to band, and 56c represented that high density is with reference to band to concentration with reference to band during 56b represented.These three indication bands are formed in the reference zone that is used for establishing reference point in the existence of determining analyte in the sample or the sxemiquantitative, are corresponding reference window 26b among the cover 10b above it.The user comes explanation results by the intensity of one or more bands among strength of signal among the visual comparison results window 22b and the reference window 26b and determines the existence of analyte in the sample, do not exist or the sxemiquantitative of concentration.
About as described in embodiment 1A and the 1B, reference window 26b downstream is to use second wedding agent as top: the contrast pad 32b of reporter conjugates dipping, but this conjugates is static when the solid support 12b unrestricted flow that becomes during by the liquid wets of sample under drying regime.Further be the another kind of reporter-conjugates binding member 56 that is anchored on the solid support 12b downstream.There is the corresponding contrast window 30a that is used for observations among the cover 10b above reporter-conjugates binding member 56.Filled up second wedding agent that 32b discharges by the liquid from sample from contrast: the reporter conjugates arrives binding member 56 and becomes and is fixed in solid support 12b, is created in observed visual signal among the contrast window 30b.Contrast pad 32b and reporter-conjugates binding member 56 are configured for producing the control zone of positive control jointly.
Far-end at device is the absorption pad 36b that absorbent material is made, and when pad 36b and solid support 12b are when wetting, described absorption pad 36b communicates by liquid with solid support 12b.Described spacer has enough porousness and ability to absorb excess liquid, and guarantees the continuous flow of through-going device.
Embodiment 4: the embodiment of Fig. 2 B
The structure and the operation of the lateral flow device that has competition/pair replacement and the horizontal video picture of test result are described below.Described lateral flow device identical with described in the top embodiment 3 is with the similar part of similar numeral indication, except the modification of reference number and additional letter " a " expression.Modify as follows: in the device of Fig. 2 B, do not have reference zone.Only according to second wedding agent: the distance that the reporter conjugates is covered reads the result from results window 60.Second calmodulin binding domain CaM of the reporter-conjugates binding member 66 among the cover 38a below the results window 60 is extended and comprises the scale 68 of second calmodulin binding domain CaM that is parallel to fixed reporter-conjugates binding member 66, and described scale 68 is calibrated with corresponding to the analyte concentration in the analyzed liquid sample.By viewed by second wedding agent along second calmodulin binding domain CaM of reporter-conjugates binding member 66 in results window 60: the area that reporter-conjugates covered is determined the existence and the concentration of analyte in the liquid.
Embodiment 5: the embodiment of Fig. 3 A
The structure and the operation of the circulation device that has competition or single replacement and vertical video picture are described below.
Fig. 3 A is used for detecting and the existence of the target analyte that definite liquid sample exists, does not exist or the side-view of the circulation device of concentration.Device comprises the hollow solid cover 80 that contains definite layer or zone, described layer or zone are to be made by the porous material that contains reagent, and be arranged so that be applied to the vertical liquid of device vertically flows through device from one deck to another layer each layer, absorbing material until the liquid contact apparatus bottom.
Comprise be used for sample application is positioned at the opening or window 84 of the vertical cover 80 of device in the sample application zone of sample application pad 82 of device near, and guiding comprises the releasable analyte analog that has with drying regime: the MIP-conjugates zone of the analyte specificity MIP 86 of the analyte specific binding site that the reporter conjugates is saturated.Analyte analog: the reporter conjugates also can comprise corresponding to target analyte, is coupled to the two polypeptide or albumen of binding partners and reporter molecule.Analyte to the avidity of the binding site of analyte specificity MIP 86 greater than analyte analog: the reporter conjugates is to the avidity of the binding site of analyte specificity MIP 86.Under the situation of type of competition device, the special agent pad assay analogue of application pad or its below: reporter conjugates dipping, and the binding site of MIP 86 is a free.In this case, the difference to the avidity of MIP is not enforceable.If target analyte is present in the liquid sample, pass analyte specificity MIP 86 migrations from sample application pad 82.When containing the liquid sample contact analysis thing specificity MIP 86 of analyte, analyte in the sample is bonded to the analyte specificity chamber of MIP, thereby and with sample in the direct proportional amount of the concentration of specific analyte replace the analyte analog-reporter conjugates that occupies these chambeies, cause the analyte analog of replacing: the reporter conjugates further is passed down through the device migration.For the type of competition device, the liquid of sample dissolves the analyte analog from sample pad or special agent pad: the reporter conjugates, and analyte analog: the analyte of reporter conjugates in sample mixes and flows until MIP zone 86, at this their competition analysis thing specific binding site.With the direct proportional quantitative analysis thing analogue of the concentration of specific analyte in the sample: the reporter conjugates not combined and to the device downstream further move.
Next be the analyte analog that is fixed in porous support or upholder 88 on the flowpaths of the sample in analyte specificity MIP 86 downstreams: reporter conjugates binding member 90, it is specifically in conjunction with being coupled to analyte analog: its binding partners of reporter conjugates.Transparent window in the solid cover 80 is positioned at analyte analog: the front of reporter conjugates binding member 90 comprises the results window 92 of device.Unconjugated analyte analog: reporter conjugates or the analyte analog that assay is replaced from analyte specificity MIP 86: the reporter conjugates migrates to analyte analog: reporter conjugates binding member 90, they are captured at this, and detectable signal is provided, in results window 92, show the existence or the concentration of analyte in the sample.
Next zone is to comprise analyte analog: separated by porous support zone 88 three of reporter conjugates binding member discrete with reference zone nonoverlapping indication band, wherein 94a represents lower concentration with reference to band, and 94c represented that high density is with reference to band to concentration with reference to band during 94b represented.Transparent reference window 96 is positioned at the front of three indication band 94a, 94b and 94c.The user comes explanation results by the intensity of the band in strength of signal in the visual comparison results window 92 and the reference window 96.
The reference zone below is porous support zone 88, then is to comprise having flooded unconjugated analyte analog: the control zone 100 of the porous support of reporter conjugates.Conjugates 100 is an immobilized under drying regime, but when the porous support unrestricted flow that becomes during by the liquid wets of sample.Downward again, what separated by another porous support zone 88 is another analyte analog: reporter conjugates binding member 102.Contrast window 104 is positioned at the front of reporter conjugates binding member 102.By the analyte analog that discharges from control zone 100 from the liquid of sample: the reporter conjugates arrives analyte analog: reporter conjugates binding member 102 also is incorporated in to carrier, is created in observable optical signal in the contrast window 104.Far-end at device is an absorbent material 106, the continuous flow that it absorbs excess liquid and guarantees through-going device.
Embodiment 6: the embodiment of Fig. 3 B
The structure and the operation of the circulation device that has competition or single replacement and the horizontal video picture of test result are described below.
Described circulation device identical with described in the top embodiment 5 with the similar part of similar numeral indication, has less following modification: do not have reference zone in the cover 108 of the device of Fig. 3 B.Only according to analyte analog: the distance that the reporter conjugates is covered reads the result from results window 114.Analyte analog: reporter-conjugates binding member 110 and results window 114 are extended.Scale 112 is parallel to the fixed analyte analog: reporter-conjugates binding member 110 is placed, the two by common calibration with corresponding to the analyte concentration in the analyzed liquid sample.By in results window 114, seen by passing analyte analog: the analyte analog of reporter-conjugates binding member: the area that reporter-conjugates covered is determined the existence and the concentration of analyte in the liquid.In contrast window 104a, observe results of comparison.
Embodiment 7: the embodiment of Fig. 4 A
The structure and the operation that have the competition/pair replacement and the circulation device of the vertical video picture of test result are described below.
Fig. 4 A be to use competition/pair replacement to detect and definite liquid sample in the target analyte that exists existence, do not exist or the side sectional view of the circulation device of concentration.Described device comprises the hollow solid cover 80b that contains definite layer or zone, described layer or zone are to be made by the porous material that contains reagent, and be arranged so that be applied to the vertical liquid of device vertically flows through device from one deck to another layer each layer, absorbing material until the liquid contact apparatus bottom.
Be used for sample application is positioned in the sample application pad 82b of device near the opening or window 84b of the vertical cover of device 80b, and guiding comprises releasable first wedding agent that has with drying regime: the MIP-conjugates zone of the analyte specificity MIP 122 of the analyte specific binding site that the analyte conjugates is saturated.First wedding agent: the analyte conjugates can comprise corresponding to by the epi-position of the target of the marking, be coupled to the synthetic peptide of binding partners.Analyte to the avidity of the binding site of analyte specificity MIP 122 greater than first wedding agent: the analyte conjugates is to the avidity of the binding site of analyte specificity MIP 122.The target analyte of finding in liquid sample passes the zone migration of MIP-conjugates from sample application pad 82, in MIP-conjugates zone, target analyte is bonded to the analyte specificity chamber of MIP 122, thereby and to replace first wedding agent that occupies these chambeies with the direct proportional amount of the concentration of specific analyte: the analyte conjugates causes first wedding agent of replacing: the analyte conjugates further is passed down through the device migration.Alternatively, if use the type of competition device, then application pad 82b or special-purpose reagent pad are flooded releasable first wedding agent: the analyte conjugates.For the type of competition device, the liquid of sample dissolves the analyte analog from sample pad or special agent pad: the reporter conjugates, and analyte analog: the analyte of reporter conjugates in sample mixes and flows until MIP zone 122, at this their competition analysis thing specific binding site.With the direct proportional quantitative analysis thing analogue of the concentration of specific analyte in the sample: the reporter conjugates not combined and to the device downstream further move.
Next one deck is porous support or upholder 88b, then be the layer of reporter-conjugates calmodulin binding domain CaM, this calmodulin binding domain CaM comprises having with drying regime, detectable, releasable second wedding agent: first calmodulin binding domain CaM of the reporter of the binding site that the reporter conjugates is saturated-conjugates binding member 124.This binding member is to being coupled to first wedding agent: the analyte and second wedding agent: the two its binding partners of reporter conjugates has specificity avidity.First wedding agent: the analyte conjugates to the avidity of the binding site of reporter-conjugates binding member 124 greater than avidity to the binding site of reporter-conjugates binding member 124.First calmodulin binding domain CaM of reporter in the reporter conjugates calmodulin binding domain CaM-conjugates binding member 124 is in conjunction with first wedding agent: the analyte conjugates, and with sample in the direct proportional amount of concentration of specific analyte replace second wedding agent: the reporter conjugates causes (replacement) or unconjugated (competition) second wedding agent of replacing: the reporter conjugates further continues migration to the device below.Alternatively, can use competition, and can be with second wedding agent before the calmodulin binding domain CaM of reporter-conjugates binding member 124: reporter conjugates drying, by sample liquids hydration again and with first wedding agent: the analyte conjugates is competed the binding site in first calmodulin binding domain CaM.In this case, do not need first wedding agent: analyte is to the bigger avidity of the binding site of reporter-conjugates binding member 124.Excessive unconjugated second wedding agent: the reporter conjugates will continue migrated downstream in the liquid-flow approach.
Next be second calmodulin binding domain CaM that is fixed on the reporter-conjugates binding member 126 on the porous support.Opening among the cover 80b is positioned at the front of reporter-conjugates binding member 126, comprises the results window 92b of device.When the liquid sample that contains analyte migrated to reporter-conjugates binding member 126, second calmodulin binding domain CaM of reporter-conjugates binding member 126 was in conjunction with second wedding agent from the unconjugated of analyte specificity MIP 122 or replacement; The reporter conjugates, and the existence that shows analyte in the sample or the detectable signal of concentration are provided in results window 92b.
Next zone is to comprise second wedding agent: three of reporter conjugates binding member discrete with reference zone nonoverlapping indication band, wherein 128a represents lower concentration with reference to band, and 128c represented that high density is with reference to band to concentration with reference to band during 128b represented.Transparent reference window 96b is positioned at the front with reference to band.The user comes explanation results by strength of signal among the visual comparison results window 92b and the band intensity among the reference window 96b.
Reference zone below is a control zone, and it comprises and has flooded unconjugated second wedding agent: the porous support 132 of reporter conjugates.Conjugates is an immobilized under drying regime, but when the porous support unrestricted flow that becomes during by the liquid wets of sample.Be downwards another reporter of visible-conjugates binding member 134 in contrast window 104b again.Filled up 132 second wedding agents that discharge by the liquid from sample from contrast: the reporter conjugates arrives reporter conjugates binding member 134 and also is incorporated in to carrier, produces optical signal in contrast window 104b.Far-end at device is absorbent material 106b, the continuous flow that it absorbs excess liquid and guarantees through-going device.
Embodiment 8: the embodiment of Fig. 4 B
The structure and the operation of the circulation device that has competition/pair replacement and the horizontal video picture of test result are described below.
Described circulation device identical with described in the top embodiment 7 is with the similar part of similar numeral indication, except the modification of reference number and additional letter " b " expression.Modify as follows: in the cover 108b of the device of Fig. 4 B, do not have reference zone.Only according to second wedding agent: the distance that the reporter conjugates is covered reads the result from results window.Fixed second wedding agent: reporter conjugates binding member 136 and results window 114b are extended.Scale 112b is parallel to fixed second wedding agent: reporter conjugates binding member 136 is placed, the two by common calibration with corresponding to the analyte concentration in the analyzed liquid sample.By in results window 114, seen by passing second wedding agent: second wedding agent of reporter conjugates binding member 136: the area that the reporter conjugates is covered is determined the existence and the concentration of analyte in the liquid.
Embodiment 9: the embodiment of Fig. 5
The structure and the operation of the combination transverse flow/circulation device that has competition and the vertical video picture of test result are described below.
Fig. 5 is used for detecting and the existence of the target analyte that definite liquid sample exists, does not exist or the side-view of the lateral flow device of concentration.Described device comprises the hollow solid cover 40 that contains specified porous pad; Sample pad 14c, with releasable first wedding agent: the conjugates pad 70 of analyte conjugates dipping, contain the MIP pad 72 of analyte specificity MIP and use second wedding agent: the reagent that the reporter conjugates floods is reported subpad 74, all pads are piled up in intimate contact, one above another, pad 74 closely contacts with solid porous upholder 12c.Pad that piles up and following calibration tape can be from transmit fluid samples wherein, and described sample can pass pad and the solid support on the liquid-flow approach moves by gravity and wicking action.Sample application pad 14c is arranged near cover 40 opening or the window 16c to be used for the using liquid sample.If target analyte is present in the liquid sample, passed conjugates pad 70 from sample application pad 14c carrying, impregnated releasable first wedding agent of liquid sample dissolving therein: the analyte conjugates, and also it mixes with analyte in the sample.When downward liquid flow when being positioned at the analyte specificity MIP in MIP zone 70, this two molecules competition analysis thing specific binding site.First wedding agent with the proportional amount of concentration of specific analyte in the sample: the analyte conjugates not combined and in the liquid-flow approach flow further downstream.Unconjugated first wedding agent: the analyte conjugates continues to flow to reporter reagent pad 74 with sample liquids, and liquid dissolves second wedding agent therein: the reporter conjugates.Contain unconjugated first wedding agent: the analyte conjugates and second wedding agent: the liquid flow of reporter conjugates is crossed pad 74 and is contacted solid porous upholder 12c, its with the further downstream transport of liquid flow to first reporter that comprises the reporter-conjugates binding member that is fixed on the solid support 12c-conjugates calmodulin binding domain CaM 54c.First wedding agent: the analyte conjugates and second wedding agent: the reporter conjugates is competed the binding site of first reporter-conjugates calmodulin binding domain CaM, stays (living) unconjugated second wedding agent: the reporter conjugates.Unconjugated second wedding agent: the reporter conjugates continues to move to liquid-flow approach downstream.
Further be at second calmodulin binding domain CaM that is fixed in the reporter-conjugates binding member 60c on the solid support 12c on the sample flow approach downstream on the solid support 12c.Second calmodulin binding domain CaM of reporter-conjugates binding member 66c in the cover 40 below the results window 60 is extended and comprises the scale 68c of second calmodulin binding domain CaM that is parallel to fixed reporter-conjugates binding member 66c, and described scale 68c is calibrated with corresponding to the analyte concentration in the analyzed liquid sample.By viewed by second wedding agent along second calmodulin binding domain CaM of reporter-conjugates binding member 66 in results window 60: the area that reporter-conjugates covered is determined the existence and the concentration of analyte in the liquid.
There is the corresponding contrast window 30c that is used for observations in the cover 40 above reporter-conjugates binding member 64c.Second wedding agent that sample liquids discharges from contrast pad 58c: the reporter conjugates arrives binding member 64c and becomes and is fixed in solid support 12c, is created in observed visual signal among the contrast window 30c.Contrast pad 58c and reporter-conjugates binding member 64c are configured for producing the control zone of positive control jointly.
Far-end at device is the absorption pad 36c that absorbent material is made, and it communicates by liquid with solid support 12c.Described spacer has enough porousness and ability to absorb excess liquid, and guarantees the continuous flow of through-going device.
Embodiment 10: material and method
Following material and method are used among the following embodiment.
A. porous solid support: the porous solid support that is used for quick diagnosis device of the present invention is the membrane filter that comprises the strong adsorption material with big surface-area, for example PuraBind TM(Whatman, USA), it is that tool is not made 100% nitrocellulose of aftertreatment.
B. represent the preparation of the synthetic peptide of the epi-position on the β subunit of TSH: the document of the binding site of the antibody from the β subunit of describing TSH (people such as Fairlie, 1995, Biochem.J.308 203-210) has obtained the aminoacid sequence (LSCKCGKCNTDY) of target epi-position.Use the 433A peptide synthesizer (Applied Biosystems, the U.S.) of automatization to prepare peptide.
The preparation of C.TSH specificity MIP: following according to Yan and Row (Int.J.Mol.Sci.2006,7, method listed in summary 155-178) prepares: with function monomer methacrylic acid (MAA) (catalog number (Cat.No.) 155721, Aldrich) and in this case be the target marking molecule of above-mentioned synthetic peptide, and with acetonitrile (catalog number (Cat.No.) 360457, Sigma-Aldrich) cross-linking monomer Ethylene glycol dimethacrylate (EGDMA) (the catalog number (Cat.No.) 33568-1 in 3% the water, Aldrich) together, with the startup factor 2,2 '-azo two (2, the different heptonitrile of 4-) (catalog number (Cat.No.) 002094, Chemos GmBH, Germany) is mixed together.With the mixture degassing and use nitrogen purging 5min, and after 40 ℃ times generation polymerizations 16 hours, obtain hard soluble polymkeric substance, and the specific binding cavity of TSH is present among the polymer network.In mechanical mortar, grind mass polymer (bulk polymer) and in water, carry out wet screening by 25 μ m sieves.By fully washing particle and extract marking molecule, dry and dry storage in a vacuum with polymer beads with methyl alcohol-acetic acid (9/1, volume ratio).
The preparation of D.TSH peptide-vitamin H conjugates: use PeptiTag-vitamin H test kit (BioSight, Israel) will synthesize peptide and be coupled to vitamin H.
The preparation in E.MIP-conjugates zone: at first the solution by the TSH specificity MIP particle that will sieve and TSH peptide-vitamin H conjugates is 37 ℃ of hatchings 24 hours down, with TSH peptide-conjugates (being peptide-vitamin H conjugates) that the binding cavity of TSH specificity MIP is saturated, then flushing is to remove excessive conjugates.In 60 ℃ of following baking ovens the MIP particle drying and the packing of flushing are advanced filter paper packet then, described filter paper packet is made by the Filtech Fabrics Ltd of India and to similar as those of tea bag.By film (the catalog number (Cat.No.) ARcare that has been coated with pressure sensitive adhesive 8570, Adhesives Research, the U.S.), will contain the MIP particulate bag of packing into and be connected on the nitrocellulose filter in the MIP-conjugates zone.
F. the preparation of reporter-conjugates: as reporter-conjugates molecule, use with the colloid gold particle coating be coupled to BSA (catalog number (Cat.No.) A3902,4-hydroxyazobenzene Sigma)-2-carboxylic acid (HABA) (catalog number (Cat.No.) 54791, Fluka).(Analytical Biochemistry (2000) 284, and 354-366) described method associates HABA to BSA according to people such as Hofstetter.The HABA azoic dyestuff is with Kd=10 -6The affinity costant of M is bonded to the vitamin H binding site of avidin.With vitamin H HABA is replaced from this binding site, described vitamin H has Kd=10 -15The affinity costant of M.Gold sol can obtain from the BioAssay Works MD of the U.S..Use Horrisberger and Clerc (Histochemistry, 1985,82,219-223A) described couling process carries out the loading of gold sol to the HABA-BSA conjugates.
G. for two zones of containing vitamin H-binding member, all preferably use NeutrAvidin TMVitamin H-conjugated protein (Pierce, the U.S.).When non-specific binding must be minimized, this albumen was the outstanding surrogate of other biological element-conjugated protein for example avidin or streptavidin.By the direct trace of NeutrAvidin is carried out the fixing of it to nitrocellulose filter.The NeutrAvidin that at room temperature will contain 20 μ g/ml in the 0.5M phosphate buffered saline buffer of pH 7.2 of the 0.5M NaCl hatching of spending the night, once also air-dry with the PBS flushing.At room temperature with the preparation the film stored dry until the device final structure.
H. by using EZ-Link sulfo group-NHS-biotin labeling test kit (Pierce, the U.S.) that the β subunit that vitamin H is connected to TSH is finished the TSH-reporter: the preparation of binding partners conjugates (vitamin H-TSH-gold).Then with gold sol coating TSH-vitamin H conjugates.As early as described in gold sol coating BSA, finishing this coating.When existing with the form of moving freely, the NeutrAvidin that this conjugates is fixed catches, and owing to the gold grain that covers this molecule obtains optical signal.
The preparation of I.TSH-reporter conjugates calmodulin binding domain CaM: TSH-reporter conjugates calmodulin binding domain CaM comprises the zone on the nitrocellulose solid support with the NeutrAvidin (as described in top relevant vitamin H-binding member zone) that is fixed on above it.
J. the preparation of reference zone: reference zone preferably includes five identical parallel bands of TSH-reporter conjugates calmodulin binding domain CaM.The solution of vitamin H-TSH-gold conjugates accurately is applied to each band.At last, five bands have the conjugates of 0.2,1.0,3.0,5.0 and the 7.5mIU/L that are bonded to them respectively.By comparing with the band colour intensity in the results window and with reference to band, the user can determine the scope of the amount of TSH in the sample.
Embodiment 11: use the mensuration of lateral flow device (two replacement) to TSH
Structure and operation that specific replacement of the present invention is measured are described below.
Structure:
Device shown is used to the TSH the liquid sample that obtains from the people is carried out quantitatively among Fig. 2 A.This device comprises a cover, and the zone that described cover contains solid support exposes the window that is used to observe.This device comprises the sample application zone of containing a pad (LF1, Whatman, the U.S.), to the liquid sample of described pad introducing whole blood, with described sample liquids and porous-film (PuraBind TM, Whatman, the U.S.) solid support (calibration tape) contact.This pad is designed to make blood plasma to separate with hemocyte.Solid support comprises definite MIP-conjugates zone, and it comprises the TSH specificity MIP that peptide-the vitamin H conjugates is saturated with TSH-.The downstream in MIP-conjugates zone is reporter-conjugates calmodulin binding domain CaM, and it comprises the NeutrAvidin with the HABA dipping that is coupled to BSA that has been coated with gold sol.Being to have the results area that comprises fixed NeutrAvidin of the concentration of MMA in the sample being carried out quantitative auxiliary scale with being used as again toward the downstream, next is to comprise the contrast pad of exsiccant HABA-BSA-gold (reporter-conjugates) and comprise the control zone that is fixed on the NeutrAvidin on the solid support.
Operation:
In order to begin to measure, a whole blood is applied to the sample application zone.The blood plasma that leaves the sample application zone contacts nitrocellulose filter and mobile to contact by the TSH specificity MIP zone of TSH-peptide-vitamin H conjugates dipping along device by wicking action.Along with the front of liquid moves through MIP-conjugates zone, be present in TSH in the sample to replace TSH-peptide-vitamin H conjugates molecule from MIP with the proportional amount of its concentration.
TSH-peptide-vitamin H conjugates molecule flow further downstream in liquid of replacing arrives reporter-conjugates calmodulin binding domain CaM, and it comprises the HABA-BSA-gold reporter conjugates that is fixed in NeutrAvidin.Because the higher avidity of vitamin H, the vitamin H of TSH-peptide-vitamin H conjugates is to replace reporter-conjugates from NeutrAvidin with the proportional amount of the concentration of TSH-peptide-vitamin H conjugates of replacing.
The further migrated downstream of reporter-conjugates of replacing contacts with NeutrAvidin binding member in the results area up to it.What be parallel to the NeutrAvidin binding member is with reference to scale, and it is titrated to allow to explain along the binding member progressive distance of results window by definite reporter-conjugates the amount of TSH in the sample.Optical signal is significantly in results area, allows the user to determine the amount of TSH in the blood sample.About 15 minutes definite results after application of samples, this is all component of the assurance device time necessary of suitably working.
After having crossed results area, liquid sample continues laterally to cross over the contrast pad and moves, with the reporter-conjugates that is immersed in the contrast pad is that the HABA-BSA-gold ribbon advances along the free-pouring state of device, and until the NeutrAvidin band that is arranged in control zone, it is captured at this.Cross over whole control zone and form the visible line, show that mensuration suitably works.Excess liq and reagent will continue laterally, and crossover device moves and is collected in the absorption pad.
Embodiment 12: use the mensuration of diagnostic circulation device (two replacement) to TSH
Structure and operation (pillar combination) that specific replacement of the present invention is measured are described below.
Structure:
Device shown is used to the TSH the liquid sample that obtains from the people is carried out quantitatively among Fig. 4 B.This device comprises a cover, and the zone that described cover contains solid support exposes the window that is used to observe.This device comprises containing and is designed to make blood plasma and the isolating pad (LF1 of hemocyte, Whatman, the U.S.) sample application zone is to the liquid sample of described pad introducing whole blood, with described liquid sample and the porosu solid upholder (Sigmacell that contains the pearl of unmodified
Figure BPA00001204794800461
Mierocrystalline cellulose, 50 types, catalog number (Cat.No.) S5504 Sigma) the zone contact.
Further be MIP-conjugates zone downstream, it comprises the usefulness TSH-TSH specificity MIP particle of packing that peptide-the vitamin H conjugates is saturated in the solid support zone of the below that is positioned at the physics contact.What further contact with MIP-conjugates zone physics downstream is reporter-conjugates calmodulin binding domain CaM, it comprises the biological plain fixed agarose pearl (catalog number (Cat.No.) VIT-H-4S, Affiland S.A. Belgium) of the Mierocrystalline cellulose of packing with the saturated NeutrAvidin coating of HABA-BSA-gold.What contact with the cellulose bead muon physics toward the downstream is results area again, and it comprises the vitamin H fixed agarose pearl with the NeutrAvidin that the packs coating of the region separation of the pearl of unmodified.The chromatogram of passing the molecule of pearl be flowing in help in the liquid to form analyte clearly with spissated zone.The visible scale is parallel to results area so that be used as and the concentration of TSH in the sample carried out quantitative auxiliary in viewing window, next is the control zone that comprises the contrast pad of exsiccant HABA-BSA-gold (reporter-conjugates) and comprise the vitamin H fixed agarose pearl of NeutrAvidin coating.Introduce sample in sample application zone and in device after the analytic sample, optical signal is significantly, allows the user to determine the amount of TSH in the blood sample, and confirms the proper property of device in results area and control zone.
Operation:
In order to begin to measure, a whole blood is applied to the vertical sample application of device zone.The blood plasma that leaves the sample application zone flows with contact by the TSH specificity MIP zone of TSH-peptide-vitamin H conjugates dipping along device by wicking action.Along with the front portion of liquid moves through MIP-conjugates zone, be present in TSH in the sample to replace TSH-peptide-vitamin H conjugates molecule from MIP with the proportional amount of its concentration.
TSH-peptide-vitamin H conjugates molecule of replacing flows below device in liquid, arrives reporter-conjugates calmodulin binding domain CaM.Because the higher avidity of vitamin H, the vitamin H of TSH-peptide-vitamin H conjugates is to replace reporter conjugates from NeutrAvidin with the proportional amount of the amount of TSH-peptide-vitamin H conjugates of replacing.
The reporter conjugates of replacing contacts with the plain pearl of the NeutrAvidin coated fibres in the results area up to it to the migration of device below.What be parallel to the binding member that contains NeutrAvidin is with reference to scale, and it is titrated to allow by determining that reporter-conjugates explains the amount of TSH in the sample along the binding member progressive distance of results window.About 15 minutes definite results after application of samples, this is all component of the assurance device time necessary of suitably working.
After having crossed results area, liquid sample continues to be moved downward to the contrast pad, with the unconjugated reporter-conjugates that is immersed in the contrast pad is the free-pouring state in HABA-BSA-gold ribbon heading device below, until the pearl of the NeutrAvidin coating that is arranged in control zone.Cross over whole control zone and form the visible line, show that mensuration suitably works.Excess liq and reagent will continue to move and be collected in the absorption pad to the device below.
Embodiment 13: the fluorescence replacement mensuration test kit that is used for determining Type B natriuresis (naturietic) peptide (BNP)
Structure:
This device comprises portable detecting unit.For convenience, this description will be referred to the general structure of the lateral flow device described in the embodiment 1-4, should be understood that through a small amount of modification, and this device can adopt the form of the circulation device described in tool such as the embodiment 5-8.
This device comprises and contains the sample application zone that is designed to make blood plasma and the isolating pad of hemocyte (LF1, Whatman, the U.S.), introduces the liquid sample of whole blood to described pad, and described liquid sample is contacted with nitrocellulose filter.MIP-conjugates zone (surveyed area) comprises BNP-peptide-specificity MIP that peptide-the vitamin H conjugates is saturated with BNP-.Reporter-conjugates is the HABA-BSA (HABA-BSA-FLUROFOR) that has been coated with luminescent dye molecule.The reporter molecule of reporter-conjugates is fluorescence dye Alexa Fluor 488 (Invitrogen, the U.S.), and by means of handheld portable fluorometric assay reader (ESE; Stockach, Germany) finish determining of result.This device is equipped with a cover that is designed to be fit to the fluorescence reader, will align with the fluorescence detection unit of reader corresponding to the zone of results area in the visual device in this way.
Operation:
The device that is used for detecting BNP is used to monitor existence and the concentration of serum BNP.Before using serum sample, with the device reader of packing into, it activates instrument and brings it into stand-by station (have only suitable packing into just to activate instrument, otherwise error message occurs).Sample application triggers instrument and carries out countdown, and it causes the measurement of fluorescence intensity about 15 minutes sample application after, and this is all component of guaranteeing mensuration time necessary of suitably working.
BNP in the sample replaces BNP-peptide-vitamin H from BNP-peptide-specificity MIP, and G-30027-vitamin H of replacing is replaced the HABA-BSA-fluor dyestuff among the NeutrAvidin from reporter-conjugates calmodulin binding domain CaM again.Reporter-the conjugates migrated downstream is until results area, and it is caught by NeutrAvidin therein.Determine the amount of BNP in the sample by the signal of the signal that relatively from sample, obtains by the fluorescence reader and internal calibration curve.The result is displayed on the LCD of instrument with pg/ml (limit of detection is 10pg/ml).
Embodiment 14: use the mensuration of lateral flow device (competition/replacement) to MMA
Structure and operation that specific replacement of the present invention is measured are described below.
Structure:
Device shown is used to the MMA the liquid sample that obtains from the people is carried out quantitatively among Fig. 2 B.This device comprises a cover, and the zone that described cover contains solid support exposes the window that is used to observe.This device comprises the sample application zone of the pad (LF1, Whatman, the U.S.) that contains useful MMA-vitamin H conjugates dipping, to the liquid sample of described pad introducing whole blood, with described sample liquids and porous-film (PuraBind TM, Whatman, the U.S.) solid support (calibration tape) contact.This pad is designed to make blood plasma to separate with hemocyte.Solid support comprises the MIP zone of determining of containing MMA specificity MIP.The downstream in MIP zone is reporter-conjugates calmodulin binding domain CaM, and it comprises the NeutrAvidin with desthiobiotin (DTB) dipping that is connected to painted polystyrene pearl (K1-030bleu (39457), Merck-Estapor, France).Being to have the results area that comprises fixed NeutrAvidin of the concentration of MMA in the sample being carried out quantitative auxiliary scale with being used as again toward the downstream, next is to comprise the contrast pad of exsiccant DTB-pearl (reporter-conjugates) and comprise the control zone that is fixed on the NeutrAvidin on the solid support.
Operation:
In order to begin to measure, a whole blood is applied to the MMA-vitamin H conjugates of sample application zone and dissolving dipping.Leave the blood plasma contact nitrocellulose filter that contains conjugates in sample application zone, and flow with contact MMA specificity MIP zone along device by wicking action.Along with the front portion of liquid moves through MIP-conjugates zone, the MMA specific binding site that is present in MMA in the sample and MMA-vitamin H conjugates molecule competition MIP, and since MMA to the good avidity in these sites, not combined with the conjugates of the proportional amount of concentration of MMA in the sample.
Unconjugated MMA-vitamin H conjugates molecule flow further downstream in liquid arrives reporter-conjugates calmodulin binding domain CaM, and it comprises the DTB-pearl reporter-conjugates that is fixed in NeutrAvidin.Because the higher avidity of vitamin H, the vitamin H of MMA-vitamin H conjugates is to replace reporter-conjugates from NeutrAvidin with the proportional amount of the amount of the MMA-vitamin H conjugates of replacing.The further migrated downstream of reporter-conjugates of replacing contacts with NeutrAvidin binding member in the results area up to it.What be parallel to the NeutrAvidin binding member is with reference to scale, and it is titrated to allow to explain along the binding member progressive distance of results window by definite reporter-conjugates the amount of MMA in the sample.Optical signal is significantly in results area, allows the user to determine the amount of MMA in the blood sample.About 15 minutes definite results after application of samples, this is all component of the assurance device time necessary of suitably working.
After having crossed results area, liquid sample continues laterally to cross over the contrast pad and moves, with the reporter-conjugates that is immersed in the contrast pad is that the DTB-pearl is brought into along the free-pouring state of device, and until the NeutrAvidin band that is arranged in control zone, it is captured at this.Cross over whole control zone and form the visible line, show that mensuration suitably works.Excess liq and reagent will continue laterally, and crossover device moves and is collected in the absorption pad.
Everybody understands, and provides in order to know that purpose also can be combined in some feature of the present invention described in the context of the embodiment of separating in independent embodiment.On the contrary, purpose is also closed discriminably or with any suitable subgroup in the of the present invention various features described in the context of independent embodiment and is provided for simplicity.
After the embodiment that does not expect to be limited below having checked, another object of the present invention, advantage and new feature will become obvious for those of ordinary skills.In addition, the various embodiments of the present invention for required protection and each embodiment in the aspect and aspect all find experiment support in the following embodiments in the above-mentioned claim part that describe and following of this paper, and its expectation illustrates but do not limit the present invention.
Though described the present invention in conjunction with its specific embodiment, be apparent that many replacement schemes, modification and variation will be significantly for those skilled in the art, for example add the condition that reagent is controlled mensuration, for example pH.Therefore, the spirit that falls into claims and all this replacement schemes, modification and the variation within the wide region are contained in expectation.

Claims (35)

1. the method for at least a analyte in the tracer liquid sample, described method comprises:
A) provide diagnostic device, described diagnostic device comprises: be fixed in the molecularly imprinted polymer with analyte specific binding site of solid support, wherein said liquid sample can along along or the flowpaths that passes described solid support move; And the sample application zone that described liquid sample is applied to described device;
B) described liquid sample is applied to described sample application zone and allow this sample along or pass described solid support and flow, and contact with described molecularly imprinted polymer; And
C) detect combining of described analyte and described molecularly imprinted polymer.
2. diagnostic device that is used at least a analyte of tracer liquid sample, described device comprises:
A) be fixed in the molecularly imprinted polymer with analyte specific binding site of solid support, wherein said liquid sample can move along the flowpaths along described solid support;
B) be used for sample application zone that described liquid sample is applied to described device and described liquid sample is contacted with described molecularly imprinted polymer; And
C) be used to detect the bonded surveyed area of described analyte and described molecularly imprinted polymer.
3. according to claim 1 or 2 each described method or devices, wherein said device comprises lateral flow device, circulation device or made up the device of transverse flow element and flow-through element, wherein said liquid sample from described sample application zone along the flowpaths of device along or the described solid support assembly that passes device flow.
4. according to each described method or device in the claim 1 to 3, wherein said analyte is replaced the analyte analog that is bonded to described molecularly imprinted polymer releasedly: the reporter conjugates, wherein said analyte analog: the replacement of reporter conjugates shows the existence of analyte described in the sample.
5. method according to claim 4 or device, wherein releasable analyte analog: the reporter conjugates is incorporated in to described molecularly imprinted polymer,
Wherein said analyte equals described analyte analog at least to the avidity of the described binding site of described molecularly imprinted polymer: the reporter conjugates is to the avidity of the described analyte specific binding site of described molecularly imprinted polymer,
Wherein when with described molecularly imprinted polymer when described analyte in the described liquid sample contacts, the combined and described analyte analog of described analyte: the reporter conjugates is replaced,
Described device also comprises results area, described results area is included in the analyte analog that is fixed in described solid support on the described flowpaths of sample: reporter conjugates binding member, described reporter conjugates binding member can be in conjunction with the analyte analog of described replacement: the reporter conjugates
Wherein said analyte analog: the concentration of analyte is proportional described in the replacement of reporter conjugates and the described liquid sample, and make the bonded of described analyte and described molecularly imprinted polymer detect the analyte that comprises described replacement: reporter conjugates and described analyte analog: the described bonded of reporter conjugates binding member detects.
6. according to each described method or device in the claim 1 to 3, wherein said analyte is replaced first wedding agent that is bonded to described molecularly imprinted polymer releasedly: the analyte conjugates,
First wedding agent of wherein said replacement: the analyte conjugates is replaced second wedding agent that is bonded to described solid support releasedly: the reporter conjugates,
Wherein said second wedding agent: the replacement of reporter conjugates shows the existence of analyte described in the sample.
7. method according to claim 6 or device, wherein releasable first wedding agent: the analyte conjugates is incorporated in to described molecularly imprinted polymer,
Wherein said analyte equals described first wedding agent at least to the avidity of the described binding site of described molecularly imprinted polymer: the analyte conjugates is to the avidity of the described analyte specific binding site of described molecularly imprinted polymer,
Wherein when with described molecularly imprinted polymer when described analyte in the described liquid sample contacts, combined and described first wedding agent of described analyte: the analyte conjugates is replaced,
Described device also comprises:
I) reporter-conjugates calmodulin binding domain CaM, it is included in the reporter-conjugates binding member that is fixed in described solid support on the described flowpaths of liquid sample, described reporter-conjugates binding member has combination detectable, releasable second wedding agent thereon: the binding site of reporter conjugates
Wherein said first wedding agent: the analyte conjugates equals described second wedding agent at least for the avidity of the described analyte specific binding site of described reporter conjugates binding member: the reporter conjugates is to described reporter: the avidity of the described binding site of conjugates binding member, wherein said first wedding agent: second wedding agent is replaced in the combination of analyte conjugates: reporter conjugates, and wherein said second wedding agent: the concentration of analyte is proportional described in the replacement of reporter conjugates and the described liquid sample; With
Ii) results area, it is included in second wedding agent that is fixed in described solid support on the described flowpaths of sample: reporter conjugates binding member, described reporter conjugates binding member can be in conjunction with second wedding agent of described replacement: the reporter conjugates
The bonded of wherein said analyte and described molecularly imprinted polymer detects and comprises described second wedding agent: reporter conjugates and described analyte analog: the described bonded of reporter conjugates binding member detects.
8. according to each described method or device in the claim 1 to 3, comprise analyte analog: the reporter conjugates, wherein said analyte and described analyte analog: the reporter conjugates is competed the described analyte specific binding site of described molecularly imprinted polymer
Wherein unconjugated analyte analog: the existing detected and show the existence of analyte in the sample of reporter conjugates.
9. method according to claim 8 or device, comprise analyte analog: the reporter conjugates, wherein said analyte equals described analyte analog at least to the avidity of the described analyte specific binding site of described molecularly imprinted polymer: the reporter conjugates is to the avidity of the described analyte specific binding site of described molecularly imprinted polymer
Wherein when with described molecularly imprinted polymer when described analyte in the described liquid sample contacts, described analyte and described analyte analog: the reporter conjugates is competed the described binding site of described molecularly imprinted polymer,
Described device also comprises results area, described results area comprises the analyte analog that is incorporated into described solid support: reporter conjugates binding member, described reporter conjugates binding member can be in conjunction with described unconjugated analyte analog: the reporter conjugates also provides detectable signal, the concentration of analyte described in the described liquid sample of described detectable signal indicating.
10. according to each described method or device in the claim 1 to 3, comprise first wedding agent: analyte conjugates, second wedding agent: the reporter conjugates and second wedding agent: acceptor conjugates binding member,
Wherein said analyte and described first wedding agent: the analyte conjugates is competed the described analyte specific binding site of described molecularly imprinted polymer, make described first wedding agent of at least a portion in the presence of described analyte: the analyte conjugates is unconjugated
Wherein said unconjugated first wedding agent: analyte conjugates and described second wedding agent: reporter conjugates competition is for described second wedding agent: the combining of acceptor conjugates binding member.
11. method according to claim 10 or device, comprise first wedding agent: the analyte conjugates, wherein said analyte equals described first wedding agent at least to the avidity of the described analyte specific binding site of described molecularly imprinted polymer: the analyte conjugates is to the avidity of the described analyte specific binding site of described molecularly imprinted polymer
Wherein when with described molecularly imprinted polymer when described analyte in the described liquid sample contacts, described analyte and described first wedding agent: analyte conjugates analyte is competed the described analyte specific binding site of described molecularly imprinted polymer, and
Wherein said unconjugated first wedding agent: the analyte conjugates flows in described liquid sample flow approach,
Described device also comprises:
I) second wedding agent: the reporter application region, it comprises second wedding agent of drying regime: the reporter conjugates;
The ii) reporter conjugates calmodulin binding domain CaM in downstream, it is included in the reporter-conjugates binding member that is fixed in described solid support on the described flowpaths of liquid sample,
Wherein said first wedding agent: the analyte conjugates equals described second wedding agent at least to the avidity of described reporter-conjugates binding member: the reporter conjugates is to the avidity of described binding site,
Wherein when with described exsiccant second wedding agent: reporter conjugates and described unconjugated first wedding agent: when the analyte conjugates contacts, described second wedding agent: reporter conjugates and described unconjugated first wedding agent: the competition of analyte conjugates combines with described reporter-conjugates binding member
Wherein said unconjugated second wedding agent: the reporter conjugates is flow further downstream in the described flowpaths of liquid sample; And
Iii) results area, it comprises and described solid support bonded second wedding agent: reporter conjugates binding member, described reporter-conjugates binding member can be in conjunction with described unconjugated second wedding agent: the reporter conjugates also provides detectable signal, the concentration of analyte described in the described liquid sample of described detectable signal indicating.
12. according to each described method or device in the claim 1 to 3, comprise first wedding agent: analyte analog, wherein unconjugated first wedding agent: analyte analog by with analyte to the competition of the binding site of molecularly imprinted polymer and by at least a produce of described analyte in the replacement of described molecularly imprinted polymer;
Described device also comprises:
Second wedding agent: reporter conjugates binding member, wherein unconjugated second wedding agent: the reporter conjugates by with described first wedding agent: the competition of analyte conjugates and by described first wedding agent: at least a generation in the replacement of analyte conjugates, wherein unconjugated second wedding agent: the existence of reporter conjugates shows the existence of analyte in the sample.
13. method according to claim 12 or device, wherein said first wedding agent: analyte analog is provided in described sample application zone, perhaps is incorporated in to described molecularly imprinted polymer;
Wherein said reporter conjugates binding member is fixed in described solid support on the described flowpaths of liquid sample, and described second wedding agent: the reporter conjugates is provided in reporter conjugates application region, perhaps be bonded to described reporter conjugates binding member releasedly
Described device also comprises results area, described results area comprises second wedding agent that is bonded to described solid support: reporter conjugates binding member, described reporter-conjugates binding member can be in conjunction with described unconjugated second wedding agent: the reporter conjugates also provides detectable signal, the concentration of analyte described in the described liquid sample of described detectable signal indicating.
14. according to each described method or device in the claim 4 to 13, at least a vitamin H that comprises in wherein said reporter conjugates or the described wedding agent conjugates, and wherein said binding member is selected from the group of being made up of avidin, NeutrAvidin and Extravidin.
15. according to each described method or device in the claim 5,7,9 or 10, wherein said reporter-conjugates-binding member flatly is fixed in the sample fluid flow approach on the described solid support,
Wherein said unconjugated reporter conjugates makes the described binding site of described reporter-conjugates binding member saturated,
Wherein said result be rasterized be advancing post, this post have with sample in the proportional length of amount of analyte.
16., also comprise at least one in following: positive control area according to each described method or device in the claim 5,7,9,11 or 13; Reference zone; The absorption region in the described results area downstream on the described flowpaths of liquid sample, described absorption region comprises the absorbent material that can absorb liquid sample; And comprise the shell that aligns with in results area, reference zone and the control zone at least one with at least one window of allowing the test result on the observing device.
17. according to claim 17 each described method or device, wherein the strength of signal in strength of signal in the described results area and the described reference zone is compared, described device is arranged and is configured to provide positive findings to show the concentration of analyte in the sample at analyte when threshold level exists to be equal to or higher than.
18. according to each described method or device in the claim 5,7,9,11 or 13, also comprise reference zone, wherein said reference zone comprises at least one discrete bands of binding member, described discrete bands comprises the described analyte analog of known quantity: the reporter conjugates, wherein the existence of sample target test thing or do not exist is to determine by the described strength of signal in strength of signal in the more described reference zone and the described results area.
19. according to claim 5,7,9,11 or 13 described method or devices, wherein said device lacks reference zone, wherein said results area also comprises and fixed reporter-parallel scale of conjugates binding member, described scale is calibrated with the analyte concentration in the analyzed liquid sample of correspondence, and wherein by by along with pass reporter in the results area-area that reporter-conjugates covered of conjugates binding member and determine the existence and the concentration of analyte in the liquid.
20. according to each described method or device in the claim 1 to 19, wherein said sample is selected from the group of being made up of the byproduct of biological liquid, environmental sample, food, toxin, production piece and industrial production step.
21. method according to claim 20 or device, wherein said analyte is selected from by cell, organism, small molecules, protein, hormone, enzyme, biomarker, the meta-bolites of biomarker, the meta-bolites of medicine, medicine, drug metabolite medicine-protein-coupled thing, drug metabolite-protein-coupled thing, VITAMIN, Drug abuse, natural or synthetic toxin, chemistry or biological warfare agent, the antibody of medicine, the antibody of infectious agent, environmental pollutant, immunoglobulin (Ig), lymphokine, cytokine, the solubility cancer antigen, somatomedin, neurotransmitter, the molecule that shows food safety or quality, the operating chemical product, the byproduct of production process, sterilant, insecticide, weedicide, fertilizer, tensio-active agent, tackiness agent and be used to make food, the group that the agent of industrial agent or chemical products is formed.
22. according to claim 21 each described method or device, wherein said analyte is selected from by MMA, homocysteine, troponin subunit I or T, CKMB, LDH and GOT/SGOT, citrozine, acetaminophen, Carbamzepine, paraxin, digitoxin, digoxin, gentamicin, kantlex, lignocaine, lithium, methotrexate, phenylethyl barbituric acid, Proprasylyte, Quinidine, theophylline, BNP, FSH, cumadine, vitamin K, aflatoxin, Ricin, 2, the 4-dichlorphenoxyacetic acid (2-4, D), atrazine, alachlor, bladex, metolachlor, simazine, suprarenin, Amphetamine, TSH, free T3 T4, PSA, steroid, estradiol, progesterone, testosterone, oestrogenic hormon, arsenic, prussiate, Strychnine, avian influenza virus, HIV virus, hepatitis virus, poliovirus, cytomegalovirus, dengue virus, Ebola virus, simplexvirus, Measles virus, EBV, rabies virus, respiratory syncytial virus, rotavirus, SARS virus, west Nile virus, yellow fever virus, campylobacter, chlamydiaceae, cholera, genus clostridium, diphtheria, intestinal bacteria, eisseria, Helicobacterium, hemophilus, Mycobacterium, Staphylococcus, Whooping cough, salmonella, Shigella, streptococcus, treponema, tetanus, Yersinia, staphyloentero-toxin, staphylococcal enterotoxin B, Ricin, Toxins, botulin, mycotoxin, tetanus toxin, Toxins,exo-, cholera, the trichothecene mycotoxin, transmissible disease vehicle there are specific antibody and the autoantibody relevant with autoimmune disorders, cancer cells, the cell of virus infection, virus, the group that bacterial cell and fungal cell are formed.
The group that 23. method according to claim 5 or device, wherein said releasable first wedding agent are selected from is conjugated protein by vitamin H, vitamin H analogue, biotin derivative, antigen, albumin A and Protein G, Mierocrystalline cellulose, hormone, toxin, lipid, lipid acid, nucleic acid, sugared conjugates, lectin, substrate and part are formed.
The group that 24. method according to claim 5 or device, wherein said releasable second wedding agent are selected from is conjugated protein by vitamin H, vitamin H analogue, biotin derivative, HABA, DTB, antigen, albumin A and Protein G, Mierocrystalline cellulose, liposome, hormone, toxin, lipid, lipid acid, nucleic acid, sugared conjugates, lectin, substrate and part or their analogue are formed.
25. according to each described method or device in the claim 1 to 24, but wherein said reporter is selected from the group of being made up of HABA, dyestuff, fluorescent agent, fluorescence dye, radio-labeling, magnetic-particle, metallic particles, semiconductor grain, quantum dot, coloured particle, fluorescent grain, metal-salt, enzyme substrates, enzyme, chemoluminescence agent, sensitizer and suspended particle.
26. method according to claim 5 or device, wherein said first wedding agent is identical or different with described second wedding agent, and be selected from separately by the avidin, streptavidin, NeutrAvidin, the ExtrAvidin compound that have the high specific avidity of vitamin H, film, acceptor, immunoglobulin (Ig), Mierocrystalline cellulose, enzyme, lectin, the sugar conjugates, complementary nucleic acid and have the group that the hydrophobic site of the high-affinity of they corresponding binding partners is formed.
27. according to each described method or device in the claim 1 to 26, wherein said solid support is selected from the group of being made up of porous material, porous-film, granulate material and absorbent material.
28. according to each described method or device in the claim 1 to 27, wherein said molecularly imprinted polymer comprises the polymkeric substance that is selected from by the following group of forming: in the presence of target analyte or target analyte-conjugates and pore former with linking agent with one or more are monomer crosslinked and the polymkeric substance that polymerization obtains, described polymkeric substance has the ability of selective binding target analyte and its conjugates; The polymkeric substance that polymerization obtains in the presence of MMA, described polymkeric substance has the ability of selective binding MMA; The synthetic peptide that contains amino acid LSCKCGKCNTDY or represent TSH the β subunit epi-position peptide in the presence of polymerization obtains in pore former polymkeric substance, described polymkeric substance has the ability of selective binding TSH.
29. the method for the TSH that exists in the tracer liquid sample, described method comprises the steps:
A) in each described device in sample and the claim 5,7,9,11,13 any contacted, and
B) in results area, detect the amount of the reporter conjugates be bonded to reporter-conjugates binding member, the amount of wherein said reporter-conjugates for the existence of TSH in the sample or amount be have tell-tale;
C) diagnosis hypothyroidism or hyperthyroidism illness.
30. a test kit comprises:
A. according in the described device of claim 1-3 any;
B. randomly, be used to extract or processed sample with one or more reagent or the composition of elution analysis thing;
C. randomly, one or more thinners; And
D. randomly, be used to put into practice and detect and the existence of definite liquid sample target test thing, do not exist or the specification sheets of the method for concentration.
31. according to each described method or device in the claim 1 to 30, the described detection of wherein said analyte comprises uses the auxiliary detection apparatus, and wherein said detector means is selected from the group of being made up of fluorescence inductor block, Magnetic Induction device, chemoluminescence inductor block, densometer, image dissector and radioactivity inductor block.
32. according to each described method or device in the claim 1 to 31, wherein said sample is pretreated to modify the characteristic of sample.
33. method according to claim 32 or device, wherein said pre-treatment are selected from by dilution, filter, distillation, concentrate, deactivation and the interpolation of reagent or the group that its combination is formed of interfering component.
34. method according to claim 16 or device, wherein said device comprise at least one porous pad in the one or more zones that place described sample application zone, described results area, described reference zone, control zone or described absorption region.
35. method according to claim 34 or device, the pretreated flow velocity that passes device with promotion or control sample of wherein said pad.
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JP2011508233A (en) 2011-03-10
WO2009083975A3 (en) 2010-03-11

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