CN101947235A

CN101947235A - The medical usage of conjugated polyelectrolytes

Info

Publication number: CN101947235A
Application number: CN2010102455198A
Authority: CN
Inventors: P·奥斯伯格; O·英格纳斯; F·安格斯
Original assignee: BIO CLAU MIX PHARMACEUTICAL Ltd
Current assignee: BIO CLAU MIX PHARMACEUTICAL Ltd
Priority date: 2007-04-18
Filing date: 2007-04-18
Publication date: 2011-01-19

Abstract

The present invention relates to the purposes of a kind of conjugated polyelectrolytes as novel treatment, its by disturbing misfolding, pathology or inferior strain form in vivo proteinic formation or capture it and work.

Description

The medical usage of conjugated polyelectrolytes

The application is that December in 2009 entered China on the 18th, international filing date is on April 18th, 2007, application number is 200780053410.0 (international application no is PCT/SE2007/050253), and denomination of invention is divided an application for the application of the protein of binding of pathological forms " use conjugated polyelectrolytes ".

Technical field

The present invention relates to conjugated polyelectrolytes and be used for following purposes: alternatively under condition optionally, separate specifically, for example capture protein misfolding, pathology or inferior strain (rogue) form, be that the protein of amyloid form, misfolding form or aggregated forms and capturing causes similarly at the accumulative protein of the not accumulative proteinic anomaly pattern of its normal form.The invention still further relates to the proteinic one step process that separates simultaneously and detect misfolding, pathology or inferior strain form.Further embodiment of the present invention relates to the purposes of conjugated polyelectrolytes as novel treatment, its by disturbing misfolding, pathology or inferior strain form in vivo proteinic formation or capture it and work.

Background technology

Can capture protein misfolding or aggregated forms by selectivity, the particularly proteinic material and the molecule of amyloid form and pathological forms, because it has as the analytical tool in clinical chemistry, the diagnosis and the potential of therapeutic agent, its development has caused great concern.Usually, with micromolecule dyestuff such as Congo red and thioflavin T staining starch sample fibril.The disadvantage of such molecular dye is to separate (separate), promptly in conjunction with, capture or emanate (isolate) goes out described misfolded proteins matter and simultaneously to its detection.

The method that a kind of protein that is used to measure amyloid, misfolding or pathological forms exists makes sample carry out Proteolytic enzyme a period of time, for example carry out Proteolytic enzyme with E.C. 3.4.21.64, the described time is enough to destroy native protein, then, use does not have antibody selective for amyloid, misfolding or pathological forms protein in the presence of native protein, carry out immunoassay, to measure existing of described misfolding formal protein.If use protease removes the native protein in comprising amyloid, misfolding or the proteinic sample of pathological forms, this will stop during the Proteolytic enzyme step and use antibody as capturing agent or detection agent, because in the presence of protease, antibody naturally can be destroyed.Therefore, before introducing antibody, must remove or the described protease of deactivation.When in the presence of native protein, capturing and detect amyloid, misfolding or during pathological forms protein, prevent this be limited in take place in the described process extremely beneficial.

Natural biological copolymer such as protein have orderly configuration usually, and as alpha-helix and beta sheet, it helps the ordered 3 D structure and the specific function of described biopolymer.Proteinic structure is that proteinic function is necessary; Many scientists have pointed out that not folding protein may not have function.The more important thing is, in recent years, recognize that gradually protein misfolding and misloading are made into for example danger of amyloid and other pathological forms.Misfolding may become non-functional, deleterious even deleterious from a kind of useful thing protein.Health relies on correct folding protein, the disease association of sedimentation and numerous protein configuration in the body of amyloid fibrils, described disease comprises Alzheimer, Huntington's disease, system's amyloidogenic disease (systemic amyloidoses) and prion disease.Prion disease, i.e. animal [for example bovine spongiform encephalopathy (BSE), the overworked disease of sheep scabies and Chronic consumptions (CWD)] and human [Creutzfeldt-Jakob disease (CJD), Jie Ciman-Si Tuosile-Shi Yinke (Gerstmann- -Scheinker) disease (GSS), Kuru disease] in infectiousness spongiform encephalopathy (TSE), with normal cell prion protein (PrP ^c) configuration conversion become to be expressed as PrP ^ScThe relevant isoform of spreading venereal diseases pathogenic disease relevant.Because different environmental stimulis, protein often changes their configuration, has reported the importance of the proteinic change of configuration that causes the pathogenicity state in a large number.Especially, make under the unsettled situation of its native state, protein can be gathered into the assembling of characteristic filamentous, is called amyloid fibrils.These the protein assembling of being rich in beta sheet has and the visibly different configuration of its native state.(infectious) of the prion protein of described misfolding or even self propagation, this is a kind of character of encoding fully in the configuration of described misfolding.The mechanism that the amyloid of proteinic misfolding and secondary forms also is not very clear.Many evidences are supported in the approach from misfolded proteins matter to amyloid, have the less oligomeric species as intermediate.These oligomer are different in morphology, may have only the part in these oligomer to cause the cytotoxicity relevant with amyloidosis.And single soluble protein can produce difference " bacterial strain " misfolding product (strains), and Sup35 confirms as the yeast Protein virus.The infectiousness that has shown different yeast Protein virus bacterial strains depends on the configuration of infectious protein matter, and a kind of protein can be taked (infectious) configuration of multiple self propagation.These configuration differences become the hereditability difference of Protein virus bacterial strain.In addition, the Protein virus bacterial strain also may have important effect aspect the decision Protein virus propagation specificity.

Chronic human diseases seriously influences healthcare system.Generally acknowledge quick precise diagnosis instrument be give to get involved in early days and treat necessary.Only have treatment such as Alzheimer at present, and these has limited therapeutic effect to symptom.At present, do not have the preceding on one's deathbed molecular diagnostic assay of Alzheimer or infectiousness spongiform encephalopathy (TSEs), and this clinical diagnosis of carrying out requires disease very serious.And, there is not effective treatment means, the have huge hope of immunization therapy as in Alzheimer, carrying out yet.Yet in treatment most protein misfolding relevant disease, lacking the reliable method that is used to capture misfolded proteins matter, monitor therapy and disease process is a serious defective.

Therefore, need below the existence: can from sample, separate, for example capture short amyloid protein (amyloidogenic proteins), as the needs of simple, the sensitive and multiduty instrument of the prion protein of misfolding and different strains prion protein.Also exist by body internal interference, combination or capture the needs of novel treatment misfolding or amyloid.

Using conjugated polymer (CPs) to study the interaction of biomolecule, biomolecule, the folding incident of biomolecule and dynamic (dynamical) probability of biomolecule requires this polymer compatible with aqueous environment.This can realize by preparation conjugated polyelectrolytes (CPEs).As in many technical presses, describing, use CPE to come the interaction of detection of biological molecule, biomolecule and the folding incident of biomolecule by several different methods.Wherein the configuration by the polyelectrolyte chain changes, and uses CPE to come the detection of biological specificity to interact, and can find in following scientific literature as the interactional example of receptor/analyte: [Nilsson, K.P.R.;

O.Nature Materials2003,2,419-424.; Ho, people such as H-A., Angew.Chem.Int.Ed.2002,41,1548.; Ho, H-A.; Leclerc, M.J.Am.Chem.S oc.2004,126,1384.; Dore, K.; Dubus, S.; Ho, H-A.; Levesque, I.; Brunette, M.; Corbeil, G.; Boissinot, M.; Boivin, G.; Bergeron, M.G.; Boudreau, D.; Leclerc, M.J.Am.Chem.Soc.2004,126,4240.; WO02/081735, WO03/096016].

Summary of the invention

Need be used to capture the protein of misfolding form or aggregated forms, particularly amyloid form or pathological forms proteinic has the straightforward procedure of improving affinity (affinity), especially, be used for separating misfolded proteins matter from sample (in ex vivo (ex vivo) or the body).Therefore, method based on conjugated polyelectrolytes has been described in the present invention, this conjugated polyelectrolytes can be captured the protein that is used for self-assembly/aggregated forms, and particularly the protein of misfolding form or aggregated forms plays the pick off effect with optical signal report capture event simultaneously.

And, the invention still further relates to the purposes of conjugated polyelectrolytes as new therapeutic agent, this novel treatment by disturb, in conjunction with or capture misfolding form, amyloid aggregated forms or pathological protein form, work as the prion protein of misfolding among amyloid-beta in the Alzheimer or the TSE.Conjugated polyelectrolytes combination and capture these protein forms, so CPEs is used for influencing pathogeny in the live organism by playing pharmacophore, promptly described CPEs is a therapeutic agent.The conjugated polyelectrolytes that can pass blood brain barrier has effect to the disease that influences brain, and this includes, but are not limited to the beta amyloid albumen pathological process in the live organism, promptly by playing the morbidity that the treatment pharmacophore is used for influencing Alzheimer.

The conjugated polyelectrolytes (CPEs) that the present invention relates to be fixed on the solid carrier or be free in the solution is used for following purposes: be used for particular separation, for example capture protein misfolding, pathology or inferior strain form, be that amyloid form, misfolding form or gathering form and cause similarly at the accumulative protein of the not accumulative proteinic anomaly pattern of its normal form.Can under selective conditions, use a kind of CPE or several CPE to carry out the described of misfolded proteins matter alternatively and capture for misfolded proteins matter form.The invention still further relates to and capture and detect proteinic one step process misfolding, pathology or inferior strain form, it can optionally carry out under the non-selective condition of misfolded proteins matter.Another aspect of the present invention is an imaging tool in the protein of CPEs as misfolding form or aggregated forms, the particularly aleuroplast of amyloid form and pathological forms.The invention still further relates to use functionalized conjugated polyelectrolytes by MRI for example to the in-vivo imaging method of misfolded proteins matter.

The present invention is used for separating by using CPE, for example capture, in conjunction with or disturbed specimen in (ex vivo or body in) misfolded proteins matter solve the shortcoming of prior art, capture and detect described protein, diagnostic novel optical method and possible therapeutic agent, all are based on conjugated polyelectrolytes.Prior art confirms the CPEs outer and amyloid fibrillation [WO2005109005] in the ex vivo tissue sample of detection bodies reliably.The physical and chemical principle that uses CPEs to capture short amyloid structure depends on the repetitive structure of described polyelectrolyte, its with provide that very the symmetrical multiple molecular target of high-affinity (amyloidosis molecule) is relevant.Therefore, can capture pathogenicity relevant and amyloid based on the method for selectivity CPE with numerous disease.According to the present invention, be used to capture the protein of misfolding form or aggregated forms, the proteinic tool and method of amyloid form and pathological forms particularly, and the treatment of diseases relevant with these protein forms is all based on conjugated polyelectrolytes.

The sample that comprises misfolded proteins matter includes, but are not limited to blood, brain, peripheral tissues, the brain that homogenizes, the peripheral tissues that homogenizes, body fluid, biopsy, urine, cerebrospinal fluid (CSF), lymph fluid, blood plasma, at the preparation of buffer that is used for pharmaceutical grade protein and biotechnology growth medium.

One aspect of the present invention is that conjugated polyelectrolytes is joined/protein, particularly amyloid fibrils of aggregated forms and the proteinic purposes of capturing agent of pathogenicity form as misloading.When capturing the reagent of misfolded proteins matter, described conjugated polyelectrolytes can be fixed on the solid carrier, on the beadlet of any particle diameter, in the pipe, in the dialysis apparatus, in the pump, perhaps provide with the free form in solution.Described CPE also can have form of hydrogels.If described CPE provides with the free form in solution, after being captured, can adopt any way to separate misfolded proteins matter by CPE, for example by precipitation, centrifugal, absorption, absorb, dry, boil or evaporate and separate.Alternatively, can functionalized described CPE with provide detection, fixing, promote to capture, increase selectivity, increase affinity or mode (means) measuring significant further feature.

What is interesting is, can use conjugated polyelectrolytes, detect misfolded proteins matter [WO2005109005] as poly-(thiophene), poly-(3,4-vinyl dioxy base thiophene) or poly-(pyrroles).Because amplification by the acquisition system reaction, pick off based on conjugated polyelectrolytes is very sensitive for very little interference, therefore, compare with the pick off based on molecule of prior art, it provides important advantage, and described polyelectrolyte also provides the direct detection of pathogenicity prion protein.According to the present invention, this direct measuring ability of CPEs is combined with the unique ability of capturing misfolded proteins matter, thereby provide than the great advantage of routine immunization histological techniques, because the method for routine immunization histological techniques need be used the one-level antibody that is used to capture usually and the secondary antibody of the misfolded proteins matter that is used to develop.Use conjugated polyelectrolytes to require polymer compatible with aqueous environment as the probability of capturing agent that is used for misfolded proteins matter, this has realized by preparing conjugated luminous polyelectrolyte.

In the present invention, the new method that a kind of CPEs of use captures misfolded proteins matter has been described.Can use CPEs to realize the separation of misfolded proteins matter in the sample, for example remove them from described sample, this was never describing before.Provide a kind of use CPEs to dye and capture misfolded proteins matter in the sample solution, follow sedimentary method.Use the method for CPEs to have other character that is different from other amyloid nutrition (amyloidotrophic) dyestuff such as Congo red and ThT, be that CPEs provides capturing of misfolded proteins matter most importantly.Therefore, expected that in the present invention use CPEs captures the several method of misfolded proteins matter.Described CPE can be fixed on the solid carrier, thereby provides a kind of misfolded proteins matter of capturing with high selectivity and high-affinity to arrive described solid carrier, thereby separates or remove its method from sample.

Of the present invention further aspect, a kind of method of capturing misfolded proteins matter is provided, comprise conjugated polyelectrolytes as defined above is exposed to sample that wherein said conjugated polyelectrolytes is optionally captured misfolded proteins matter in the sample.

On the one hand, the present invention relates to be used for capturing the method for the sample misfolded proteins pledge kind on the solid carrier, it may further comprise the steps:

-at least a conjugated polyelectrolytes (CPE) is fixed on the solid carrier

-sample is contacted with described CPE

-alternatively, add competitor

-from sample, remove described solid carrier or from sample, separate described solid carrier, or

-wash described solid carrier, and do not remove the misfolded proteins matter capture mutually from CPE

-alternatively, detect the misfolded proteins matter of capturing by preferable methods, then, primary sample solution does not comprise or comprises seldom misfolded proteins matter thus.

On the other hand, the present invention relates to be used for to capture the method for misfolded proteins pledge kind in the sample of solution, it may further comprise the steps:

-sample is combined with at least a conjugated polyelectrolytes (CPE)

-alternatively, add competitor

-from sample, separate the misfolded proteins matter capture

In the further embodiment of the present invention, competitor joined in the solution CPE that captures misfolded proteins matter with increase and for example combine the natural or distinctiveness of folding proteinic CPE not of non-specific binding.Such reagent includes, but are not limited to detergent, ion, salt, chelating agen and solvent.

If carry out the detection of misfolded proteins matter that CPE is captured, in the method for the invention the wavelength that has of the radiation of Shi Yonging for about 100nm between about 2000nm.In one embodiment, the radiation of use in visible range.It also is possible using a plurality of photon excitations, therefore, uses the radiation of 2x or 3x (be respectively two-photon and three-photon excites) to replace the exciting radiation of x nm.

On the other hand, the present invention relates to implement the device of the method according to this invention.Such device is provided with the parts that make the contact of CPE and sample that are fixed on the solid carrier or in the solution, be used for adding alternatively the parts of competitor, alternatively from sample remove CPE or from sample separation of C PE parts, wash that CPE can remove the parts of the misfolded proteins matter of capturing mutually from described CPE mutually and not alternatively, the parts of the misfolded proteins matter of capturing by the preferable methods detection alternatively and alternatively collection be exposed to the parts of the sample solution of described CPE.

Preferably, the conjugated polyelectrolytes of Shi Yonging comprises the copolymer or the homopolymer of thiophene, pyrroles, aniline, furan, phenylene, ethenylidene, fluorenes, vinyl dioxy thiophene and replacement form thereof in the present invention, described conjugated polyelectrolytes can have one or more ion side chain degrees of functionality, as the derivant of aminoacid, amino acid derivativges, neurotransmitter, monosaccharide, nucleic acid or its combination and chemical modification.Described ionic functional degree can comprise one or more aniones and cationic side chain degree of functionality.

Used in the present invention CPE also can have the group of end-functionalization; wherein said functional group can for, but be not limited to DNA; RNA; peptide; aminoacid; histidine (histidin); histidine 10 (histidin10); protein; peptide support (peptide scaffolds); biotin; avidin; streptavidin; chelating agen; active group; antibody; enzyme; part; receptors ligand; steroid; biomolecule or other molecule; nano-particle; microgranule; gold nano grain; the gold microgranule; magnetic bead; the beadlet of protein coating; the beadlet of peptide coating; super magnetic (supramagnetic) beadlet or granule; the gadolinium nano-particle; gadolinium ion; the nano-particle of doping lanthanide series; the Gadolinia. nano-particle of doping lanthanide series; the lanthanide series granule.

In the further embodiment of the present invention, use DNA; RNA; peptide; aminoacid; histidine; histidine 10; protein; peptide support (peptide scaffolds); biotin; avidin; streptavidin; chelating agen; active group; antibody; enzyme; part; receptors ligand; steroid; biomolecule or other molecule; nano-particle; microgranule; gold nano grain; the gold microgranule; magnetic bead; the beadlet of protein coating; the beadlet of peptide coating; super magnetic bead grain or granule; the gadolinium nano-particle; gadolinium ion; the nano-particle of doping lanthanide series; the Gadolinia. nano-particle of doping lanthanide series; the functionalized CPE of lanthanide series granule can be for covalency in fact or non-covalent.

A kind of method is provided in one aspect of the invention, it detects from the native form protein of the back of barrier such as tube wall, film, skin etc. misloading joins/protein of aggregated forms, the particularly proteinic formation of amyloid fibrils and pathogenicity form, this method comprises injection, flow into, pump into, suck, import or by alternate manner sample is exposed to after the barrier, and by suitable detection part (means of detection) detect after the described barrier or within the target protein of described misfolding.The parts that are used to detect with administration CPEs can change, and this depends on that according to side-chain of the present invention or end-functionalization its other parts in description illustrate.

Another aspect of the present invention is provided for relating to misloading joins/protein of aggregated forms, particularly the Therapeutic Method and the therapeutic agent based on conjugated polyelectrolytes of the disease that forms of the protein of amyloid fibrils and pathogenicity form, Creutzfeldt-Jakob disease (vCJD), secondary amyloidosis, type 2 diabetes mellitus, the infectiousness spongiform encephalopathy (TSE is as CWD, the overworked disease of sheep scabies, GSS and Kuru disease, bovine spongiform encephalopathy (BSE)) of described disease such as Alzheimer, Creutzfeldt-Jakob disease (CJD), variation.

Another aspect of the invention is bioassay, it relies under the existence of native protein, use the comprehensive differentiation during CPE captures the proteinic step of amyloid form, misfolding form or pathologic form, detect by suitable method subsequently, as method based on colorimetric, absorbance or fluorescence.Avoid false positive and avoid false negative result extremely important.The selectivity Proteolytic enzyme of native protein can be but be unessential, obtains a kind of method of this point.

Another aspect of the present invention relates to the uniqueness of CPEs, it is not only captured misloading and joins form, misfolding form, aggregated forms or the proteinic of pathogenicity form and capture, and can work to capture agent, simultaneously if necessary, it can report described capture event, and converts it into optical signal and detectable signal.

The misfolded proteins matter that may wish the various ways identified means that aspect further, the present invention also can implement with the form of microarray, and this microarray requires detection system fixing and medelling from the teeth outwards.Then, in solution or on solid carrier, use the capture agent of conjugated polyelectrolytes of the present invention as misfolded proteins matter, and if expectation or needs, use it as the detection agent that is used for described capture event.

Four corner of the present invention defines for the accessory claim book.

Description of drawings

Fig. 1: based on poly--thiophene main chain poly-with mixed type-some of the different CPEs of thiophene-benzene main chain select the chemical constitution of example.Can make up the main chain size that side chain changes and defines.These have provided following simple name: POWT, PTAA, POMT, PT2, tPOWT, tPTMl, f-PONT, L-POMT and D-POMT.

Fig. 2: functionalized CPEs.R1 and the R2=end/end is functionalized.R1 and R2 can be identical or different.The Rs=side chain.

Fig. 3: use the CPE of end-functionalization or do not have end-functionalization CPE any amyloid polymer capture sketch map.This figure is not to describe in proportion, and can not represent the size relationship between CPE and the amyloid.In the present invention, the monomeric quantity of amyloid can be at one between a plurality of in misfolded proteins matter.And CPEs can be fixed on the solid carrier, and this example is presented in the figure of back.The quantity of functionalized or not functionalized CPE molecule can be an any situation in a plurality of.In the diagram, side chain (Rs) does not show.

Fig. 4: show and use the CPE that is fixed on the solid carrier to capture the sketch map of misfolded proteins matter.Sample is exposed among the functionalized or not functionalized CPE that is fixed on the solid carrier, and described thus CPE captures misfolded proteins matter in the described sample.This figure is not described in proportion, and can not represent the size relationship between CPE and the misfolded proteins matter.The quantity of functionalized or not functionalized CPE molecule can be an any situation in a plurality of.In the diagram, side chain (Rs) does not show.

Fig. 5: show to use CPE on suitable solid carrier such as microscope slide, bead, filtered matrix, separation matrix etc., to capture the sketch map of misfolded proteins matter.Sample is exposed among the functionalized or not functionalized CPE that is fixed on the solid carrier, and described thus CPE captures misfolded proteins matter in the described sample.This figure is not described in proportion, and can not represent the size relationship between CPE and the misfolded proteins matter.The quantity of functionalized or not functionalized CPE molecule can be an any situation in a plurality of.In the diagram, side chain (Rs) does not show.

Fig. 6: the operating weight sedimentation detects the principle of the painted PrP-amyloid of CPE PTAA.The size of crystal structure that also provides PrP is as the particle diameter reference of PrP content in the small aggregation.

Fig. 7: show and use the sketch map of capturing misfolded proteins matter with the magnetic bead of CPE coating.With functionalized CPE, for example the CPE with biotin functionalization is coated with magnetic bead, for example the magnetic bead that is coated with avidin.Sample is exposed to the magnetic bead that contains CPE, and described thus CPE captures misfolded proteins matter in the described sample.This figure is not described in proportion, and can not represent the size relationship between CPE and the misfolded proteins matter.Can use Magnet as collection/concentration step.The quantity of CPE molecule can be an any situation in a plurality of.In the diagram, side chain (Rs) does not show.After capturing and using Magnet collection/concentration step, can comprise optional detection step.

Fig. 8: show the CPE of use biotin functionalization and the sketch map of the surface trapping misfolded proteins matter that avidin is coated with.Described functionalized CPE can be an ortho states or on beadlet.Sample is exposed to CPE, and wherein said CPE captures misfolded proteins matter in the described sample.The surface that can use the avidin coating is as collection/concentration step.This figure is not described in proportion, and can not represent the size relationship between CPE and the misfolded proteins matter.The quantity of CPE molecule can be an any situation in a plurality of.In the diagram, side chain (Rs) does not show.Capture and surface collection/concentration step after, can comprise optional detection step.

Fig. 9: before and after filtering by 0.22 μ m filter, the fluorescence spectrum of the 0.5mg/ml PTAA that comprises insulin in the phosphate buffer of 20mM pH 8.

Figure 10: after filtering, in the phosphate buffer of 20mM pH 8, comprise the fluorescence spectrum of the 0.5mg/mlPTAA of 0,5,50 and 100% fibriilar insulin by 0.8 μ m filter.

Figure 11: after contrary-filtration (back-filtration), in the phosphate buffer of 20mM pH 8, comprise 0,5,50 and 100% fibriilar fluorescence spectrum with 0.5mg/ml PTAA of insulin by 0.8 μ m filter.

Figure 12: use the centrifugal fibril of capturing of PTAA-fibril complex, then carry out fluorescence measurement.

Figure 13: fluorescence spectrum, it confirms the CPEs of some selections and the interaction between the Gd203 nano-particle.Described measurement is carried out in double distilled water.

The specific embodiment

Generally speaking, the present invention relates to a kind of proteinic new method of using conjugated polyelectrolytes to capture the protein that misloading is joined form, misfolding form or aggregated forms, particularly amyloid fibrils and pathogenicity form.Described misfolded proteins matter is exposed in the conjugated polyelectrolytes (CPE), thus CPE capture, in conjunction with or the described target misfolded proteins matter of emanating.Compare with protein,, can have capturing of high-affinity or high selectivity for misfolded proteins matter for natural folding.Conjugated polyelectrolytes is in conjunction with the protein of the change of misfolding, as PrP therein ^ScOr under the condition of A-beta protein, carry out described capturing, preferably, but not necessarily, wherein such conjugated polyelectrolytes can be in conjunction with their the normal native form of non-gathering in conjunction with these anomaly patterns.Non-covalent combination between expectation CPE and the target misfolded proteins matter takes place with sufficiently high affinity with under selected condition and has enough selectivitys, captures from sample or the protein of the change of accumulative, the misfolding of emanating to be of value in mensuration.

According to the present invention, (capture), combination (binding) or segregation (isolation) captured in term is that general term separates the subclass of (separation).Can use CPEs to realize the separation of misfolded proteins matter in the sample, for example remove them from described sample, this never described before being.For example, sample solution is flow through has the described solid carrier of the CPE/CPEs that is fixed on the solid carrier or flows into its inside.The effect of described CPE/CPEs is to capture misfolded proteins matter from sample, also separates misfolded proteins matter simultaneously from sample.

" capture (capture) " as the term that uses in this application and refer to a kind of probe (probe), it can be in conjunction with misfolded proteins matter in ex vivo or body, thereby in the presence of the described protein of non-gathering normal form, with its separation (separate), segregation (isolate) or fixing.

The present invention is based on to use with the interactional conjugated polyelectrolytes of described misfolded proteins matter and comes non-covalent property to capture misfolding, unusual, that misloading is joined, accumulative or pathogenic protein.Described interaction takes place under the covalent bonding not having, it is based on the dipole-dipole bonding between conjugated polyelectrolytes and the misfolded proteins matter, hydrogen bonding, static-and apolar interaction, this paper is called non-covalent bonding, and it further comprises the natural non-covalent bonding of any kind.

Some aspect of the present invention can provide the covalently bound of conjugated polyelectrolytes and some entity, described entity such as protein, misfolded proteins matter, peptide, biomolecule, other molecule or surface.

Therefore, an aspect of of the present present invention is a kind of method, wherein in the presence of native form proteinic, uses conjugated polyelectrolytes to separate, for example selectivity capture, in conjunction with or the protein of segregation misfolded proteins matter, accumulative anomaly pattern or pathologic form.With sample be exposed to be fixed on the solid carrier or the beadlet of virtually any size on conjugated polyelectrolytes in, described beadlet is hydrogel or is solution to be provided, described thus conjugated polyelectrolytes captures, in conjunction with or any misfolding of emanating, accumulative, that misloading is joined or pathogenic protein.Described method can be carried out under such condition: wherein said high-affinity (avidity) conjugated polyelectrolytes can optionally be captured described misfolded proteins matter, and its form for the described misfolding in the proteinic sample of the form of protein that comprises native form and normal form and misfolding has high selectivity.

Described conjugated polyelectrolytes can be fixed on the solid carrier of adequate types and applicable shapes, and there is the conjugated polyelectrolytes of sample in the surface of wherein said solid carrier.The shape and size of described solid carrier can be any types, for example circular, pancake or beadlet shape, the surface can be with conjugated polyelectrolytes with non-covalent mode modification, or in described solid support structure covalent bonding or be attached to the surface of described solid carrier.Also constitute pipe, dialysis apparatus, pump, insulin pump, the proteinic device of administration, syringe and pin according to solid carrier of the present invention.In the time of on being fixed on solid carrier, described conjugated polyelectrolytes can be any suitable form, for example is hydrogel, polymeric film, free CPEs, support or lamellated.When capturing the reagent of misfolded proteins matter, described CPEs also can provide with the free form in solution.If described CPE provides with the free form in solution, can adopt any method to remove misfolded proteins matter after CPE captures, for example sedimentation, centrifugal, absorption, absorb, dry, boil or evaporate.Alternatively, described CPE can functionalised, and captures, increases selectivity to be provided for detecting, to fix, to promote, increases affinity or for the method for measuring significant other thing.

Describedly capture, in conjunction with or the segregation condition can comprise and have the competitor that offers sample that wherein compare with conjugated polyelectrolytes, competitor has littler binding affinity for the protein of the form of misfolding.Usually, if use competitor, it has still less affinity or selectivity than conjugated polyelectrolytes.

Alternatively, can be coated on conjugated polyelectrolytes on the solid carrier surface or bonding thereon.

In scientific literature, reported the protein of amyloid form, or the misfolded proteins matter of protease sensitive form may interact at least with polyanion.These electronegative polymer may with the positively charged regional interaction of misfolded proteins matter structure, and may have multiple interaction with accumulative protein.We find that conjugated polyelectrolytes can be in conjunction with misfolded proteins matter aggregation, the perhaps little conjugate of misfolded proteins matter, described conjugate even can be as small as two monomers of amyloid, its structure has the affinity more much higher than these polyanions, therefore, itself in addition can replace described polyanionic compound.A possible mechanism of the high-affinity of CPEs is that CPEs can form polyion and hydrophobic interaction simultaneously with the misfolded proteins matter with charged substituent group and hydrophobicity conjugated main chain.Therefore, the conjugated polyelectrolytes that is fixed in surface or the solution can be captured abnormal protein especially.The native protein right and wrong are accumulative, can not have the high-affinity interaction with polyanion usually.Can select described condition determination, make, capture specificity by competing further to improve with fixed CPE or the CPE in solution than the anion of low affinity such as the existence of detergent Sa Kaoxi (Sarkosyl).

Can coupling use the separation of CPE, for example capture, in conjunction with or the segregation sample in protein misfolding, that assemble anomaly pattern or pathological forms, capture to change described CPE response, in conjunction with or the character of the described misfolded proteins matter of emanating.Can be by optical means for example, as fluorescence or absorb the qualitative change that detects CPE, then, use it to determine when described capture event takes place.

Other detection method

According to aspects of the present invention, can functionalized described conjugated polyelectrolytes, with realize effectively or expectation be used to detect isolating method.Detect bonding or contact other method of the conjugated polyelectrolytes of described misfolded proteins matter, described suitable method such as two-photon or multiphoton spectroscopy, nuclear magnetic resonance (MRI), PET etc. by suitable method.Describe an example of the interaction imaging of CPE after making barrier and misfolded proteins matter at this, but had other conspicuous method, described barrier such as pipe, film, pipe, skin etc.For example can use two-photon or multiphoton spectroscopy or nuclear magnetic resonance (MRI) behind described barrier, to detect conjugated polyelectrolytes or functionalized conjugated polyelectrolytes in some modes, use it to determine when then described separation has taken place, for example capture or binding events.

CPE as therapeutic agent

The invention still further relates to the purposes of conjugated polyelectrolytes as new therapeutic agent, wherein a kind of model of action is for disturbing the formation of amyloid in the body.When injection entered organism, described CPE can change the pathological process of amyloid, that is, and and by working the morbidity of the pharmacophore function influence disease for the treatment of.Therefore, when preceding diagnosis and treatment on one's deathbed has tremendous influence and great interests for public health, expected this therapeutic agent in the present invention, described therapeutic agent is for being used for the therapeutic agent based on CPE of protein misfolding disease, particularly Alzheimer and prion disease.

In one embodiment, the invention provides a kind of method, wherein conjugated polyelectrolytes is directly captured described misfolded proteins matter.In this case, described being trapped in do not have to take place under the covalent bonding, it is based on dipole-dipole bonding between conjugated polyelectrolytes and the biomolecule, hydrogen bonding, static-and nonpolar interaction, this paper is called non-covalent bonding, and it further comprises the natural non-covalent bonding of any kind.

" directly capture " as the term that uses in this application and to refer to a kind of probe (probe), its can be by the described misfolded proteins matter of Direct Bonding directly capture misfolded proteins matter, thereby it from sample segregation or be fixed on the sample, and is not needed other macromolecular compound.

Its common implication should be taked in term " affinity (avidity) " as used in this article, refers to the whole bond strength of many binding sites of molecule and multivalence bonding agent.This should form contrast with " affinity (affinity) ", and it is the bond strength of each independent binding site and molecule and binding agent.

For example, might suitably functionalized or not functionalized conjugated polyelectrolytes be implemented as the active component of trap setting by described conjugated polyelectrolytes is fixed on the substrate.Described trap setting comprises the suitable containers that is used for described substrate, and the complex of conjugated polyelectrolytes and misfolded proteins matter forms on this substrate.Alternatively, described CPE is used to detect the capture event when it occurs.

Yet other make is possible, and for example described conjugated polyelectrolytes can provide with solution, and passes flow cell, simultaneously protein solution is mixed with the complex solution fluid.Can capture by various analytical technology monitorings.

Especially, the invention enables to use and capture misfolded proteins matter with disease association with the interactional conjugated polyelectrolytes of misfolded proteins matter.Be used for diagnosing the method for the disease that relates to misfolded proteins matter in research, capturing of misfolded proteins matter is to remove misfolded proteins matter from sample, detect in the separation of misfolded proteins matter and necessary based on the various treatment technologies of conjugated polyelectrolytes in sample behind the barrier.

The example of conjugated polyelectrolytes that demonstrates the feature of above-mentioned discussion is poly-(3-[(S)-5-amino-5-carboxyl-3-Evil amyl group]-2,5-sulfo-phenylene hydrochlorate) (POWT), polythiophene acetic acid (PTAA), poly-(3-[(S)-5-amino-5-methoxyl group carboxyl-3-Evil amyl group]-2,5-sulfo-phenylene hydrochlorate) (POMT), poly-((3,3 " two [(S)-5-amino-5-carbonyl-3-Evil amyl group]-[2,2 '; 5 ', 2 "])-5; 5 "-uncle sulfo-phenylene hydrochlorate) (tPOWT), poly-((1,4-two (3-[(S)-5 amino-5-carbonyl-3-Evil amyl group]-thiophene-2-yl)-benzene) hydrochlorate) (f-PONT), poly-(3-[(R)-5-amino-5-methoxyl group carboxyl-3-Evil amyl group]-2,5-sulfo-phenylene hydrochlorate) (D-POMT), poly-(3-[(S)-5-amino-5-methoxyl group carboxyl-3-Evil amyl group]-2,5-sulfo-phenylene hydrochlorate) (L-POMT) and other material, referring to Fig. 1.In the present invention, can comprise L and D enantiomer for the variant of the conjugated polyelectrolytes that comprises these and other type such as pyrroles, vinyl dioxy thiophene, fluorenes etc.The length of CPE can be fixed, variable, monodispersed or polydisperse.

The functional group who joins side chain (Rs) or end (R1 and R2) is presented among Fig. 2.These can be, but are not limited to charged anionicly, cationic or zwitterionic, have the pKa at different pH, and this makes these polythiofuran derivatives be suitable for and electronegative or positively charged oligomer and polymer formation compound polyelectrolyte.In addition, described ionic group has been set up multiple hydrogen bonding pattern with different molecules.Capture the ability of the uniqueness of misfolded proteins matter for CPEs, one of reason of proposition is an affinity, but the present invention should not be subjected to the constraint of this proposal.A method of observing misfolded proteins matter can be to be seen as the polyion molecule alternative or multiple CHARGE DISTRIBUTION that has along molecular axis.Special CPEs of the present invention can form very strong noncovalent interaction with misfolded proteins matter, thereby captures them, referring to schematic view illustrating among Fig. 3.Can provide functional group wherein to be used for for the conjugated polyelectrolytes of following end-functionalization that the surface is fixing, secondary is captured or be used for according to the detection part behind the barrier of the present invention: described functional group can for, but be not limited to DNA, RNA, peptide, aminoacid, histidine, histidine ₁₀Protein; the peptide support; biotin; avidin; streptavidin; chelating agen; active group; antibody; affinity peptide support; scFV; FAB; enzyme; part; receptors ligand; steroid; biomolecule or other molecule; nano-particle; microgranule; gold nano grain; the gold microgranule; magnetic bead; the beadlet of protein coating; the beadlet of peptide coating; super magnetic bead grain or granule; the gadolinium nano-particle; the Gadolinia. granule; gadolinium ion; the nano-particle of doping lanthanide series; the Gadolinia. nano-particle of doping lanthanide series; the lanthanide series granule.

CPE can be selected so that it has following ability: under non-selective condition, have and capture misfolding, that assemble change or the protein of inferior strain form and the proteinic ability of non-accumulative normal form simultaneously, with under selective conditions, selectivity is captured the ability of misfolded proteins matter.If necessary, can be under the non-selective condition and change under the selective conditions of its optical property, be fixed on CPE on the solid carrier or in the solution, so, then might distinguish described CPE is to capture misfolded proteins matter or native protein.Can by regulate sample, according to the selection of the reactant liquor of pH or cleaning mixture, ionic strength, buffer, perhaps obtain suitable non-selective conditioned disjunction selective conditions analysis condition by adding salt, ion, metal ion, detergent, polyelectrolyte, protein, granule or chelating agen.

And, it is possible adding one or more reagent in sample, reactant liquor or cleaning mixture, described reagent can increase in conjunction with the CPE of misfolded proteins matter and with the difference of the interactional CPE of normal protein matter, in such a way, increased the selectivity of capturing misfolded proteins matter in the given sample.In this case, CPE can provide to be fixed on the solid carrier or in the solution.Such reagent comprises detergent such as Triton-X, saponin, SDS, Sa Kaoxi, just-lauryl creatine acid (laurosylsarcosine), the fatty acid sarcosine, CHAPS, Brij, octyl group-b-glucosides, polysorbas20, Nonidet P40 (Nonidet) P-40 or other variant, ion and salt such as metal ion, molecular ion and organic ion, chelating agen such as EDTA, EGTA, 2,2 '-bipyridyl, dimercaptopropanol, BAL, ionophore (lonophores), nitrilotriacetic acid, neighbour-phenanthroline, salicylic acid and triethanolamine, solvent such as water, alcohol, organic solvent, chlorinated solvent, amination solvent and sulfonation solvent and other reagent, as polymeric material, polyelectrolyte is (zwitterionic, anionic or cationic), saccharide (comprising polysaccharide), has the organic acid that surpasses a coordinating group, the lipid sterin, aminoacid and related compound, peptide, phosphate, nucleotide, tetrapyrrole, ironweed amine, ionophore is as Gramicidin, monensin and valinomycins bakelite.Other reagent comprises protein, as trypsin, E.C. 3.4.21.64, antibody, serum albumin etc.

Described conjugated polyelectrolytes can be used suitably as the active part of device, described device captures, separates, in conjunction with or remove misfolded proteins matter in the sample.This can pass through, and for example described conjugated polyelectrolytes is fixed on the substrate, is exposed to sample by suitable method then and realizes (referring to the Figure 4 and 5 that are used for two embodiment).Suitably, described device comprises the suitable containers that is used for described substrate, and the interaction of conjugated polyelectrolytes and misfolded proteins matter forms on this substrate, thus its capture, separate, in conjunction with or remove misfolded proteins matter in the sample.Described conjugated polyelectrolytes can be to use those identifications or identify misfolded proteins matter (PrP for example ^Sc, PrP ^Res, amyloid-β, fibril form A β (1-40), A β (1-42) or A β (1-43), amyloid-β (A β) peptide, the beta-2-microglobulin molecule in dialysis-relevant amyloidosis (DRA), IAPP, the alpha-synapse nucleoprotein in Parkinson's disease or the Huntington protein in Huntington's disease) the part of system of antibody, described misfolded proteins matter is or will be captured by CPE.Alternatively, can use the optical probe of capturing (referring to the Figure 4 and 5 that are used for two embodiments) of conjugated polyelectrolytes as report misfolded proteins matter.Yet alternate manner is possible, and for example described conjugated polyelectrolytes can provide in solution, and misfolded proteins matter is used for capturing, dyes in sample or body simultaneously.The antibody that can be used in then in the solution or on the solid carrier is identified or the misidentify unfolded protein.

Following detailed description of the invention will relate separately to conjugated polyelectrolytes, misfolding, unusual, accumulative, inferior strain or pathogenic protein, relate to the disease of described misfolded proteins matter, capture method, the fixing and array of conjugated polyelectrolytes on solid carrier of described misfolded proteins matter from sample solution.At last, the present invention adopts a plurality of experiments to confirm its practicality for example.

The I conjugated polyelectrolytes

The present invention relates to multiple conjugated polyelectrolytes, it has minimum three unit, and described unit is derived from following monomer: the form of thiophene, pyrroles, aniline, furan, phenylene, ethenylidene, fluorenes, vinyl dioxy thiophene or its replacement, its homopolymer and the copolymer of formation.Described conjugated polyelectrolytes can be monodispersed, forms by polyelectrolyte chain with well-defined chain length, or polydisperse, form by polyelectrolyte chain with different chain length.And the monomer with anionic, cationic or zwitterionic side chain degree of functionality comprises within the scope of the invention.Described side chain degree of functionality derives from, but is not limited to aminoacid, amino acid derivativges, neurotransmitter, monosaccharide, nucleic acid, or the L and the D enantiomer of its combination and its chemical modification, or derivatives thereof.Conjugated polyelectrolytes of the present invention can comprise single side chain functional group, perhaps can comprise two or more different side chain functional groups.Under different pH, the functional group of the conjugated polyelectrolytes of band anion or cationic charge makes these polyelectrolyte derivants be suitable for and electronegative or positively charged oligomer and the strong compound polyelectrolyte of polymer formation.In addition, described ionic group has been set up multiple hydrogen bonding pattern with different molecules.

Some aspect of the present invention can provide the covalently bound of conjugated polyelectrolytes and some entity, described entity such as protein, misfolded proteins matter, peptide, biomolecule or other molecule.

In Fig. 1 or Fig. 2, the functional group who joins on side chain (Rs) or the end (R1 and R2) can be, but be not limited to have charged anion, cation or amphion functional group at the pKa of different pH, this make these polythiofuran derivatives be suitable for with electronegative or positively charged oligomer and polymer formation compound polyelectrolyte.In addition, described ionic group has been set up multiple hydrogen bonding pattern with different molecules.Capture the ability of the uniqueness of misfolded proteins matter for CPEs, one of reason of proposition is an affinity, but the present invention should not be subjected to the constraint of this proposal.A method of observing misfolded proteins matter can be to be seen as the polyion molecule alternative or multiple CHARGE DISTRIBUTION that has along molecular axis.Special CPEs of the present invention can form very strong noncovalent interaction with misfolded proteins matter, thereby captures them, referring to schematic view illustrating among Fig. 3.Can provide functional group wherein to be used for for the conjugated polyelectrolytes of following end-functionalization that the surface is fixing, secondary is captured or be used for according to the detection part behind the barrier of the present invention: described functional group can for, but be not limited to DNA, RNA, peptide, aminoacid, histidine, histidine ₁₀Protein; the peptide support; biotin; avidin; streptavidin; chelating agen; active group; antibody; affinity peptide support; scFV; FAB; enzyme; part; receptors ligand; steroid; biomolecule or other molecule; nano-particle; microgranule; gold nano grain; the gold microgranule; magnetic bead; the beadlet of protein coating; the beadlet of peptide coating; super magnetic bead grain or granule; the gadolinium nano-particle; the Gadolinia. granule; gadolinium ion; the nano-particle of doping lanthanide series; the Gadolinia. nano-particle of doping lanthanide series; the lanthanide series granule.

II misfolded proteins matter

Conjugated polyelectrolytes of the present invention is captured target misfolded proteins matter.In the present invention, term misfolded proteins matter should be with its broad understanding, the protein that comprises protein, pathological forms or inferior strain form that misloading is joined, be the protein of amyloid form, misfolding form or aggregated forms, as plaque, preferably, described misfolded proteins matter can be the amyloid of enjoying the insoluble fibre shape protein aggregation of specificity structure feature.

Described being trapped in do not have under the covalent bonding to occur, and it is based on the dipole-dipole bonding between conjugated polyelectrolytes and the misfolded proteins matter, hydrogen bonding, static-and apolar interaction.Described conjugated polyelectrolytes can interact with normal native protein and misfolded proteins matter.Described conjugated polyelectrolytes also can be trapped in the amyloid or the misfolded proteins matter of external preparation.Not bound by theory, at present hypothesis is under suitable condition determination, and conjugated polyelectrolytes can be specifically and captured misfolded proteins matter with having high-affinity.Can the described misfolded proteins matter of chemical modification, so that its selecteed conjugated polyelectrolytes is captured.The method of protein of derivatization different range is known.For example, easily the modified amino acid side chain so that its group that comprises polarity and non-polar group or have the hydrogen bonding ability.Described misfolded proteins matter can be in solution, on the solid phase, in vivo, external or in tissue sample, capturing of misfolded proteins matter can be in aqueous solution, in the organic solvent, in the body fluid, carry out on the solid phase or in the tissue sample.

Can comprise in the body of misfolded proteins matter or external target sample includes, but are not limited to blood, brain, peripheral tissues, the brain that homogenizes, the peripheral tissues that homogenizes, body fluid, biopsy, urine, cerebrospinal fluid (CSF), lymph fluid and other suitable sample.The mensuration of capturing based on misfolded proteins matter has great commercial interest, huge diagnostic value, can be used as therapeutic agent and is used for capturing the selective determination of blood (screening blood products) or other complex media misfolded proteins matter and from the dyeing of the tissue slice of brain, muscle, fat, mucus, nerve, blood vessel or other tissue.

The disease of III and misfolded proteins qualitative correlation

The disease that is characterized as amyloid is the corresponding embodiment that is used to illustrate with the disease of misfolded proteins qualitative correlation, wherein amyloidosis be called as a kind of can heredity or erworbene Krankenheit.The amyloidosis of noting acquiescence is often referred to the AA amyloidosis, but wherein having the settled any disease relevant with amyloid of amyloid all is amyloidosis.For example CJD, vCJD, Alzheimer and diabetes nearly all are not called as amyloidosis.

In this section, some examples of relevant amyloidosis have so far been pointed out.Primary amyloid degeneration comprises sudden change and the AL amyloidosis (light chain immunoglobulin is as seeing) in lysozyme, transthyretin, apolipoprotein B, the Fibrinogen in multiple myeloma.Secondary amyloidosis comprises AA amyloidosis (serum amyloid sample A protein, a kind of because the acute phase protein that chronic inflammation causes) and gelsolin amyloidosis (pGSN fragment).The familial amyloid sample become or hereditary amyloidosis modal be that sudden change by transthyretin causes, but under few cases, also can cause by ApoA 1, gelsolin, Fibrinogen and lysozyme sudden change, it mainly is to be caused by heredity, is considered to the sharpei calentura (Shar Pei fever) of amyloidosis high probability autosomal dominant, that be passaged to the offspring, A Balaqiya (Appalachian) type and the amyloidosis in sharpei (Shar Peis).The example of organ specificity amyloidosis is a type 2 diabetes mellitus (dextrin, be also referred to as IAPP), the neurological, Alzheimer (A β 39-42), Parkinson's disease (alpha-synapse nucleoprotein), Huntington's disease (Huntington protein), infectiousness spongiform encephalopathy (prion protein, PrP), some example is Creutzfeldt-Jakob disease (PrP in brain), Kuru disease (sedimentary diffuse PrP in brain), fatal familial insomnia (PrP in thalamus) and bovine spongiform encephalopathy (PrP in the cow brain), have a liking for Congo red sample angiopathy (Congophilic angiopathy) (amyloid-beta).The heart amyloidosis comprises congestive heart failure; Some example (PrP in heart or transthyretin).Inclusion body myositis.Another kind of important example is iatrogenic disease, and as the insulin amyloidosis, it is considered to because the drug administration by injection insulin causes.

Some non-disease amyloid is coat protein, spider silk, mammal melanosome (pMel), tissue plasminogen activator (tPA), the hematodinamics factor, the calcitonin and the protein of the native starch shape albumen in organism, curl escherichia coli (Curli E.coli) protein (curlin), yeast Protein virus [Sup35], goose Podospora (Podospora Anserina) Protein virus Het-s, malaria and the peptide that manufactures and designs amyloid.

Described prion disease [for example bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob disease (CJD)] and Normocellular prion protein (PrP ^c) configuration conversion to being expressed as PrP ^ScThe isoform of infectious disease-relevant relevant.The protein PrP of the infectious form of described misfolding ^ScBe the reason of one group of rare, mortality encephalopathy, described mortality encephalopathy is called as prion disease, and it influences the mankind and mammal.Described prion disease is also referred to as infectiousness spongiform encephalopathy (TSE), and they comprise the bovine spongiform encephalopathy (BSE, or " crazy cow " disease) in the cattle; The overworked disease of sheep scabies in the sheep; Chronic consumptions in deer and elk; With disease [Creutzfeldt-Jakob disease (CJD), the Jie Ciman-Si Tuosile-Shi Yinke (Gerstmann-among the mankind

-Scheinker) disease (GSS), Kuru disease].Conjugated polyelectrolytes of the present invention means the method that is used to capture with the pathogenicity Protein virus of these disease associations.

The IV detection method

As already noted, the present invention be based on use conjugated polyelectrolytes, functionalized conjugated polyelectrolytes or its to make up from sample to capture, in conjunction with, separate or remove misfolded proteins matter.Can observe described capture event by the following method of indefiniteness: fluorescence, Foster Resonance energy shifts (FRET), cancellation emission light, absorption, two-photon or multiphoton spectroscopy, nuclear magnetic resonance (MRI), dynamic light scattering (DLS), electric or magnetic reaction, electrochemistry, amperometry, coulometry, gravimetry, polarization or anisotropy, fluorescence correlation spectroscopy (FCS), quartz crystal microbalance, the quartz crystal microbalance with dissipation or other technology and physical property.Can write down emissive porwer by exometer, strengthen the photon that flows in the detector and can increase sensitivity.This can use lamp or laser to obtain as excitation source.Also can detect the variation of fluorescence by using fluorescence microscope or confocal microscope.The combination that excites or launch optical filter can be used, and CCD-photographing unit, video camera, ordinary camera or polaroid camera document image can be passed through.Then, can be by image processing software, image correlated spectroscopy (ICS) or the alternate manner analysis image in computer.

V conjugated polyelectrolytes and proteinic fixing

The conjugated polyelectrolytes of Shi Yonging in the present invention; biomolecule; misfolded proteins matter; antibody; protein; peptide; misfolded proteins matter or other molecule can be fixed on the multiple solid carrier; described solid carrier includes, but are not limited to silicon wafer; glass (microscope slide for example; bead); vitreous carbon; silicone rubber; polystyrene; polyethylene; polypropylene; Teflon (politef); the silica gel bead; gold; tin indium oxide; filter paper (nylon for example; cellulose and NC Nitroncellulose); standard copy paper or variant and spacer medium; other chromatography media; magnetic bead or granule; super magnetic bead grain or granule; gadolinium; useful other solid carrier among Gadolinia. granule or the present invention.Can pass through for example to use, but be not limited to dip-coating, spin coating, contact print, silk screen printing, ink-jet technology, spraying, dispersion and pass through to use soft lithography or BIACORE ^TM(Sweden) the microfluid printing of system (microfluidic printing) obtains the transfer of conjugated polyelectrolytes to solid carrier for Biacore, Uppsala.

Conjugated polyelectrolytes fixing can or be covalently bond to solid carrier by physical adherence and obtain, and it can at high temperature carry out, and perhaps undertaken by being embedded in the hydrogel matrix.Expectation conjugated polyelectrolytes of the present invention fixing can improve they the use easiness, be fitted into device (for example array), stability, robustness, fluorescence reaction with the process that meets the high flux screening (HTS) that uses microtitration plate and the mode of other expectation.During being fixed to solid carrier, the solvent that is used for conjugated polyelectrolytes of the present invention and prion protein can be, but be not limited to water, buffered aqueous solution, methanol, ethanol and combination thereof.In this step, also can add the carrier polymer of other type.Biomolecule, misfolded proteins matter, antibody, protein, peptide, misfolded proteins matter or other molecule also can be fixed on the solid carrier or microtiter well (microtiter wells) in.Have identification, evaluation or the trapping antibody of selectivity ability or other biomolecule also can with himself, fixing with conjugated polyelectrolytes (that is, mixing) with polyelectrolyte solution, perhaps the CPE of misfolded proteins matter has been captured in identification subsequently.When biomolecule, misfolded proteins matter, antibody, protein or peptide and conjugated polyelectrolytes of the present invention were fixed on the solid carrier, they formed complex by noncovalent interaction and described polyelectrolyte.This complex forms under the covalent chemical not having, and it is based on hydrogen bonding between conjugated polyelectrolytes and the prion protein, static-and apolar interaction.

VI array and line

According to the present invention, the big array of the identical or different conjugated polyelectrolytes that produces in each point or line can overcome single-sensor or based on the defective of the method for solution.Described array or parallel lines method have been developed with simple method with the parallel analysis of a kind of or different conjugated polyelectrolytes to one or different sample, described sample can comprise misfolded proteins matter, and wherein at least one step of this experimental design uses CPE to capture misfolded proteins matter.The main purpose of using array is use easiness, portability, quantitative, the selectivity that is increased in other character and the feature.Use this method, we can probe into and measure multicomponent sample and use the optionally ability of sensor points of part.The chance that this has given to analyze two or more target sample simultaneously and has carried out measuring in the chip.By conjugated polyelectrolytes and/or biomolecule, misfolded proteins matter, prion protein, antibody, protein or peptide are fixed on the solid carrier of arbitrary dimension and optional pattern (as array, line, point, post), can make up little, portable, easy reading and explicable device.

The instructions for use of multiple array can be simultaneously detects more or less a large amount of samples.This carries out with the form of microarray usually, wherein many independently detecting elements (or probe) is incorporated on the little surface area, detects to be used for large-scale parallel.We are verified can to print conjugated polyelectrolytes and conjugated polyelectrolytes/protein complex by the micro-contact printing that uses the elastomer imprinter.Also can pass through point sample conjugated polyelectrolytes solution,, perhaps be undertaken in the lip-deep transmission of microarray by other above-mentioned method perhaps by the ink-jet polyelectrolyte solution.These steps are that many pixels of preparation microarray is necessary.

Embodiment

The PrP-amylaceous in the solution is captured and detected to embodiment 1. use conjugated polyelectrolytes Albumen.Utilize the surface of fluorescence microscope to detect

Comprise PrP and PrP-amyloid sample in the test tube, the latter is PrP ^Sc-or PrP ^Res-sample albumen.The 1mg/mlrecHuPrP90-231 that is dissolved in the 3M guanidinesalt hydrochlorate by the dialysis to water prepares the PrP-amyloid.Another testing regulations be by under the high salt concentration condition in test tube jolting prepare the PrP-amyloid.In this solution that comprises PrP and PrP-amyloid, add conjugated polyelectrolytes PTAA (10 μ g/ml),, make PTAA capture, in conjunction with and the PrP-amyloid of dyeing.Then, leave standstill this PTAA/PrP-amyloid and form thing, then, can easily it be separated from sample, in the time of if necessary, detect to the bottom that is settled down to test tube.Other method of collecting the PTAA/PrP-amyloid is, but be not limited to filter, centrifugal, on the surface of capturing, having covalently bound PTAA on the hydrophobic surface, capture, use functionalized granule, salt precipitation and immunoprecipitation.Be used to detect or observe the method for PTAA/PrP-amyloid comprise, but be not limited to fluorescence microscope, the fluoroscopic examination in plate reader or spectrofluorimeter, the absorption detecting in plate reader or spectrometer, array fluorescence reader, photodiode, fluorescence polarization or anisotropy, circular dichroism etc.This method also provides a kind of capture, remove, remove or filter PrP-amyloid, PrP from given sample in solution ^Sc, PrP ^ResMethod.In this special example, the PTAA/PrP-amyloid is formed thing be collected on the glass substrate, and use fluorescence microscope observation, referring to Fig. 6.Use this instrument, its resolution can be identified the independently PrP-amyloid granule less than 1 μ m * 1 μ m * 1 μ m, and it is equivalent to be less than the PrP of 2pg.

Embodiment 2: filter, capture

For separating natural with the fibril insulin, thereby this fibril insulin of purification carries out the filtration of polymer-insulin sample.The sample of measuring is natural insulin and fibril insulin, and it contains or do not contain PTAA.At first, the concentration of PTAA is 0.5mg/ml, and concentration of insulin is 2mg/ml, and sample comprises 10 μ l PTAA+25 μ l insulins in the phosphate buffer of the 20mM of 1ml pH8.For all samples, before and after filtering, all to carry out fluorescence measurement and open light filter, observation is checked.The filter that uses is 0.22 μ m Millex-gu, and every kind of sample filtering of 1ml is passed filter.

Pass in filtration before and after the filter of 0.22 μ m, the fluorescence spectrum of PTAA-natural insulin (PTAA-ins) and PTAA-fibril insulin (PTAA-fib) is presented among Fig. 9.Before filtering, spectrum looks as expection.After filtration, the sample that clearly contains PTAA and fibril insulin is retained in filter (seeing that fluorescence reduces), does not pass through and the sample that contains natural insulin can impact polymer.Therefore, can use filtration to remove the natural insulin that is bonded to PTAA.After filtration, the observation inspection of filter is shown, on filter, have the aggregation (seeing red aggregation) of PTAA-fibril insulin, and the filter that contains the PTAA-natural insulin does not demonstrate aggregation or change in color.

Also carried out detecting the measurement of the PTAA-fibril insulin complex substance existence of capturing in the described filter.Preparation only contains the sample and the sample that contains natural insulin of PTAA as mentioned above, and described natural insulin comprises 0,5,50 and 100% fibril insulin.Along with the increase of fibril insulin content, the content of natural insulin reduces, and causes that the insulin total amount in all samples is identical.Sample filtering is passed 0.8 μ m Millex-aa filter, and measure the fluorescence of filtrate.Then, the buffer by extruding 1ml oppositely passes filter and contrary-filters described filtrate.Afterwards, measure the fluorescence of contrary-filtering filtrate.

The fluorescence spectrum of filtrate is presented among Figure 10 for the first time.Increase fibril content and can cause that more PTAA-fibril insulin complex substance is retained in the filter, and PTAA and natural insulin can freely pass.Contrary-filtering sample is presented among Figure 11.Though most of complex still are trapped in the filter, clearly after contrary-filtration, increase fibriilar amount and cause that more PTAA-fibril insulin complex substance is dissolved in the solution.Therefore, PTAA can be used for capturing the fibril insulin.

Use filtration to capture PTAA-fibril complex and be not limited to above-mentioned filter and example.Can use filter, substrate, buffer, concentrate and the additive of any other type, and can pass through acquisitions such as fluorescence measurement, absorbance measuring, observation inspection, microscopic method, fluorescence polarization or anisotropy, circular dichroism and analyze and detect.

Embodiment 3:

Insulin-the amyloid in the solution is captured and detected to the use conjugated polyelectrolytes.Sharp Solution with fluorescence measurement detects

In test tube, will contain the sample of natural insulin and the 20mM phosphate buffer pH 7.0 of PTAA (0.05mg/ml, 10 μ l) and 965 μ l and mix, described natural insulin comprises 0,1,5,50 and 100% fibril (2mg/ml, 25 μ l).By under 65 Celsius temperatures, produced the fibril insulin in 8 hours at pH 2 incubation natural insulins.With sample centrifugal 5 minutes with 10000rpm, remove supernatant, add new buffer, and repeat this process once.Described PTAA is in conjunction with the fibril insulin, and in the centrifugal bottom that is deposited to test tube down.Then, use fluorescence microtest plate reader measuring samples.Measured again do not have centrifugal PTAA-natural insulin and PTAA-fibril insulin with reference to sample.The result is presented among Figure 12.

Described PTAA-fibril aggregation is deposited to the bottom of test tube, and it can easily be collected by removing supernatant after centrifugal to be used for detection.By dissolving described precipitate, can detect PTAA-fibril complex by fluorescence measurement afterwards.Be used for detecting or observe the method for PTAA/PrP-amyloid include, but are not limited to fluorescence microscope, at the fluoroscopic examination of plate reader or spectrofluorimeter, absorption detecting, array fluorescence reader, photodiode, fluorescence polarization or anisotropy, circular dichroism etc. in plate reader or spectrometer.This method also provides a kind of and captures from given sample in solution, removes, removal or sifting starch shape albumen and fibriilar mode.Just as shown in this embodiment, increase fibriilar amount and obtain more PTAA-fibril complex.From 1% fibril to 100% fibril, fluorescence intensity increases, and demonstrates the combination between PTAA and the fibril, because after centrifugal, does not have PTAA and natural insulin.

Embodiment 4: CPEs's is fixing on solid carrier

This embodiment has described and has scribbled on the glass surface of APTES fixedly PTAA.Described silane APTES is the amido that is used for chemical Coupling in order to have.EDC (or EDAC) is used to be coupled the zero-length cross-linking agent of carboxyl to primary amine.This cross-linking agent can be used for multiple application, as form amido link in peptide is synthetic, hapten is bonded to carrier protein to form immunogen, with 5 ' phosphate labeling nucleic acid and amine-reactive NHS-ester of setting up biomolecule.

At first, can activate have carboxyl ,-CPEs (as PTAA, POWT, tPTAA or tPOWT) of COOH, then it is coupled to any kind of, as the amine surface of functionalized magnetic bead.The CPEs (as POMT, tPOMT and PTT) that can will not comprise any carboxyl but have an amido is coupled to activatory carboxyl surface.If the magnetic bead with carboxyl is arranged, as long as put it in the EDC/NHS solution.

Testing regulations, PTAA's is fixing

Use TL1 washings (5 parts of H ₂O, 1 part 25% NH ₃, 1 part 28% H ₂O ₂, be heated to 85 ° degrees centigrade 5 minutes, wash with water) and TL2 washings (6 parts of H ₂O, 1 part 37% HCl, 1 part 28% H ₂O ₂, be heated to 85 ° degrees centigrade 5 minutes, wash with water) cleaning glass microscope slide, removing Organic substance and inorganic matter, and make its possess hydrophilic property.Use N ₂Dry.

Microscope slide is placed vaporizer.The APTES of 200 μ l is placed described chamber,, cured 60 minutes at 150 ° degrees centigrade 60 ° of degrees centigrade of evaporations 10 minutes.In dimethylbenzene, wash, and be stored in the dimethylbenzene.For different vaporizers, the amount of APTES may be different.

Preparation is at H ₂The mixture of the EDC of 2mM among the O and the NHS of 1mM.

1 part of EDC/NHS is mixed with 1 part PTAA.

The PTAA-EDC/NHS of 100 μ l is administered on the microscope slide, and in the dark incubation is 30 minutes.

With phosphate buffer pH 8 washing surfaces of 20mM 3 times.Use N ₂Dry.

PTAA is covalently bond to the surface of thin layer, and it can be used for the combining target molecule.Can use some improvement, as the material of concentration, pH, buffer, incubation time and use to testing regulations.Selected the concentration of EDC/NHS to activate sub-fraction carboxyl among the PTAA, be bonded to the amount of the PTAA on surface with increase, and keep PTAA in conjunction with character.Preferably, should only activate a side chain of described polymer.Whether the concentration of not estimating this report is best.The buffer composition that can change pH and be used to wash.In the testing regulations of the Covalink microtitration plate that is used for Nunc, described lavation buffer solution is NaCl, the 10g MgSO of 116.9g ₄* 7H ₂O, 0.5ml polysorbas20,1 liter of PBS.Can change incubation time to improve the result.Can use sulfo--NHS to replace NHS, because it can be more stable a little.

Testing regulations, POMT's is fixing

Because POMT does not comprise the carboxylic acid side group, the method that is used for fixing is opposite with the method for PTAA.Should use the surface such as the PEG substrate that comprise carboxyl to replace the APTES surface.Use BrCH ₂COOH and NaOH handle described PEG substrate to introduce the COOH group, afterwards, before adding POMT EDC/NHS are applied to described surface to activate described carboxyl.Also can use other surface.Do not estimate this method.Referring to document " Photografted Poly (ethylene glycol) Matrix for AffinityInteraction Studies ", Biomacromolecules, the 8th volume, the 1st phase, 287-295 page or leaf, the method explanation in 2007.

Testing regulations, POWT and PTT's is fixing

Because POWT and PTT comprise carboxyl and amido simultaneously, use EDC/NHS can cause crosslinked between the polymer chain, rather than crosslinked between polymer and the surface, this does not expect.Yet,, might produce and be covalently bond to lip-deep thicker crosslinked polymer layer, even obtain better result by adjusting the concentration of EDC/NHS and polymer.Can use APTES surface or comprise the surface of carboxyl.Do not estimate this method.

The surface

In the above-described embodiments, used microscope slide to fix.Also can use other surface, as silicon wafer, bead, microtitration plate (Covalink of Nunc with APTES surface pre-coating), or any other surface with suitable character.

Other technique for fixing

Also can use other technique for fixing.For covalent bond, can use functionalized polymer with biotin or other end group.For non-covalent combination, can use protein layer physical absorption from the teeth outwards.Polymer for example can be, is coated on the Nunc Maxisorp microtitration plate that is coated with asuro.Also can use any other can with polymer-coated to the surface technology.

Sample is added to the surface

Can in a different manner sample to be evaluated or that capture be joined polymer-coated surface.Can be simply the sample of certain volume be joined the surface, time that incubation is suitable and washing.The sample that can use flow-catheter (Flow canal) guiding appropriate amount is to the surface, and it obtains more repeatably system.If the use beadlet can join it in sample solution, incubation, and use centrifugal, Magnetic Isolation or any other isolation technics that it is separated from solution.

Embodiment 5: with the lanthanide series nano-particle of conjugated polyelectrolytes coating

This embodiment has shown CPE and lanthanide series nano-particle such as Eu ₂O ₃, Gd ₂O ₃Or Nd ₂O ₃Between specificity to interact be possible.These granules can be any granule of particle diameter from several nanometers to the hundreds of nanometer, and it is in turn intended for in-vivo imaging misfolded proteins matter and according to of the present invention and the disease misfolded proteins qualitative correlation.The particulate modification of lanthanide series is possible, as the multiple metal that mixes, as Fe ³⁺, Eu ³⁺, Tb ³⁺Deng.In coating or before in conjunction with CPEs, might pass through the described lanthanide series granules of pre-coating such as biological example molecule, diethylene glycol, alkane, carboxyl, citric acid, amido, further add other molecule afterwards.

For CPEs and the Gd that confirms some selections ₂O ₃Interaction between the nano-particle has been write down the fluorescence of CPEs, referring to Figure 13.Excitation wavelength is set at 400nm, and the measurement concentration of CPE is set at 2.5 μ g/mL, and uses the stock solution available from Sigma-Aldrich to dilute 200 times the Gadolinia. granule.The measurement that in redistilled water, occurs herein, but it also can carry out in the buffer solution of many types or complex media.

Claims

1. pharmaceutical formulation, it comprises optional functionalized CPE and optional pharmaceutically suitable carrier.

2. preparation according to claim 1; wherein said conjugated polyelectrolytes has one or more ion side chains and/or terminal degree of functionality; wherein said ion side chain and/or terminal degree of functionality are selected from the group of being made up of following: aminoacid; amino acid derivativges; neurotransmitter; monosaccharide; nucleic acid; DNA; RNA; peptide; histidine; histidine 10; protein; the peptide support; biotin; avidin; streptavidin; chelating agen; active group; antibody; enzyme; part; receptors ligand; steroid; biomolecule or other molecule; nano-particle; microgranule; gold nano grain; the gold microgranule; magnetic bead; the beadlet of protein coating; the beadlet of peptide coating; super magnetic bead grain or granule; the gadolinium nano-particle; gadolinium ion; the nano-particle of doping lanthanide series; the Gadolinia. nano-particle of doping lanthanide series; lanthanide series granule, or the derivant of its combination and chemical modification.

3. conjugated polyelectrolytes, it uses in the treatment of diseases method that the gathering by misfolded proteins matter causes, and described method comprises the separation of patient's body fluid being carried out aggregation misfolded proteins matter.

4. conjugated polyelectrolytes, it uses in the treatment of diseases method that the gathering by misfolded proteins matter causes, described method comprises the CPE of patient's effective dose that administration suffers from described disease with the further gathering that suppresses misfolded proteins matter or in conjunction with the misfolded proteins matter of intermediate forms, and is further removing these materials in the reaction.

5. according to claim 3 or 4 described conjugated polyelectrolytes; wherein said conjugated polyelectrolytes has one or more ion side chains and/or terminal degree of functionality; wherein said ion side chain and/or terminal degree of functionality are selected from the group of being made up of following: aminoacid; amino acid derivativges; neurotransmitter; monosaccharide; nucleic acid; DNA; RNA; peptide; histidine; histidine 10; protein; the peptide support; biotin; avidin; streptavidin; chelating agen; active group; antibody; enzyme; part; receptors ligand; steroid; biomolecule or other molecule; nano-particle; microgranule; gold nano grain; the gold microgranule; magnetic bead; the beadlet of protein coating; the beadlet of peptide coating; super magnetic bead grain or granule; the gadolinium nano-particle; gadolinium ion; the nano-particle of doping lanthanide series; the Gadolinia. nano-particle of doping lanthanide series; lanthanide series granule, or the derivant of its combination and chemical modification.