Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• • 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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders

Definitions

• This invention relates to methods, assays, and components for the rapid and sensitive detection and analysis of peptide aggregation associated with neurodegenerative diseases. Such methods and assays can be employed for clinical testing as well as to facilitate drug discovery by high-throughput drug screening.
• Neurodegenerative diseases including Alzheimer's,
• Parkinson's Gertsmann-Strausseler-Scheinker Syndrome, Fatal Familial Insomnia, Huntington's Chorea, Kuru, and Familial amyloid polyneuropathy and transmissible spongiform encephalopathies such as Creutzfeldt Jakob, Scrapie, and Bovine Spongiform Encephalopathy (BSE, Mad Cow), are characterized by ordered protein fibrils, aggregates that form in the brain. Although the proteins that make up these fibrils share no sequence homology, or even conserved motif, the fibrils themselves share certain morphological features. See Lansbury, Proc. NatL Acad. Sci. USA, 96:3342 (1999).
• peptides related to the pathogenic state which are normally soluble, undergo a conversion of their 3 -dimensional structure via a mutation of the native peptide or a physical association with altered peptides, to an insoluble, ordered polymerized state that is characteristic of the abnormal protein deposits that are found in the brain in neurodegenerative diseases.
• This ordered polymerizing or aggregation may result from seeding with indogenous or exogenous agents or peptides.
• these fibrils are made up of ⁇ -amyloid protein that has undergone a conformational change, from soluble monomers, to insoluble, ⁇ -sheet oligomers.
• concentration of these fibrils in the brain has been correlated to the progression of clinical disease.
• the growth profile of the characteristic fibrils is extremely non-linear, which could explain why its victims can appear asymptomatic for years, then suddenly undergo a rapid degeneration into dementia.
• Fibril formation in vitro is peptide concentration dependent.
• Short synthetic peptides, derived from the ⁇ -amyloid (A ⁇ ) protein can be made to form fibrils in vitro to mimic fibril formation that is characteristic of Alzheimer's disease.
• a ⁇ 1-42 (with an extended hydrophobic C-terminus) has been shown to form fibrils at a faster rate than A ⁇ 1-40. See J. Jarrett J et al, "The carboxy terminus of the ⁇ -amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer's disease," Biochemistry 32: 4693-4697 (1993). While A ⁇ 1-40 can form fibrils or aggregates on its own, solutions containing A ⁇ 1-40 undergo accelerated fibril formation if they are "mixed” with the less soluble 1-42 peptide or are "seeded” with pre-formed peptide fibrils.
• a ⁇ 1 -40 is the predominant protein in neuritic plaque, these studies indicate that the rate of fibril formation may depend on the ratio of the concentration of 1-42 to 1-40. Consistent with these findings, all forms of early onset AD involve higher expression levels of the 1-42 peptide.
• AFM Atomic force microscopy
• Drug candidates that act at a pre-symptomatic stage of the disease would have a greater efficiency in inhibiting plaque/fibril formation and preventing symptomatic disease.
• small fibril aggregates need to be efficiently detected.
• these aggregates are too small to be detected by nearly every detection method. Although they can be detected by AFM, this technique does not lend itself to clinical diagnostics or drug screening protocols. Therefore, at present, it has not been possible to screen for drugs that would act on the smaller fibrillar species. Additionally, screening for drugs to inhibit fibril formation at any stage has been severely limited.
• the rate of aggregate and fibril formation is an extremely non-linear function of the concentration of converted or misfolded peptide, such as mutant neuro peptide or converted prion peptides, which are aggregate-forming or fibril-forming species.
• concentration of the aberrant species reaches a critical concentration, the reaction rate proceeds too quickly to be affected by drug treatment. Therefore, for a drug to be effective at inhibiting plaque formation, thus making it a preventative therapeutic rather than a palliative one, it would necessarily have to act at an early stage.
• Current state of the art technology is not capable of detecting small aggregates or fibrils in a manner that is compatible with parallel drug screening methods or non-invasive diagnosis. This means that: 1) drugs to treat the early disease state cannot be readily identified; 2) pre-symptomatic patients cannot be identified; and 3) the effectiveness of potential drug candidates that exist, or will be identified in the future, cannot be accurately assessed.
• Congo Red and Thioflavin-T require mechanical intervention during the process. That is, the process requires transfer of fluid from one container to another, and the like which disturbs the assay in a non- reproducible way accelerating fibral formation. Additionally, addition of the Congo Red and Thioflavin components quenches the reaction and stops the aggregation process, thus several time points cannot be taken of a single assay in solution.
• the present invention provides a series of compositions, articles, kits, and methods associated with neurodegenerative disease.
• Techniques and components are provided for diagnosis of disease, as well as for screening of candidate drugs for treatment of neurodegenerative disease.
• the techniques are simple, extremely sensitive, and utilize readily- available components.
• the techniques and components of the invention are able to very sensitively detect neurodegenerative disease aggregate-forming or fibril-forming species, which typically are very small, e.g., between 1 and lOOnm in length and have heretofore been difficult or impossible to detect with existing technology and in screening for drugs to treat neurodegenerative diseases.
• the present invention therefore contemplates the ability to detect and monitor fibrils or pre-fibrils that are characteristic of early stages of these neurodegenerative diseases.
• the present invention also contemplates the ability to study the rate of aggregate formation in response to treatment with putative drug candidates.
• the present invention provides methods for the detection of aggregates and fibrils which can be used to screen for peptide sequences that are involved in the phenotype of neurodegenerative diseases.
• selected peptide sequences are synthesized and are fastened to or adapted to be fastened to particles and subjected to assays of the invention to see whether particle agglomeration occurs or other binding involving particles as described herein indicative of the peptide sequence's potential for participating in neurodegenerative disease processes.
• random peptide sequences are genetically engineered into a phage, which can be bound to a particle and participate in an assay of the invention.
• the methods of the present invention provide for detection of fibrils and peptide aggregates and can be used for the diagnosis of these diseases, since it has been shown that fibrillar species circulate in the cerebro spinal fluid (CSF) and other bodily fluids that can be readily sampled.
• CSF cerebro spinal fluid
• the present invention contemplates a detection system in which biological samples can be rapidly and sensitively analyzed for the presence of aggregates, fibrils, and pre-fibrils (aggregate-forming or fibril-forming species) that are characteristic of neurodegenerative diseases, including transmissible spongiform encephalophathis.
• the invention provides a series of kits that can be used for disease detection or drug screening.
• a kit includes a first article having a surface, a second article having a surface, and a plurality of binding species, at least some of which are fastened to or adapted to be fastened to the surface of the first article, and at least some of which are fastened to or adapted to be fastened to the surface of the second article.
• the binding species are capable of binding a neurodegenerative disease aggregate-forming or fibril-forming species.
• Each article can be a fiuid-suspendable, isolatable article, each being a colloid particle or one being a colloid particle and the other being a magnetic bead or the like, or one article can be a surface of a larger article such as an electrode, a surface plasmon resonance (SPR) chip, or other macroscopic article, and the other article can be a fiuid- suspendable particle as described above.
• SPR surface plasmon resonance
• kits of the invention includes an article having a surface (a variety of articles are contemplated), and a plurality of binding species at least some of which are fastened to or adapted to be fastened to the surface.
• the surface has a chemical functionality that substantially inhibits non-specific binding of binding species that are not fastened to or adapted to be fastened to the surface, or aggregate-forming or fibril-forming species.
• binding species means binding species capable of binding a neurodegenerative disease aggregate-forming or fibril-forming species.
• kits of the invention includes an article having a surface and a plurality of binding species fastened to or adapted to be fastened to the surface, and in this embodiment, the article is specifically not an SPR component.
• This embodiment allows for very sensitive, non- SPR analyses such as electronic analyses of diseased samples or drug activity.
• the invention provides a series of components, systems, and articles that can be used in conjunction with various kits and methods of the invention.
• One article of the invention includes a surface at which a binding species is fastened.
• a signaling element also is fastened to the surface of this article.
• a composition of the invention includes a binding species, and an electronic signaling entity immobilized with respect to the binding species.
• Another composition of the invention includes a binding species fastened to a moiety that can coordinate a metal.
• One system of the invention comprises at least two particles immobilized relative to a neurodegenerative disease fibril or aggregate.
• Another system of the invention includes at least two particles immobilized relative to a neurodegenerative disease aggregate-forming or fibril- forming species. These systems typically are the result of assays designed to detect diseased or pre-diseased states, or candidate drug screening mixtures.
• the invention provides a series of methods.
• One method involves providing a binding species, fastened to or adapted to be fastened to a surface of an article, and converting the binding species from a state in which it is not a neurodegenerative disease aggregate-forming or fibril-forming species to one that is an aggregate-forming or fibril-forming species. This method finds significant use in highly sensitive assays, as will be apparent from the detailed description below.
• a method of the invention involves allowing a binding species to interact with a neurodegenerative disease aggregate-forming or fibril-forming species and thereby to be converted to a neurodegenerative disease aggregate-forming or fibril-forming species.
• the converted species thereafter participates in agglomeration characteristic of the presence of neurodegenerative disease aggregate forming or fibril-forming species. This agglomeration then is detected.
• This method also represents a significant advance in sensitivity of disease state or pre-disease state screening.
• a method of the invention is provided which, while not intended to be limited to this purpose, can find use in drug screening and represents a significant advance in that arena.
• the method involves providing a binding species fastened to or adapted to be fastened to a surface of an article, optionally allowing the binding species to fasten to the surface (if unfastened initially), and allowing the binding species to bind a neurodegenerative disease aggregate-forming or fibril-forming species.
• the binding can occur before or after fastening of the binding species to the surface, and the binding species is not converted to an aggregate- forming or fibril-forming species during binding.
• a second binding species, fastened to or adapted to be fastened to a surface of a second article, is allowed to bind the aggregate-forming or fibril-forming species.
• the binding of the second binding species to the aggregate-forming or fibril-forming species can occur at any point in time relevant to other steps according to this method.
• Another method of the invention involves an article having a surface and a plurality of binding species fastened to or adapted to be fastened to the surface.
• the surface of the article is exposed to a sample suspected of containing neurodegenerative disease aggregate-forming or fibril-forming species.
• a variety of individual techniques can accompany this method, for example, for detection of disease state in a sample, as will be apparent.
• Another method of the invention involves first forming neurodegenerative disease aggregates or fibrils in a sample that contains aggregate-forming or fibril-forming species.
• the aggregates or fibrils are exposed to an article including a plurality of binding species fastened to or fastenable to the surface of the article.
• the binding species are capable of binding the aggregates, fibrils, aggregate-forming species, or fibril-forming species.
• the invention provides a technique for determining effectiveness of candidate drugs for inhibition of neurodegenerative disease.
• the technique involves exposing a cell adapted to secrete neurodegenerative disease fibril- or aggregate-forming species to a candidate drug and determining the aggregation potential of material secreted from the cell.
• the cell can be adapted to secrete aggregate- or fibril-forming species by transcription with an overexpression plasmid of, for example, A ⁇ fibril-forming species.
• the potential of the material secreted from the cell for aggregation characteristic of neurodegenerative disease can be monitored by exposing the cell environment, after exposing the cell to the candidate drug, to any assays of the invention, for example, assays designed to exhibit colloid agglomeration indicative of the presence of aggregate-forming or fibril-forming species.
• Another aspect of the invention is a series of methods for forming a self-assembled monolayer on a surface. These methods can be used to form monolayer-coated articles that can be used in conjunction with other aspects of the invention.
• One method involves forming a self- assembled monolayer on a surface by exposing the surface to a medium containing self- assembled monolayer-forming molecular species and a surfactant.
• a surface is exposed to a medium containing self-assembled monolayer-forming molecular species and a carboxylate.
• surfactant and carboxylate can be included together in the medium.
• Surfactants that can be used in accordance with this aspect of the invention to deposit a self-assembled monolayer onto a species include essentially any of a wide variety of surfactants.
• Carboxolates used to assist in self-assembled monolayer formation include preferably salts of carboxilic acids, including sodium citrate.
• Another method for self-assembled monolayer formation involves forming a self- assembled monolayer on a surface of a colloid particle. The formation does not occur during formation of the colloid particle itself.
• the monolayer can be formed on a fully pre-formed colloid particle.
• a self-assembled monolayer is formed on a surface of a colloid particle in suspension in a fluid, where the particle is not present at any fluid- fluid interface.
• peptides or other binding species capable of incorporating into, or binding, aggregates plaques and fibrils are modified with signaling moieties and then mixed with a biological sample that may contain disease- associated peptide aggregates/fibrils or agents capable of inducing aggregate formation.
• Other species, capable of incorporating into plaques and fibrils, modified with recruitment moieties that can be differentially attracted to a sensing surface, are also mixed into the same sample. After a suitable incubation period, the sample solution is subjected to conditions that differentially attract the recruitable peptides to a detection apparatus.
• signaling and recruitable moieties into pre-existing plaques or fibrils provides a sensitive and efficient method for labeling the aggregates and then concentrating them into a detection area.
• the sequences of the binding species attached to signaling and recruitment species can be chosen such that they can readily incorporate into an existing fibril or aggregate, but in the absence of a pre-existing fibril, would not readily aggregate.
• Peptides can be linked to signaling moieties by direct chemical bond attachment to labeled dendrimers or polymers. Alternatively. peptides can be linked to signaling moieties through association with a particle-like material, including colloids, that simultaneously present both binding species and signaling group.
• versions of species that can participate in fibril formation can be added, free in solution (labeled or unlabeled), to accelerate the aggregation process and thus act as amplifiers.
• Binding species can be linked to recruitment moieties by direct chemical attachment, such as attaching peptides to magnetic polymers that can be recruited to a detection area by an electromagnetic field or stationary magnet. Additionally, binding species can be linked to recruitment moieties by association with particle-like materials, such as magnetic beads. A- sepharose beads, (which present a surface functionality including a portion of protein A that will bind antibodies), or charged particles, that can then be differentially attracted to a sensing area or surface.
• Binding species can also be linked by direct chemical bond or by association with particle-like materials to recruitment moieties that are recruitable by virtue of a biological recognition unit, such as a peptide linked to a metal binding tag (such as a histidine tag) which then binds to a metal coordinated by a surface-bound chelate, such as nitrilotriacetic acid (NT A) which can be immobilized at a surface as part of a self-assembled monolayer (SAM) at a surface.
• Binding species or any other species can also be linked to a surface with use of a Strept tag, commercially available from Biometra, attached to self-assembled monolayer-forming species such as a thiol. Strept tag forming part of a self-assembled monolayer will readily fasten biotin- modified moieties such as biotin-modified binding species. Binding species can be linked to surfaces in other ways, as described more fully below.
• Figure 1 depicts peptides capable of participating in fibril or plaque formation attached to metal-containing compounds, such as ferrocene, via dendrimers or polymers. These derivatized peptides become associated with magnetic particles presenting similar peptides through their simultaneous incorporation into neuro peptide aggregates or fibrils. After recruitment to a SAM-coated electrode via a magnet, the presence of the aggregates is determined by detecting the presence of incorporated electroactive signaling moieties, like ferrocene, using techniques such as cyclic voltammetry (CV) or alternating current voltammetry (ACV).
• CV cyclic voltammetry
• ACV alternating current voltammetry
• Figure 2 depicts peptides capable of participating in fibril formation and a metal complex, such as a ferrocene derivative, attached to a colloid particle. These peptides become associated with magnetic particles bearing similar peptides when both are incorporated into aggregates of abnormal versions of these prion-type (or fibril forming) peptides in the solution. After recruitment to a SAM-coated electrode via a magnet, the presence of the aggregates is electronically detected.
• a metal complex such as a ferrocene derivative
• Figure 3 is a bar graph showing the detection of fibril formation by spectrophotometric means.
• Figure 4 is a photocopy of a photograph of a negative control in one assay of the invention.
• Figure 5 is a photocopy of aggregates, easily visibly identifiable to the unaided human eye, formed of an assay of the invention.
• Figure 6 is a photocopy of a photograph of small aggregates visible under a microscope, formed in an assay of the invention from 50 picomolar A ⁇ fibrils (figs. 4-6 all are magnified 40x).
• Figure 7 A is a photocopy of a photograph of a 40x magnified large colloid agglomeration, readily visibly identifiable to the unaided human eye, formed in accordance with another assay of the invention.
• Figure 7B is a photocopy of a photograph of the negative control of the assay of Figure 7A.
• Figure 8 is a photocopy of a photograph of an ELISA plate showing the results of a drug screening assay of the invention.
• Figure 9 is an overlay of four ACVs showing electronic detection of a fibril in another assay of the invention.
• Figure 10 shows the results of a light-scattering fibril assay of the invention.
• Figure 11 is the negative control of the light-scattering experiment of Figure 10.
• Small molecule means a molecule less than 5 kiloDalton. More typically less than 1 kiloDalton.
• neurodegenerative disease aggregates or fibrils refers to complexes of the homologous molecules (e.g. fibril aggregates) as well as mixtures of molecules (e.g. interacting molecules such as binding partners), typically proteinecous molecules, that are characteristic and expected to cause neurodegenerative disease.
• Aggregates of the present invention are typically of such a size (by virtue of the colloid interaction) that they can be visualized by eye, under a microscope, by light scattering, or by color changes.
• the phrase "render the fibril visible” refers to techniques whereby a non-visible fibril is made visible to the eye, visible under a microscope, or visible by absorbance. A variety of approaches are contemplated, including but not limited to attaching or decorating fibrils with colored particles.
• candidate drug refers to any medicinal substance used in humans or other animals. Encompassed within this definition are compound analogs, naturally occurring, synthetic and recombinant pharmaceuticals, hormones, antimicrobials, neurotransmitters, etc. This includes any substance (whether naturally occurring, synthetic or recombinant) which is to be evaluated for use as a drug for treatment of neurodegenerative disease or prevention thereof. Evaluation typically takes place through activity in an assay, such as the screening assays of the present invention. A variety of types of particles can be used in the invention.
• fluid suspendable particle means a particle that can be made to stay in suspension in a fluid in which it is used for purposes of the invention (typically an aqueous solution) by itself, or can be maintained in solution by application of a magnetic field, an electromagnetic field, agitation such as stirring, shaking, vibrating, sonicating, centrifuging, vortexing, or the like.
• a "magnetically suspendable” particle is one that can be maintained in suspension in a fluid via application of a magnetic field.
• An electromagnetically-suspendable particle is one that can be maintained in suspension in a fluid by application of an electromagnetic field (e.g., a particle carrying a charge, or a particle modified to carry a charge).
• a “self-suspendable particle” is a particle that is of low enough size and/or mass that it will remain in suspension in a fluid in which it is used (typically an aqueous solution) for at least 1 hour. Other self-suspendable particles will remain in suspension, without assistance, for 5 hours, 1 day, 1 week, or even 1 month, in accordance with the invention.
• a "binding species capable of binding a neurodegenerative disease aggregate-forming or fibril-forming species” includes any species such as a protein, peptide, sequence of either, antibody, Congo red, Thioflavin-T, or any species capable of the binding so described. In the case of antibodies, this binding is site-specific. In other cases it can be nonspecific. In the case of proteins or peptides, the binding typically involves non-specific ⁇ -sheet/ ⁇ -sheet interactions. Binding species also can include peptides, fragments, or whole proteins that are homologous to naturally-occurring neurodegenerative disease aggregate- or fibril-forming species.
• Proteins and “peptides” are well-known terms in the art, and are not precisely defined in the art in terms of the number of amino acids that each includes. As used herein, these terms are given their ordinary meaning in the art. Generally, peptides are amino acid sequences of less than about 100 amino acids in length, but can include sequences of up to 300 amino acids. Proteins generally are considered to be molecules of at least 100 amino acids.
• a "metal binding tag” refers to a group of molecules that can become fastened to a metal that is coordinated by a chelate. Suitable groups of such molecules include amino acid sequences including, but not limited to, histidines and cysteines ("polyamino acid tags"). Metal binding tags include histidine tags, defined below.
• chelate coordinating a metal or metal coordinated by a chelate, refers to a metal coordinated by a chelating agent that does not fill all available coordination sites on the metal, leaving some coordination sites available for binding via a metal binding tag.
• “Signaling entity” means an entity that is capable of indicating its existence in a particular sample or at a particular location.
• Signaling entities of the invention can be those that are identifiable by the unaided human eye, those that may be invisible in isolation but may be detectable by the unaided human eye if in sufficient quantity (e.g., colloid particles), entities that absorb or emit electromagnetic radiation at a level or within a wavelength range such that they can be readily detected visibly (unaided or with a microscope or the like), or spectroscopically, entities that can be detected electronically, such as redox-active molecules exhibiting a characteristic oxidation/reduction pattern upon exposure to appropriate activation energy (“electronic signaling entities”), or the like.
• Examples include dyes, pigments, electroactive molecules such as redox-active molecules, fluorescent moieties, up-regulating phosphors, or enzyme-linked signaling moieties including horse radish peroxidase and alkaline phosphatase.
• fastened to or adapted to be fastened in the context of a species relative to a surface of an article, means that the species is chemically or biochemically linked via covalent attachment, attachment via specific biological binding (e.g., biotin/streptavidin), coordinative bonding such as chelate/metal binding, or the like.
• fastened in this context includes multiple chemical linkages, multiple chemical/biological linkages, etc., including, but not limited to, a binding species such as a peptide grown on a polystyrene bead, a binding species specifically biologically coupled to an antibody which is non-specifically biologically bound to a protein such as protein A, which is covalently attached to a bead, a binding species that forms a part (via genetic engineering) of a molecule such as GST or Phage, which in turn is specifically biologically bound to a binding partner covalently fastened to a surface (e.g., glutathione in the case of GST), etc.
• a binding species such as a peptide grown on a polystyrene bead
• a binding species specifically biologically coupled to an antibody which is non-specifically biologically bound to a protein such as protein A, which is covalently attached to a bead
• a binding species that forms a part (via genetic engineering) of a molecule such as
• a moiety covalently linked to a thiol is adapted to be fastened to a gold surface since thiols bind gold very strongly, perhaps covalently.
• a species carrying a metal binding tag is adapted to be fastened to a surface that carries a molecule covalently attached to the surface (such as thiol/gold binding) which molecule also presents a chelate coordinating a metal.
• a species also is adapted to be fastened to a surface if a surface carries a particular nucleotide sequence, and the species includes a complementary nucleotide sequence.
• fastened includes some nonspecific binding, namely, intentional adherence of a species to a surface for purposes of a technique of the invention.
• Non-specific binding falling under the definition of "fastened” in the invention will result in attachment that withstands routine washing steps associated with assays of the invention.
• a non-specifically bound species is "fastened” to a surface if it will withstand at least one routine washing with an aqueous wash solution typically including a surfactant such as Tween-20 and a buffer, such as a phosphate buffer for maintaining the fluid at physiological pH.
• Specifically fastened or “adapted to be specifically fastened” means a species is chemically or biochemically linked to a surface as described above with respect to the definition of "fastened to or adapted to be fastened”, but excluding all non-specific binding.
• Non-specific binding is given its ordinary meaning in the field of biochemistry.
• colloids as used herein, is given its ordinary meaning in the field of biochemistry. Typically, colloid particles are of less than 250 nm cross section in any dimension, more typically less than 100 nm cross section in any dimension.
• a "moiety that can coordinate a metal”, a used herein, means any molecule that can occupy at least two coordination sites on a metal atom, such as a metal binding tag or a chelate.
• a component that is "immobilized relative to" another component either is fastened to the other component or is indirectly fastened to the other component, e.g., by being fastened to a third component to which the other component also is fastened, or otherwise is translationally associated with the other component.
• a signaling entity is immobilized with respect to a binding species if the signaling entity is fastened to the binding species, is fastened to a colloid particle to which the binding species is fastened, is fastened to a dendrimer or polymer to which the binding species is fastened, etc.
• Neurodegenerative disease aggregate-forming or fibril-forming species means biological species associated with neurodegenerative disease having sufficient binding capacity to bind to other molecules associated with neurodegenerative disease (including like molecules), to form fibrils or aggregates characteristic of neurodegenerative disease.
• Such aggregate- forming or fibril-forming species typically are characterized by a change in molecule conformation, relative to sequence-homologous, healthy counterparts, allowing them to bind more readily to like or similar molecules.
• aggregate-forming or fibril-forming species have the capability to convert binding species from non-aggregate-forming or fibril-forming conformation into aggregate-forming or fibril-forming conformation.
• Protofibrils have been reported to bind other molecules associated with neurodegenerative disease to form fibrils for aggregates characteristic of neurodegenerative disease. To the extent that this is the case, protofibrils are included in the definition of neurodegenerative disease aggregate-forming or fibril-forming species as used herein.
• the aggregate- or fibril-forming species typically include amino acid sequences from 1-38 to 1-44.
• “Diverse biological species” means different animals, such as mouse and hamster, mouse and goat, etc.
• sample refers to any cell, tissue, or fluid from a biological source (a "biological sample”, or any other medium that can advantageously be evaluated in accordance with the invention including, but not limited to, a biological sample drawn from a human patient, a sample drawn from an animal, a sample drawn from food designed for human consumption, a sample including food designed for animal consumption such as livestock feed, an organ donation sample, or the like.
• a biological sample drawn from a human patient, a sample drawn from an animal, a sample drawn from food designed for human consumption, a sample including food designed for animal consumption such as livestock feed, an organ donation sample, or the like.
• sample suspected of containing a particular component means a sample with respect to which the content of the component is unknown.
• a fluid sample from a human suspected of having neurodegenerative disease, but not known to have neurodegenerative disease defines a sample suspected of containing neurodegenerative disease aggregate-forming or fibril-forming species.
• sample in this context includes naturally-occurring samples, such as physiological samples from humans or other animals, samples from food, livestock feed, etc., as well as “structurally predetermined samples", which are defined herein to mean samples, the chemical or biological sequence or structure of which is a predetermined structure used in an assay designed to test whether the structure is associated with neurodegenerative disease.
• a "structurally predetermined sample” includes a peptide sequence, random peptide sequence in a phage display library, and the like.
• Typical samples taken from animals include blood, urine, ocular fluid, saliva, cerebro-spinal fluid, fluid or other samples from tonsils, lymph nodes, needle biopsies, etc.
• the term "poly-histidine tract” or “histidine tag” or “HIS-tag” refers to the presence of two to ten histidine residues at either the amino- or carboxy-terminus of a peptide or protein. A poly-histidine tract of six to ten residues is preferred for use in the invention.
• the poly-histidine tract is also defined functionally as being a number of consecutive histidine residues added to a protein of interest which allows the affinity purification of the resulting protein on a metal chelate column, or the identification of a protein terminus through the interaction with another molecule (e.g. an antibody reactive with the HIS-tag). Histidine-tagged ligands and His-tagged putative binding partners can be incubated together with NTA/Ni(II) presenting colloids. A visible reticulum will result if the two components are binding partners. Alternatively, the putative binding partners can be GST fusion proteins that would bind to glutathione presented on the colloid. Other linkers useful for attaching a binding species or other participant in assays of the invention to a surface include affinity tags. Affinity tags are well-known species used widely in biology, biochemistry, etc.
• FIG. 1 Shown in Figure 1 is an article 20, specifically an electrode including a SAM 22 on a surface thereof.
• SAM 22 includes "molecular wires" 24, in embodiments where it is desirable that SAM 22 be relatively electrically conductive.
• a mixed SAM is formed of a relatively non-conducting species such as an alkyl optionally terminating in a non-specific binding inhibitor such as polyethylene glycol, mixed with molecular wires.
• Molecular wires as used herein, means wires that enhance the ability for a fluid encountering a SAM-coated electrode to communicate electrically with the electrode. This includes conductive molecules or, as mentioned above and exemplified more fully below. molecules that can cause defects in the SAM allowing fluid contact with the electrode.
• a non- limiting list of additional molecular wires includes 2-mercaptopyridine, 2- mercaptobenzothiazole, dithiothreitol, 1 , 2-benzenedithiol, 1 , 2-benzenedimethanethiol, benzene-ethanethiol, and 2-mercaptoethylether. Conductivity of a monolayer can also be enhanced by the addition of molecules that promote conductivity in the plane of the electrode.
• Conducting SAMs can be composed of, but are not limited to: 1) poly (ethynylphenyl) chains terminated with a sulfur; 2) an alkyl thiol terminated with a benzene ring; 3) an alkyl thiol terminated with a DNA base; 4) any sulfur terminated species that packs poorly into a monolayer; 5) all of the above plus or minus alkyl thiol spacer molecules terminated with either ethylene glycol units or methyl groups to inhibit non specific adsorption. Thiols are described because of their affinity for gold in ready formation of a SAM. Other molecules can be substituted for thiols as known in the art from U.S. Patent No. 5,620,820, and other references.
• Molecular wires typically, because of their bulk or other conformation, creates defects in an otherwise relatively tightly-packed SAM to prevent the SAM from tightly sealing the surface against fluids to which it is exposed.
• the molecular wire causes disruption of the tightly-packed self-assembled structure, thereby defining defects that allow fluid to which the surface is exposed to communicate electrically with the surface.
• the fluid communicates electrically with the surface by contacting the surface or coming in close enough proximity to the surface that electronic communication via tunneling or the like, can occur.
• a particle 26, specifically a magnetic bead is provided along with a plurality of binding species, capable of binding a neurodegenerative disease aggregate-forming or fibril-forming species, fastened to or adapted to be fastened to the bead.
• a plurality of binding species 28 also are provided fastened or fastenable to signaling entities 30 which, in the embodiment illustrated, comprise dendrimers 32, each carrying a plurality of individual signaling entities 34.
• a plurality of target molecules 36, which are neurodegenerative disease aggregate-forming or fibril-forming species, are introduced by, for example, being included in a physiological sample suspected of containing the molecules.
• Binding species 28 bind to aggregate-forming or fibril-forming species 36 to define a linkage including bead 26 with attached binding species, signaling entity 30 with attached binding species, and aggregate or fibril-forming species 36 immobilized with respect to each.
• This arrangement can be drawn to a surface of electrode 20 via a magnet 38 under the electrode and, where signaling entities 34 are electronic signaling entities, their proximity near electrode 20 can be detected indicating presence of the aggregate or fibril- forming species 36 in the sample.
• electronic signaling entity 34 can be a redox-active molecule such as a metallocene, specifically ferrocene.
• Proximity of a ferrocene as signaling entity 34 near electrode 20 can be determined by a cyclic voltametric technique such as alternating current voltammetry (ACV).
• ACV alternating current voltammetry
• Binding species 28 need not be fastened to signaling entity 30 and/or bead 26 initially, but a mixture can be provided including a sample suspected of containing species 36, binding species 28, signaling entity 30, and bead 26, where binding species 28 is adapted to be fastened to signaling entity 30 and/or bead 26 if not already fastened.
• species 28 can be adapted to be fastened to signaling entity 30 and/or bead 26 by including a chemical or biological binding partner of a molecule fastened to signaling entity 30 or bead 26.
• Bead 26, if a polymeric bead can include a variety of covalently-attached linking molecules.
• entity 30 can be modified in this way.
• Bead 26 also can be coated with a surface layer of a material that facilitates formation of a SAM thereon, such as gold, and a SAM forming molecule (such as a thiol in the case of the gold surface) carrying a linking molecule can be formed thereon.
• Linkages between a linking molecule on bead 26 or signaling entity 30 and binding species 28 can include metal binding tag/metal/chelate linkages, complementary nucleic acid sequences, biotin/streptavidin, etc.
• a chelate can form part of a SAM on bead 26, and can be covalently attached to signaling entity 30.
• the chelate coordinates a metal, but leaves at least two open coordinate sites on the metal.
• a metal binding tag such as a polyamino acid tag can be incorporated into binding species 36, giving species 36 the ability to fasten to the bead or signaling entity by coordination of the metal binding tag to the metal.
• suitable chelates include nitrilotriacetic acid, 2,2'-bis(salicylideneamino)-6,6'-demethyldiphenyl, or l,8-bis(a-pyridyl)-3,6-dithiaoctane.
• binding species 36 can carry a terminal cysteine and fasten thereby to a gold surface of bead 26.
• binding species 36 can be selected.
• the binding species can be a peptide, protein, sequence from a protein, small molecule such as Congo red or Thioflavin-T, or sequence homologous to sequence derived from aggregate-forming species, RNA, DNA, nucleoside derivatives, or antibody to the aggregate-forming or fibril-forming species 36.
• a variety of surfaces can form components of kits or systems or participate in methods of the invention.
• Surfaces such as surfaces of beads, colloids, electrodes, ELISA plates, other multi-well plates, and the like can be used.
• aggregate- binding, but aggregate-formation-resistant binding species can be used.
• Aggregate-formation- resistant species can easily be selected by those of ordinary skill in the art from among species such as antibodies, fragments of proteins or peptides, etc. that will bind but not form aggregates.
• the surface of the article preferably has a chemical functionality that substantially inhibits non-specific binding of aggregate-forming or fibral-forming species. This can be accomplished in one embodiment by forming a SAM on the surface for presentation of a desired functionality for the assay, the SAM being a mixed SAM including other non-specific-binding inhibitors.
• NSB inhibitors include polyethylene glycol-terminated SAM-forming species.
• a SAM coating the colloid inhibits colloid/colloid self-aggregation.
• This can be accomplished by using a sufficient amount of SAM-forming species preventing charges moieties and/or carboxy-terminated species outward in the SAM.
• the charged moieties and/or carboxy-terminated species should be present in an amount of about 10 percent or more of the SAM, or preferably at least about 30 percent or at least about 45 percent of the SAM.
• species 28 is immobilized relative to or adapted to be immobilized relative to individual signaling entities 34 by way of each of species 28 and signaling entities 34 being fastened to or adapted to be fastened to a colloid particle 40.
• Species 28 and signaling entities 34 can be adapted to be fastened to colloid particle 40 by any of a variety of chemical or biological techniques known in the art or described herein. It is particularly convenient, with the use of gold colloid particles 40, to attach signaling entities 34 and binding species 28 to a thiol which will bind to the surface of colloid 40.
• a SAM can be formed coating a surface of colloid 40 including the signaling entities 34 and binding species 28.
• Electronic detection of signaling entities 34 near electrode 20 is routine using ACV or the like.
• FIG. 1 and 2 The assays illustrated in Figures 1 and 2 can be used for a variety of purposes.
• a kit including bead 26, signaling entity 30 ( Figure 1 ) or colloid particle 40 ( Figure 2), and binding species 28 fastened to or adapted to be fastened to bead 26, entity 30, or colloid particle 40 is provided and mixed in solution with the sample. If aggregate or fibril-forming species 36 is present in the sample, then this is indicated by the proximity of signaling entity 34 near electrode 20 after mixture and activation of magnet 38. If aggregate of fibril-forming species 36 is not present, then signaling entity 34 should not be drawn to the electrode upon activation of the magnet.
• aggregate or fibril-forming species 36 can be provided in known quantity in the sample in addition to a candidate drug for treatment of neurodegenerative disease.
• different candidate drugs will have different effects upon the binding or signaling entity 30 or colloid particle 40 to bead 26 via the aggregate or fibril-forming species 36.
• Candidate drugs that inhibit this binding are candidates for treatment of neurodegenerative disease.
• the assay can be established in such a way that a signal (proximity of signaling entity 34 to electrode 20) is proportional or otherwise related not only to the concentration of aggregate or fibril-forming species 36 in the sample, but to the degree of aggregation or fibril formation occurring within the sample.
• the signal will be proportional or related to the degree to which a particular drug candidate prevents aggregation or fibril formation characteristic of neurodegenerative disease from aggregate or fibril-forming species 36 present in the sample.
• binding species 28 bind aggregate or fibril-forming species 36 without conversion of binding species 28 to an aggregate- forming or fibril-forming species, to maintain a 1 : 1 correlation between binding events and signal. This can be done by selecting binding species 28 and aggregate or fibril-forming species 36 from diverse biological species, e.g. from hamster and mouse, respectively, or a sequence derived from the same species that binds 1 : 1 with target.
• binding species 28 from species that simply will bind neurodegenerative aggregate or fibril-forming species 36, but are not aggregate or fibril- forming species themselves, into aggregate or fibril-forming species with the ability to convert other binding species to aggregate or fibril-forming species.
• binding species 28 from species that simply will bind neurodegenerative aggregate or fibril-forming species 36, but are not aggregate or fibril- forming species themselves, into aggregate or fibril-forming species with the ability to convert other binding species to aggregate or fibril-forming species.
• amplifier species 29 can be provided in solution and mixed with a sample. In such an arrangement, even though a very small amount of aggregate or fibril-forming species 36 may be present in a sample, amplifier species 29 can be converted thereby to aggregate or fibril-forming species thereby rendering aggregate or fibril-forming species present in sufficient concentration to be detected using the assay. If no aggregate or fibril-forming species 36 is initially present in the sample, then amplifier species 29 will remain unconverted and little or no signal will be detected.
• amplifier species 29 amplify the concentration of aggregate or fibril-forming species 36 present in a sample to detectable levels, but it can facilitate massive aggregation of species 36 characteristic of neurodegenerative disease which in turn can form macro structures including many hundreds or thousands of beads, colloids, and/or signaling entities bound to many thousands of aggregate or fibril-forming species in a single macro structure. These structures can be easily detected electronically, and often visibly.
• Amplifier species 29 can be selected among all of the species listed above for binding species 28, but need not be the same as binding species 28 in a particular assay.
• binding species 28 in a particular assay can be a binding species, but without the ability to be converted to an aggregate or fibril-forming species
• amplifier species 29 can be convertible to define an aggregate or fibril-forming species. The latter situation (binding species 28 is not convertible but amplifier species 29 is convertible to a fibril or aggregate-forming species) it is useful in preventing particle/particle aggregation in the absence of a positive result (in the absence of aggregate or fibril-forming species 36 in a sample), but amplification of species 36, when it is provided in a sample, occurs, sensitizing the assay.
• a kit can be provided including colloid particles 40, binding species 28 fastened to or adapted to be fastened to colloid particles 40, and amplifier binding species 28. Such a kit can be mixed with a sample suspected of containing aggregate or fibril-forming species 36.
• species 36 can convert amplifier binding species 28 to aggregate or fibril-forming species, causing massive agglomeration and the formation of macro structures easily detectable, for example, visible by the unaided human eye, or visible under a microscope, detectable via a light scattering, absorbance or the like.
• colloid particles 40 need not necessarily carry any auxiliary signaling entities.
• binding species 28 be selected, and provided in concentration on surfaces of beads 26 and colloid particles 40 (where colloid particles are used), such that in the absence of auxiliary, non- surface immobilized aggregate-forming or fibril-forming species 36, particle agglomeration on particle/particle exposure is hindered within a time frame allowing comparison of (1) agglomeration in the absence of auxiliary aggregate or fibril-forming species with (2) agglomeration in the presence of auxiliary aggregate or fibril-forming species.
• a control can include no sample, and the rate and/or agglomeration level of the sample can be compared to that of the control.
• the methods and assays of the present invention be limited to only a particular fibril associated with a particular neurodegenerative disease. Indeed, the present invention contemplates detecting fibrils associated with a host of such diseases (e.g. Alzheimer's, Parkinson's, Creutzfeldt Jakob, etc.).
• the present invention provides a variety of assays and components for assays, for detection of disease or determination of effectiveness of drugs for treatment of such disease.
• the assays can involve electronic interaction of a sample or drug screen assay with a sensing electrode, or can involve aggregation, which can be detected by a variety of techniques, including but not limited to visual inspection, density scanning, light transmission, absorbance, color change and/or light scattering.
• binding species that can bind aggregate-forming or fibril-forming species or can incorporate into pre-existing aggregates or fibrils, are attached to colloids (e.g. gold colloids) or other particles (e.g. fluorescent beads). These particles, optionally along with free binding species (species that are not attached to colloids but are suspended or dissolved in a fluid in which the colloids are suspended) are mixed with solutions that contain aggregates or fibrils (e.g. patient samples). As the particles (e.g. colloids) become incorporated into the fibril, they render the fibril optically detectable.
• colloids e.g. gold colloids
• particles e.g. fluorescent beads
• repellant groups - such as charged moieties (COOH, PNA, charged peptides) - can be fastened to surfaces of the colloids. This causes the immobilized peptides to interact with the fibril rather than with each other and thus reduces the occurrence of false positives in a diagnostic assay.
• flexible groups, such as glycol units can be chemically inserted between the nitrilo tri-acetic (NTA) moiety and the thiol portion. The introduction of more degrees of freedom would slow the rate of interaction by increasing the entropic cost of the reaction. It is not intended that the present invention be limited by the method of detection.
• a signaling entity such as a dye
• a signaling entity can be fastened to one end of a peptide capable of incorporating fibrils and plaques.
• the signal-modified peptides incorporate into the fibril, they render it "visible” by concentrating the dye on its surface.
• fluorescent moieties and quenching moieties are attached separately to the ends of peptides. At low concentration in solution, they do not interact frequently with each other; however, when co-localized on a fibril, a quenching reaction takes place, causing a change in the fluorescence of the solution. These changes can be analyzed using standard ELISA plate readers.
• two separate compounds are attached to the N-terminus end of a peptide capable of incorporating into a fibril or plaque.
• the compounds are designed such that one compound modifies the second to render it active or detectable.
• the chemical modification is such that without the modification the compound does not fluoresce but after modification it does. Statistically, the reaction will happen orders of magnitude more frequently when the two compounds are co- localized on a fibril or plaque.
• electronic interaction serves as a detection technique, for example, where the presence of a redox-active molecule near an electrode surface can be detected, indicating binding of a species immobilized relative to the redox-active molecule to a species immobilized relative to the electrode, or relative to a recruitable particle that can be drawn to the electrode, such as a magnetic bead.
• an image of a sample can be digitized, then pattern recognition software is used to determine whether the sample contains aggregates.
• a sample as shown in Figure 8 can be digitized using a CCD camera, and pattern recognition can be used to determine color, aggregate size, aggregate spatial distribution, relative amount of aggregation, etc. This allows automation of both diagnostic screening and drug screening.
• Techniques of the invention can be carried out using individually spatially- addressable regions, such as different wells of a multi-well plate (see Figure 8) and/or a plurality of electrodes.
• a plurality of electrodes can be arranged in individual wells of a multi-well plate. or the like.
• Techniques of the invention can be carried out using individually spatially-addressable regions, such as different wells of a multi-well plate (see Figure 8) and/or a plurality of electrodes.
• a plurality of electrodes can be arranged in individual wells of a multi-well plate, or the like.
• the assays of the present invention can be readily adapted to screen drug libraries for compounds that inhibit aggregate or fibril formation.
• a drug-screening assay one can attach binding species to colloids (or other particle) and incubate with solutions containing aggregate-forming or fibril-forming species and a drug candidate.
• the solutions may or may not be agitated to accelerate the incorporation of peptide into the fibrils.
• the binding species are incorporated into the aggregates or fibrils, or aggregate-forming or fibril-forming species that then form aggregates or fibrils, they bring the attached colloids close to each other, which causes the colloid solution to change color (e.g. from a pink suspension to a dark blue precipitate in a clear solution). This transition is clearly visible by eye.
• absorption spectrophotometry the peak at 569 nm degrades as the colloids aggregate.
• solutions that contain 1 ⁇ M ⁇ -amyloid (A ⁇ ) fibril 46 "seeds *" and His-tagged ⁇ -amyloid peptides attached to gold colloids, and control solutions that did not contain the fibril seeds are subjected to absorption spectrometry. Both show the major absorption peak at 569 nm.
• the reaction containing means e.g. a cuvette, tube, microwell, etc.
• the colloids do not precipitate out of solution or change color, but act to decorate the fibril with visible particles. Dark red clusters appear in a cleared solution and are clearly visible by eye.
• the method of the present invention employs peptides (that can form fibrils characteristic of neurodegenerative diseases) which are modified with signaling and/or recruitment moieties and are incubated with a biologically-derived sample (or an in vitro mimic of such a sample) potentially containing converted or aggregated peptides indicative of the disease state.
• a biologically-derived sample or an in vitro mimic of such a sample
• the existence of peptide plaques, aggregates, or fibrils is detected via the incorporation of the labeled peptides.
• This assay can be done in a variety of ways, as described below, in a system that allows fast and efficient detection of fibrillar or aggregated species.
• one element can be a magnetic particle that bears a peptide that participates in fibril or plaque formation.
• the second element can be in the form of a colloid derivatized with a heterologous SAM that presents both a peptide that participates in fibril or plaque formation and a metal-containing compound, such as ferrocene, which acts as a signaling moiety.
• Peptides that participate in fibril or plaque formation can catalyze the misfolding and spontaneous aggregation of other peptides, such as those attached to the beads and colloids.
• the presence of these fibrillar or converted species of peptide can be detected in a sample by detecting the association of peptides on magnetic beads with peptides on signaling colloids through their simultaneous incorporation into fibrils or aggregates.
• an electromagnetic field can be applied that attracts the complex, which now contains magnetic particles as well as signaling colloids.
• magnetic particles provide a method of mixing biological recognition particles through a large sample volume and then rapidly recruiting the labeled targets to an electrode for detection.
• an oscillating potential is applied to the electrode, a current peak at the oxidation potential that is characteristic of the included metal indicates the presence of the aggregate at the electrode and, consequently, the presence of the aggregated peptides.
• free non-converted peptides can be added into the measurement solution to increase the rate of aggregation and/or the amount of material aggregated, thus acting as aggregation amplifiers.
• Aggregate formation can also be accelerated by varying the incubation and measurement conditions, including using frequency pulses and changes in temperature and electrical and magnetic fields.
• the complexes can be detected using a variety of techniques, however, the preferred detection method is alternating current voltammetry (ACV). This detection method can be supplemented by the use of additional analysis techniques such as higher order harmonic analysis. These complexes may also be able to be detected by optical means such as surface plasmon resonance (SPR). In using the latter technique, large shifts in optical properties are caused by recruitment of the complexes to the detecting surface.
• ACV alternating current voltammetry
• SPR surface plasmon resonance
• the aggregated state of these disease related peptides can also be detected by attaching potentially pathogenic peptides (binding species or aggregate or fibril-forming species) to either end of a linker, such that upon introduction of a polymerization seed or aggregate (aggregate or fibril-forming species optionally able to convert binding species to aggregate or fibril-forming species), the chains would form aggregates whose detectable properties would be altered from the original, non-aggregated state, e.g. by being visibly identifiable, rendered able to scatter light in light-scattering detection, change in viscosity in a flow detection system, electronic detection where an electronic signaling entity is linked to a component that forms part of the aggregate, etc.
• pathogenic peptides can be attached to functionalized hydrogels or to ordered "holes" in gel-like materials. Since the introduction of a seed or protein aggregate would cause the conversion of the presented, soluble peptides into polymers or aggregates, that have altered optical properties, these aggregates could be optically detected.
• the magnetic particle can be gold-coated and derivatized with a SAM that presents the desired peptide either by direct attachment of a peptide-thiol, or indirectly such as a DNA-peptide binding to a DNA-thiol incorporated in a SAM, or a histidine-tagged peptide binding to an NTA -thiol incorporated into a SAM.
• the colloid described herein may be a cluster of metal atoms, the preferred metal being gold.
• the magnetic particle can also be gold-coated and derivatized with a SAM that includes
• NTA-thiols for the capture of histidine-tagged peptides and peptide fragments.
• the SAMs can also be derivatized with DNA-thiols that are bound to a complementary DNA sequence that is also covalently attached to a peptide.
• This linker or spacer can be made of a variety of materials, including glycols or amino acids.
• the length of the spacer can be varied to promote a shift in the oxidation potential of the electroactive signal detection substance by creating an increased hydrophobic environment due to the proximity of the aggregates.
• the magnetic particle can also be replaced by a second colloid of larger dimensions and these larger colloids can be electromagnetically attracted to the electrode surface. However, they will only signal if decorated with satellite colloids that present signaling moieties. Alternatively, either colloid can be decorated with charged moieties that alter its mobility when an electromagnetic force is applied. All of the colloids can also be of similar size and an electromagnetic force can then be used to separate aggregated colloids from individual particles.
• the first particle could be a heavier particle or colloid that can be mechanically mixed during incubation and subsequently allowed to accumulate on the electrode surface by sedimentation when measurements are taken.
• magnetic beads can present pathogenic peptides and a transition metal complex.
• pathogenic peptides in solution interact with peptides on beads to form aggregates, the local environment of the transition metal changes such that it will oxidize at a higher potential.
• the presence of pathogenic aggregates or fibrils are detected as a shift in the oxidation potential of the transition metal or as the appearance of a second oxidation peak at a lower potential, when the system is electrochemically analyzed.
• An alternative one-particle system includes magnetic particles that display the normal peptide and amplifying peptides in solution that carry transition metal complexes. These transition metals can be linked to dendrimers or polymers, either conducting or non-conducting. As pathogenic peptides in the test sample promote aggregate formation, the metal-containing compounds become intertwined with peptides attached to magnetic particles resulting in an electroactive and magnetic complex that can be recruited and electronically detected.
• This assay system can also be constructed without using beads.
• Peptides with metal- containing compounds or peptide-thiols and transition metal complex thiols can be immobilized on or incorporated into SAMs on electrodes.
• Amplifying peptides may or may not be added to the measurement solution. If the test sample contains pathogenic or infectious peptides, then they attach to the surface immobilized peptides. This changes the local environment of the metal complexes and thus changes their oxidation potential. Amplifying peptides in the measurement solution may carry metal complexes that may be linked to dendrimers or polymers. If the test sample contains pathogenic peptides, then the aggregated species act to bridge the metalated peptides and the surface immobilized peptides.
• peptides incorporated into SAMs formed on electrodes by either direct thiol attachment or by binding a His-tagged peptide to a pre-formed NTA-SAM, can be introduced to a sample solution that contains pathogenic peptides and also colloids that present both peptide and signaling moieties such as ferrocenes. Again, their integration into fibrils/aggregates brings the signaling moiety (in this case a colloid) close to the electrode and a signal is transmitted.
• peptides derivatized with NTA groups can be attached to surfaces via an interaction with a peptide that contains a six histidine stretch.
• Amplifying peptides may or may not be added to the measurement solution. These techniques can also be adapted to screening for potential drug candidates to treat these neurodegenerative diseases. Specifically, in all of the described variations, a drug candidate can be added to the solution and its ability to inhibit aggregate formation as a function of time and concentration, which would translate into dosage, measured. Various parameters, such as a loss of signal or a shift in the position of the current peak can be indicative of a positive, and therefore therapeutically useful, effect.
• the drug candidates can be attached to beads bearing metal-containing compounds, which can be colloids coated with SAMs either with or without NTA-thiols, that oxidize at a different potential than those metal- containing compounds attached to the colloids having peptides attached to them.
• the signaling metal-containing compound can also be removed from the colloid that has peptides attached and the single current peak from the drug-containing colloids measured.
• a multiplexing drug screening device can be designed in which the drug candidates are added to microarrays and the whole array is flipped upside down onto a SAM-modified electrode lid for analysis.
• This competitive inhibition assay could also be performed in a system in which the signal detection compound is on a colloid containing drug candidates.
• a magnetic bead without metal-containing signal detection compounds carries peptides. Free peptides in solution can interact with the peptides bound to these magnetic beads. Putative drug candidates are then placed on colloids having metal-containing signal detection compounds. The metal-containing signal detection compounds on the colloids would signal if they interrupted the aggregation process and interacted directly with the peptide on the magnetic beads. One could also obtain a signal if the drug candidates interacted with aggregates. The latter information could be useful, but would likely require a subsequent multi-step assay to determine where the drug was acting.
• Drug candidates are added to solutions containing pre-formed fibrils, peptides attached to electronic signaling colloids, and peptides attached to magnetic beads. Following an incubation period, magnetic beads are attracted to a sensing electrode and analyzed by ACV. A loss of signal indicates that the drug candidate has inhibited fibril formation.
• the meta-stable protofibril can be selectively formed and purified to homogeneity by size exclusion chromatography, then used as the target fibril species for drug screening.
• alpha-synuclein the protein that incorporates into fibrils characteristic of Parkinson's disease, can be His-tagged and attached to electronic signaling colloids and magnetic particles. These particles are incubated with synuclein fibrils in solution or from a Parkinson's patient sample, then magnetically attracted to a sensing electrode for electronic analysis.
• peptides or proteins that are attached to surfaces are presented away from the surface (separated from the surface) so that the interactions of interest to occur.
• the particle or surface can sterically interfere with binding processes necessary for the assay.
• a flexible linker can be inserted between the protein or peptide and the His-tag.
• the length of the carbon chain, to which the NTA group is attached can be varied, and a number of linker groups can be inserted between the NITA group and the carbon chain.
• HS-(R)n-(X)m-NTA where S is sulfur, R is any molecule that can incorporate into a SAM such as CH 2 , X is a linker unit such as ethylene glycol, and NTA is nitrilo tri-acetic acid. Pulsing the .solutions with electromagnetic fields or mechanical agitation is used to accelerate the incorporation process.
• the method does not employ a sensing surface. Rather, the method involves aggregation, which can be detected by a variety of techniques, including but not limited to visual inspection, density scanning, light transmission, absorbance, color change and/or light scattering.
• Peptides which can incorporate into preexisting fibrils, are attached to colloids (e.g. gold colloids) or other particles (e.g. fluorescent beads). These peptide-presenting particles, along with free peptides, are mixed with solutions that contained fibrils (e.g. patient samples). As the particles (e.g. colloids) become incorporated into the fibril, they render the fibril optically detectable.
• a convenient approach is the 96-well ELISA plate format.
• the following reagents can be added to the ELISA plate in phosphate buffer (lOmM phosphate, pH 7.4, 100 mM NaCl) and the following concentrations can be conveniently used: a synthetic A ⁇ (1-40) peptide, with an N-terminal (His) 6 tag, at either 58.2 ⁇ M or 14 ⁇ M, 30 ⁇ L NTA/Ni ++ -presenting gold colloids (preparation described below) and 0.6 ⁇ M of A ⁇ -amyloid fibril "seeds".
• phosphate buffer pH 7.4, 100 mM NaCl
• concentrations can be conveniently used: a synthetic A ⁇ (1-40) peptide, with an N-terminal (His) 6 tag, at either 58.2 ⁇ M or 14 ⁇ M, 30 ⁇ L NTA/Ni ++ -presenting gold colloids (preparation described below) and 0.6 ⁇ M of A ⁇ -amyloid fibril "seeds
• the ELISA plate can then be incubated at 37°C, without agitation, for a desired period (e.g. 30 minutes to 2 hour). At 1 hour, large dark red aggregate structures are clearly visible in the wells that contained 58.2 ⁇ M (His) 6 -A ⁇ peptide. After a desired period (e.g. between 1 and 4.5 hours), the plate can be agitated briefly and then visually inspected. The aggregate structures are clearly visible in all wells that contained both (His) 6 -A ⁇ peptide and A ⁇ fibril "seeds". These aggregate, fibrillar-like structures are also able to form in solutions that contained up to 50% fetal bovine serum (FBS), albeit at a slower rate.
• FBS fetal bovine serum
• fibril seed concentrations as low as 10 picomolar are visibly distinguishable from solutions that contained colloids and A ⁇ peptide, but did not contain seed fibrils.
• the colorimetrics techniques described herein do not disrupt the assay process.
• a single fluid mixture is prepared and, without any transfer or other agitation required, the mixture can be observed to determine aggregate formation or lack thereof.
• a particular assay can be exposed to agitation or other form of energy. For example, it may be desirable in certain instances to determine whether introduction of energy into a particular system affects aggregate formation.
• Energy can be introduced in the form of agitation such as stirring, shaking, vibrating, sonicating, or other mechanical agitation, exposure to electromagnetic radiation such as infrared, ultraviolet, or visible light, exposure to radio frequency energy, microwave radiation, or essentially any electromagnetic radiation at any portion of the spectrum.
• agitation such as stirring, shaking, vibrating, sonicating, or other mechanical agitation
• electromagnetic radiation such as infrared, ultraviolet, or visible light
• radio frequency energy microwave radiation
• microwave radiation or essentially any electromagnetic radiation at any portion of the spectrum.
• Example 1 Spectrophotometic Detection of Fibrils
• 100 ⁇ L afiquots that contained 14 ⁇ M His-A ⁇ (1-40) peptides and 30 ⁇ L of NTA-thiol colloids were incubated with either 1 ⁇ M of A ⁇ fibril seeds, 50 pM A ⁇ , fibril seeds or no fibrils.
• the mixtures were incubated at 37°C for 0.5 hours then transferred to a cuvette and placed in a Hitachi U-2000 Spectrophotometer.
• the examples involve SAM formation. collagen coating, cell growth, colloid formation, and Alternating Current Voltammetry (ACV).
• SAM formation glass microscope slides were sputtered with a layer of Ti followed by a layer of Au. Each electrode was incubated at RT for 0.5 hours with 300 ⁇ L of a DMF solution that contained 10% methyl-terminated thiol (HS-(CH2)15 CH3), 40% tri-ethylene glycol- terminated thiol, HS(CH 2 ) n (CH 2 CH2) 3 OH, (formula) and 50% poly (ethynylphenyl) thiol (C 16 H 10 S).
• Gold colloids were derivatized with nitrilo tri-acetic acid/nickel self-assembled monolayers (NTA-Ni-SAMs), for the capture of histidine-tagged proteins, as described in Example 2. Colloids were coated with a low density of NTA-Ni (40 ⁇ M NTA-thiol in a total thiol concentration of 1000:M) to inhibit the aggregation of nearest neighbor peptides, immobilized on a common colloidal particle. 30 ⁇ L aliquots of the derivatized colloids were placed in wells of a 96-well plate.
• NTA-Ni-SAMs nitrilo tri-acetic acid/nickel self-assembled monolayers
• Histidine-tagged ⁇ -amyloid peptides (amino acids 1-40) were dissolved in phosphate buffer pH 7.4, then added to the colloid solutions such that the final concentration in a lOO ⁇ L volume was 14 ⁇ M.
• Pre-formed fibrils made from synthetic non-histidine-tagged ⁇ -amyloid peptides, were serially diluted and added to the solutions such that the final concentration of fibrils varied from 330 nanomolar to 50 picomolar. For every assay (each well), a negative control assay, in which no fibril was added, was performed ( Figure 4). Solutions were incubated at 37° C for 1 hour and care was taken not to agitate the solutions.
• colloids bearing 40 ⁇ M NTA-Ni SAMs were prepared as described in Example 2. 30 ⁇ L aliquots of colloids were added to wells of a 96-well plate. Histidine-tagged ⁇ -amyloid peptide (1-40) was added to achieve a final concentration of 20 ⁇ M in a final volume of 1 OO ⁇ L. Pre-formed ⁇ -amyloid fibrils were added such that the final concentration was 20 ⁇ M in a final volume of lOO ⁇ M. Pre- formed ⁇ -amyloid fibrils were added such that the final concentration was l ⁇ M. The sample was incubated at 37 degrees C for 20 minutes then rapped sharply three times to accelerate aggregation.
• Colloids agglomerated onto fibrils in the wells also enabling the identification of wells that contained drugs that inhibited fibril formation by noting the lack of visible aggregates in the wells. 40-fold magnification enhanced the ability to discriminate between the action of drugs in different wells.
• Figure 8 is a photocopy of a photograph of a small molecule library, rack 1 after 72 hours incubating at 37 degrees C.
• the wells on the right-most side of the plate (column 12) are negative control wells that remained pink, in which His-tagged GST replaced the ⁇ -amyloid peptide.
• column number 1 1 was the positive control that contained His-tagged ⁇ - amyloid and no drug candidate.
• Well G9 remained bright pink, indicating that the compound within that well inhibited fibril formation.
• Example 7 Using peptides free in solution to amplify
• Colloids presenting NTA-Ni-SAMs were prepared as described in example 2; 40 ⁇ M NTA- thiol in a total thiol concentration of lOOO ⁇ M was used. Histidine-tagged ⁇ -amyloid (1-40) peptides were added to achieve a final concentration of 58.2 ⁇ M in lOO ⁇ L final volume. As a negative control, an irrelevant histidine-tagged peptide, GST, was added to colloid solutions in place of the ⁇ -amyloid peptide. Solutions were incubated at 37 degrees C. Within 15 minutes large aggregates were clearly visible in solutions that contained histidine-tagged ⁇ -amyloid peptide. No structures were visible in control solutions.
• Our strategy to electronically detect ⁇ -amyloid fibrils was to first incorporate histidine- tagged ⁇ -amyloid peptides (1-40) into pre-formed fibrils made from ⁇ -amyloid 1-42 peptides. Colloids bearing NTA and a ferrocene derivative, for electronic signaling, were added so that at least some of the His-tagged ⁇ -amyloid peptides were attached to the colloidal particles. Our aim was to then add magnetic beads that bore binding ligands that would bind to the colloid-decorated fibrils and magnetically recruit them to the working electrode.
• Colloids bearing NTA-SAMs were prepared as described in Example 2 (with the exception that lOO ⁇ M octamethyl ferrocene-thiol was used in place of a standard ferrocene-thiol). 30 ⁇ L of colloids were added to each assay solution. To facilitate alternating current voltammetry (ACV) analysis, without having to pipet solutions after peptide addition (which would accelerate fibril formation of peptides in the absence of pre-formed fibrils), assay solutions were mixed in a 1ml. capacity silicon gasket clamped over a gold-coated glass slide that had been derivatized with a self- assembled monolayer as described in Example 3, over a stationary magnet.
• ACCV alternating current voltammetry
• Histidine-tagged ⁇ - amyloid peptide (amino acids 1 -40) were added to a final concentration of 14 ⁇ M in a lOO ⁇ L volume. Pre-formed fibrils made up of ⁇ -amyloid peptide (1-42) were added to achieve a final concentration of 15 ⁇ M. Negative control solutions were: (1) colloids, histidine-tagged GST plus fibrils, but no His-tagged ⁇ -amyloid peptide (1-40); (2) colloids. His-tagged ⁇ -amyloid peptide (1- 40) but no fibrils; and (3) all the components expected to give a positive result, but measured at time zero, before His-tagged peptides could incorporate into the fibril. Solutions were incubated at 37 degrees C for 20 minutes.
• NTA-Ni and ferrocene plus pre-formed fibrils that had been incubated at 37 degrees C for 20 minutes, produced a current peak of 2.0 ⁇ Amps at the characteristic oxidation potential (220 mVolts) of the ferrocene derivative (octamethyl ferrocene) that was attached to the colloidal particle, see Figure 9, trace A.
• a commercially available light scattering device that quantitates the average diameter of particles in solution, was used to analyze solutions that contained NTA-Ni-SAM coated colloids and histidine-tagged ⁇ -amyloid (1-40) peptides in the presence or absence of pre-formed fibril.
• Colloids were prepared as described in Example 2, histidine-tagged ⁇ -amyloid was added such that the final concentration was 10.2 ⁇ M, in a final volume of lOO ⁇ L.
• Pre-formed fibrils were added such that the final concentration was 2 ⁇ M.
• Baseline measurements, of average particle diameter were taken of the reagents alone to ensure that none of the components were aggregating in the absence of ⁇ - amyloid fibrils. Baseline Measurements:
• colloids presenting histidine-tagged A ⁇ 1 -40 peptides, mixed with 1 ⁇ M ⁇ -amyloid peptide
• FBS fetal bovine serum
• Negative control solutions which contained His-tagged A ⁇ 1 -40 immobilized on colloids but did not contain pre-formed A ⁇ fibrils, remained pink; no structures were observed under 40- or 100-fold magnification.
• Example 11 Using the Extent of Visible Fibrillar Structure to Diagnose Alzheimer's Disease in CSF Samples
• colloids presenting NTA-Ni are added to a solution containing a His-tagged PrP protein in its soluble (unconverted form) such that at least some of the His-tagged peptides are attached to the colloids.
• a sample suspected of containing infectious PrP is added.
• the infectious PrP will convert (induce aggregation that includes a radical conformational change from alpha helix to pleated beta sheet that is, unlike normal PrP, resistant to Proteinase K digestion) the normal PrP protein, both on colloids and free in solution, such that they can then convert other PrP proteins.
• This sort of signal amplification, or conversion amplification induces massive aggregation that can be readily detected as a colorimetric (pink to purple) change and the generation of large protein aggregates that cab be detected with the unaided eye or by light scattering devices.
• the added PrP His-tagged or not
• the added PrP must be derived from the same species as the PrP that is expected in the sample, or from a species whose PrP can be converted by the infectious sample.
• an assay that is quantitative and does not include the signal amplification described above or an assay can detect prion diseases of unknown species origin.
• a peptide or protein derived from the prion protein (PrP) such as sequences homologous to 1 19-141 (GAVVGGLG GYMLGSAMSRPMMHF) which was excised from Syrian hamster PrP would be histidine-tagged , preferably at the N-terminus and added to colloids presenting NTA-SAMs as described in Example 2.
• This sequence has been shown to bind to multiple sites on converted or infectious PrP and thus inhibit its ability to "convert" other normal PrP proteins.
• a sample suspected of containing infectious PrP would be added to the colloids and peptides.
• the presence of prion disease in a sample can also be electronically detected.
• Colloids presenting NTA-Ni and octamethyl ferrocene are added to a solution containing a His-tagged PrP protein in its soluble (unconverted form) such that at least some of the His-tagged peptides are attached to the colloids.
• Magnetic particles are also added to the assay that either present PrP or an antibody against it.
• a sample suspected of containing infectious PrP is added.
• the infectious PrP will convert the normal PrP both on the colloid and free in solution, causing them to also participate in converting other proteins which results in massive aggregation.
• Massive aggregation results which incorporates both magnetic particles and colloidal particles into the same aggregates, which are then magnetically drawn to an electrode that can be SAM-coated and analyzed by ACV.
• ELISA Enzyme- linked Immunosorbent Assays
• the plate is washed and the presence of the second species is detected by binding to it a "secondary" antibody that also has a signaling capability;
• the secondary antibody is usually conjugated to an enzyme, typically horseradish peroxidase (HRPO), alkaline phosphatase (AP), or a fluorescent tag which is capable of performing a reaction on an added substrate that results in a color change (detected by a spectrophotometer), or a fluorescence labeling tag that can be detected by a fluorimeter (see, for e.g., "Localization of a passively transferred human recombinant monoclonal antibody to herpes simplex virus glycoprotein D to infected nerve fibers and sensory neurons in vivo", Sanna PP, Deerinck TJ, and Ellisman MH ,1999, Journal of Virology Oct. Vol 73 (10 8817-23)).
• HRPO horseradish peroxidase
• AP alkaline phosphatase
• fluorescent tag which is
• the presence of the immobilized species is detected by binding to it a ligand attached to a colloid that also presents a plurality of horseradish peroxidase (HRPO) or alkaline phosphatase (AP).
• HRPO horseradish peroxidase
• AP alkaline phosphatase
• the enzyme can be conveniently linked to the colloid by a variety of means, including a histidine-tag attached directly to the enzyme, or by binding a mouse-anti-goat enzyme- conjugated antibody to a goat antibody that is attached to the colloid via a histidine-tagged protein G (Akerstom, B., Nielson, E., Bjorck, L. Jounal of Biological Chemistry, 1987 Oct. 5 Vol. 262 (28); pgs. 13388-91 and Fahnestock, S.R., Alexander, P., Nagle, J. and Filpula, D. (1986) Journal of Bacteriology Vol. 167, 870-880).
• a histidine-tag attached directly to the enzyme
• a goat antibody that is attached to the colloid via a histidine-tagged protein G
• the ratio of signaling molecules to binding events is increased by orders of magnitude.
• the binding of one antibody or ligand on the colloid to a presented antigen on an ELISA plate indirectly results in the binding of thousands or millions of enzymes.
• a known species can purposely be attached to wells of a 96-well plate so that one can probe with colloids that each present a separate drug candidate along with the signaling enzymes.
• each drug candidate can not be conjugated to an enzyme.
• the natural ligand for the immobilized target can be presented on the colloid along with the signaling enzymes and drug candidates added to each well of the plate to disrupt the interaction. Unbound colloids and thus their signal are lost in a wash step.
• the target for the antibody- or ligand-presenting colloids can be a cell or a protein bound directly or indirectly (via another antibody or ligand) to an ELISA plate.
• the advantage to this modification of an ELISA is not only sensitivity, but also efficiency. Because several hundred signaling enzymes remain bound via the colloids to one antigen, substrate hydrolysis will occur more quickly, and less time will be needed for an adequate reading.
• a binding species that binds a neurodegenerative disease aggregate or fibril-forming species, or an antibody to a fibril or aggregate-forming species, proto fibril or aggregate is immobilized in a well of an ELISA plate.
• the antibody then is exposed to a sample suspected of containing an aggregate-forming or fibril-forming species, and mixed with the sample is a binding species carrying a metal-binding tag (histidine-tag) optionally amplifier species, as described above, and colloid particles with a chelate coordinating a metal (NTA) and a non-electronic signaling entity each fastened to the particle.
• the nonelectronic signaling entity could be a fluorescent tag, horse radish peroxidase, alkaline phosphatase, or the like. Where one wants to screen for drugs using this assay, the same procedure is carried out but in the presence of a candidate drug for inhibiting neurodegenerative disease.
• Example 15 Cell-Based Screening Assay for Candidate Drugs for Inhibition of Neurodegenerative Disease at a Variety of Stages of Biochemical Progression of the Disease
• a candidate drug for activity against cellular processes involved in production of neurodegenerative disease aggregate-forming or fibril-forming species such as the enzymes ⁇ -secretase and the ⁇ -secretase.
• a cell In a well of an ELISA plate, a cell is placed which can secrete a fibril-forming or aggregate-forming species, specifically, the A ⁇ -fibril-forming species.
• the cell is transfected with an over-expression plasmid for A ⁇ (LaBlanc, et al., JNeurosci Res., April, 1992, 31(4):635-45).
• an over-expression plasmid for A ⁇ LaBlanc, et al., JNeurosci Res., April, 1992, 31(4):635-45.
• the well After a period of time sufficient to allow the cell to overexpress the A ⁇ fibril- forming species, into the well is introduced components of one of the above-described assays for drug screening.
• colloids and binding species fastened to or adapted to be fastened to the colloids and, optionally, amplifier species are introduced into the well.
• the ability of the drug to inhibit A ⁇ fibril-forming species (which drug is introduced prior to overexpression
• This example represents yet another aspect of the invention involving exposing a candidate drug for inhibition of neurodegenerative disease to a cell adapted to secrete neurodegenerative disease fibril- or aggregate-forming species, and determining the effect of the candidate drug on the aggregation potential of material secreted from the cell.

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