Methods for screening a biological fluid sample for an analyte associated with proteinopathy

Abstract

Disclosed herein is a method for screening a biological fluid sample for an analyte associated with proteinopathy which relies on a signal that is quadratically proportional to a mass difference generated by molecular interactions of a plurality of first molecular recognition elements (10) and a plurality of second molecular recognition elements.

Description

[0001] METHODS FOR SCREENING A BIOLOGICAL FLUID SAMPLE FOR AN ANALYTE ASSOCIATED WITH PROTEINOPATHY

[0002] Field of disclosure

[0003] The present invention lies in the field of analysis of biological fluid samples and in particular in the field of proteinopathy. The invention relates to a method for screening a biological fluid sample for an analyte which is associated with proteinopathy.

[0004] Background, prior art

[0005] Proteinopathies are a group of disorders which can affect the entire body. In the brain or in the peripheral nervous system, they are characterized by the appearance of pathological extracellular or intracellular protein accumulations. The pathology can be analyzed postmortem in the brain or in biopsies in peripheral tissues and the temporospatial distribution and abundance of the proteinopathy is used for disease staging and postmortem confirmation of the clinical diagnosis. In some neurodegenerative disorders, the sequence of appearance of proteinopathy follows neuroanatomical connections throughout the entire nervous system. This observation gave rise to the hypothesis that certain pathogenic species of the involved proteins (in some instances called ‘pathogenic seeds’) may spread in a time-dependent and region-specific manner between neurons leading to a neuron-to- neuron propagation and development of pathology. Some of the proteins and different proteoforms thereof generated during the pathogenic process can reach body fluids such as the cerebrospinal fluid or the blood. It is hypothesized that pathogenic seeds are also present in different body fluids. The neuropathological hallmarks are associated with synaptic and overall neuronal dysfunction and ultimately neuronal loss. Neuropathology is also accompanied by abnormally regulated local innate immune cells, a process which in some instances may appear as neuroinflammation. The concomitant and continuous breakdown of brain structure and function can occur over several decades and is reflected by a currently unstoppable worsening of non-motor functions (i.e., cognitive and overall social behavior) and in some disorders also motor functions. The early stages of neurodegeneration and the development of a proteinopathy often develop unnoticed for several years. Because often the early symptoms are subtle or not recognized, initially not specific to one disorder, and no so-called ‘cardinal symptoms’ can be observed, the disease stage before being diagnosed is considered ‘pre-clinical’ or prodromal, if a certain higher likelihood to develop a disorder is achieved based on preclinical symptoms. Clear detection of the preclinical stage and distinction of potential disease trajectories early on would help identify patients and improve diagnosis towards upcoming novel therapies for neurodegenerative disorders. In addition, it would be desirable to allow for better categorizing and stratification of the patients into clinical subtypes, and for monitoring disease progression and treatment efficacy.

[0006] The brains of a patient suffering from Alzheimer's disease, Parkinson's disease, Huntington's disease, or other related proteinopathies of the nervous system is interspersed with abnormal proteinaceous cellular inclusion bodies. In some disorders, similar inclusions can be found in the peripheral nervous system as well and some proteins or peptide fragments thereof accumulate in the extracellular space of the brain into so called plaques (for instance, in Alzheimer’s disease they are called senile or amyloid plaques). Analytically and therapeutically, such pathology forming proteins are drug targets and diagnostic markers since several decades. Analysis of brain tissue and exploration of disease mechanisms biochemically, in cell culture and in in vivo model systems suggest that disease-associated proteins undergo an abnormal metabolism and post-translational modifications and accumulate into poorly soluble moieties, which can be complex biochemical materials, reach micrometers dimension, and become crowded with poorly degradable material, often with oligomeric, proto-fibrillar, fibrillar or aggregated protein assemblies. Cellular inclusions of these proteins can contain collapsed organelles, lipid vesicles and other unspecified structures and material of cellular origin. Thus, these pathological features, which may be analytes associated with proteinopathy, are highly diverse and heterogeneous. The analysis of the composition of cellular inclusions and plaques of the brain is only possible postmortem. The totality of data from human tissue and model systems suggests multifactorial contributions to the development of a proteinopathy, including maturation over a longer period of time based on certain intrinsic factors (i.e. , genetic susceptibility, aging processes) or due to extrinsic triggers (i.e. viral or bacterial infection, toxins), and accumulation of specific proteins due to overproduction, or due to dysfunctional clearance mechanisms or lipid turn-over. Abundant proteins can reach high local or subcellular concentrations and thus have per se a higher chance to accumulate and become part of pathological hallmarks of different proteinopathies. The multifactorial causes, the development of multiple proteinopathies within the same disease spectrum and the inability to derive a biological sample most proximal to the neurodegenerative process (i.e., directly from the brain in a living human being) aggravate proper diagnosis and selection of treatment options on an individual patient level. Brain imaging with specific radioactive tracers (i.e., by positron emission tomography, PET) during the disease course allows so far for the detection of only one type of proteinacious inclusion (i.e., Tau tangles) and of extracellular amyloid-beta plaques. Some proteinopthay causing proteins or proteoforms thereof can be specifically detected and quantified by immunoassays or mass spectrometry in body fluids such as blood or cerebrospinal fluid. Only for very few and only for certain monomeric p-amyloid peptides and Tau proteoforms this has turned into a biomarker-based support of the clinical diagnosis so far. Detection by immunoassays of individual aggregated forms of proteins are so far not robust enough for proper validation and qualification or lack sufficient sensitivity and specificity to serve as diagnostic markers. Other methods propose to detect aggregated proteins in biological fluids by employing a so-called seed amplification assay. In this method, a biological fluid is suspected to contain protein aggregates that are associated with a particular proteinopathy and that can act as a seed to trigger aggregation of the monomeric recombinant form of the same protein, which is added as substrate to the amplification process. The in vitro amplified aggregates become typically detectable by an amyloid-specific fluorescent dye. So far, this assay shows low reproducibility between labs, assay performance allows only for a qualitative readout and the assay requires several days of handling and incubation. Together, this makes the current seed amplification difficult to develop into a diagnostic assay.

[0007] From a technical point of view, a prerequisite of currently known methods to screen for proteinopathies are molecular entities which selectively stain or label abundant hallmark proteins. The detection of aggregated proteins requires molecules which are selective for such. Aggregates as they underlie proteinopathies can be unforeseeable complex and host other proteins, lipids as well as collapsed cellular organelles as mentioned above. This fact makes it difficult, especially in Parkinson Disease, to reach selectivity for aggregates over monomers in bio fluids. The fact that PET tracer programs known so far have failed and that biomarkers are missing, supports the view that known methods cannot sufficiently well-

[0008] 5 distinguish between aggregated and non-aggregated matter.

[0009] The dissociation constants of antibodies targeting intrinsically disordered proteins typically have nM (nano-molar) affinity. Structured epitopes and / or targets with multiple binding sites enable to reach pM (pico-molar) affinity, well-structured oligomers and coherently assembled fibrils are such examples. All antibodies tested in Parkinson’s Disease (PD)0 targeting aSYN aggregates were not capable to discriminate between patients and control groups in biological fluids, at least with the established detection principles. This can be related with 1) unfavorable concentration ratios of aggregates and monomers in the investigated groups, 2) unspecific binding and lack of detection sensitivity in bio fluids by the applied physical detection principle, 3) a generally low abundance of targeted epitopes, 5 and / or 4) the wider spatial structure and constitutional complexity of the targeted aggregate. Published data suggest that the concentration range, which here means the concentration or amount of targeted epitopes, is matching the affinity-selectivity limit of all so far established recognition elements. The seed amplification assay is the only approach, which enables to distinguish between PD patients and healthy control groups. Interestingly, this0 assay does not require antibodies or other recognition elements in the first place, because it measures the nucleation potential of a large fluid volume, and it is known that traces of a seed can be sufficient to start the amplification processes.

[0010] Ideally, however, aggregates and co-aggregates that are associated with a proteinopathy would be detected as analytes in easy to access biological fluids without the need for5 complex tissue sampling (in particular human brain tissue, which is sampled mostly postmortem) and a method would be applied to investigate and identify the analytes in the undisturbed biological fluid.

[0011] Summary of disclosure It is the general object of the present invention to advance the state of the art in the field of screening methods for proteinopathies and preferably to overcome the disadvantages of the prior art fully or partly.

[0012] In advantageous embodiments, a method is provided which allows to screen for a proteinopathy in a body fluid sample having been obtained from a patient. In particular favorable embodiments, a method is provided which allows for an early detection of a proteinopathy. In advantageous embodiments, a method is provided which can be performed on a relatively easily obtainable biological fluid sample, such as blood or urine.

[0013] In other advantageous embodiments, a method is provided which allows to identify patients who are at higher risk of developing a disease or who have a specific subtype of the disease and thus allows for more personalized treatment and monitoring. In some favorable embodiments, a method is provided which allows for improved categorization and stratification of the patients into clinical subtypes, and for monitoring disease progression and treatment efficacy.

[0014] In further advantageous embodiments, a method is provided which allows to monitor disease progression and treatment response, in particular during clinical trials.

[0015] In further advantageous embodiments, a method is provided which has a high signal / noise ratio.

[0016] The general object is achieved by the subject-matter of the independent claims. Further advantageous embodiments follow from the dependent claims and the overall disclosure.

[0017] According to aspects of the invention, the invention relates to a method for screening a biological fluid sample for an analyte associated with proteinopathy. The method may in a first aspect comprises the steps of: a. Providing a sensing device, which comprises an evanescent illuminator being configured for generating an evanescent field from a beam of coherent light with a predefined wavelength on a first surface of the evanescent illuminator. The first surface of the evanescent illuminator, respectively the evanescent illuminator, comprises one or more sensing spots. Each sensing spot comprises a first recognition grating with a plurality of first unit cells and a second recognition grating with a plurality of second unit cells. The first recognition grating and the second recognition grating are interdigitated with each other such that the plurality of first unit cells are arranged in an alternating manner with the second unit cells and vice versa. A plurality of first molecular recognition elements which are configured to bind an analyte associated with proteinopathy, is bound to the first unit cells. Furthermore, a plurality of second molecular recognition elements, which are different from the first molecular recognition elements, is bound to the second unit cells. In addition, the one or more sensing spots are each configured such that i) at least a portion of coherent light of the evanescent field is scattered at the plurality of first unit cells to generate first constructive interference beams at a detector of the sensing device, wherein the first constructive interference beams have a first phase; ii) at least a portion of coherent light of the evanescent field is scattered at the plurality of second unit cells to generate second constructive interference beams at the detector, wherein the second constructive interference beams have a second phase which is inverse to the first phase of the first constructive interference beams; iii) the generated first constructive interference beams interfere at the detector with the second constructive interference beams to generate a mass difference dependent, and optionally time dependent, signal at the detector wherein the signal is quadratically proportional to a mass difference generated by molecular interactions of the plurality of first molecular recognition elements and the plurality of second molecular recognition elements; b. Providing a biological fluid sample to the one or more sensing spots of the evanescent illuminator; c. Generating a beam of coherent light with the predetermined wavelength at a predefined beam generation location; d. Measuring a signal for each of the one or more sensing spots at the detector. Each signal is quadratically proportional to the mass difference generated by molecular interactions of the plurality of first molecular recognition elements and the plurality of second molecular recognition elements of the corresponding sensing spot.

[0018] The method according to the invention uses recognition elements, such as for example antibodies or other elements as mentioned in the embodiments herein, but it bypasses most central limits of established detection principles. The sensing device reaches selectivity for aggregates over monomers likewise easily, because the underlying detection principle transduces the higher mass coming along with captured aggregates with quadratic signal intensity. A wider consequence is that the signal intensity is not anymore proportional or limited by the amount of epitopes, which may be rare or poorly accessible on the analyte associated with proteinopathy, i.e. its surface. This is particularly true if the analyte forms an aggregate. The entire mass of the analyte is transduced independently of the secondary peripheral or general chemical constitution of the analyte. The spatial lock-in detection principle of the sensing device is suppressing environmental noise, and unspecific binding is filtered out. Taken together, the method according to the invention preferably represents an approach to measure the mass of analytes, in particular aggregates, which come along with targeted epitopes, and in the presence of monomers; even if the analyte concentration is below the affinity limit of the molecular recognition element. The detection principle applied in the particular field of proteinopathies, where aggregated and high molecular weight analytes are required to be differentiated from monomers which are exposing similarly constituted epitopes, go beyond the concept of established immunosensors. The sensitivity for aggregates can be much higher than for the monomer, even, if the same amount of binding events is in fact counted. Therefore, the method of the invention can essentially weight and differentiate aggregates in proteinopathies.

[0019] It is understood that the referencing of the steps by letters a., b., c., d., etc. as well as i., ii., ill., etc. does not imply a specific order of steps, but that these letters serve as reference letters to identify a specific step of the claimed method. Although it may be the case that the method is performed sequentially step by step, starting from step a. until step d., it is also encompassed by the claimed invention that the steps are performed in another order or that at least some of the steps are performed simultaneously. As an example, it is also encompassed by the invention that step c., i.e. the generation of the beam of coherent light is performed before, after and / or during step b., i.e. the provision of the biological fluid sample.

[0020] It is generally understood herein that the term “comprising” is interpreted as meaning that it includes those features following this term, but that it does not exclude the presence of other features, as long as they do not render the claim unworkable. On the other hand, if the wording "consist of' is used, then no further features are present apart from the ones following said wording.

[0021] The term “predetermined wavelength” denotes the wavelength of the coherent light which is known in advance and which is typically a single wavelength (meaning that the coherent light is monochromatic).

[0022] The term “predefined beam generation location” denotes the location where the beam of coherent light is generated, and is also known in advance. Of course, in case the sensor is tunable (for example within very small ranges as regards to the exact location of the light source, as regards to the direction of impingement of the beam of coherent light, as regards to the predetermined wavelength of the coherent light, or as regards to the exact location of the detector), the predefined beam generation location is allowed to vary to an extent such that it is within the tuning range of the sensing device.

[0023] The term “unit cell” is not limited to any specific dimension, but merely serves to identify a location or region in which either the first or the second molecular recognition elements are bound. This does not mean that the unit cell must necessarily be 3-dimensional although this may in some embodiments be the case. Furthermore, the unit cell can have any desired shape, which may be a regular or an irregular shape. In other words, a first unit cell may also be referred to as “first region” and a second unit cell as “second region”. Furthermore, the plurality of first unit cells form together the first recognition grating and the plurality of second unit cells form together the second recognition grating.

[0024] Furthermore, it is understood by the skilled person that a beam of coherent light generated in step c. and the first unit cells are typically configured such that a portion of the coherent light being scattered at the plurality of first unit cells generates the first constructive interference beams at the detector of the sensing device having the first phase. Similarly, it is understood by the skilled person that the beam of coherent light generated in step c. and the second unit cells are typically configured such that a portion of the coherent light being scattered at the plurality of second unit cells generates the second constructive interference beams at the detector of the sensing device having the second phase being inverse to the first phase.

[0025] It is further understood that the sensing device comprises the detector. The detector may in some embodiments comprise one or more different detector sub-units. Preferably the detector may comprise one detector sub-unit per sensing spot of the evanescent illuminator. In particular embodiments, each detector sub-unit may be associated with a single sensing spot. The detector is typically an optical detector.

[0026] It is understood by the skilled person that the detector is arranged at a predefined detection location, respectively that each detector sub-unit is arranged at a predefined detection location. The term “predefined detection location” denotes the location where the first constructive interference beams interfere with the second constructive interference beams and where the signal mentioned in step d. is measured. The predefined detection location is known in advance. Again, in case the sensing device is tunable, the predefined detection location may vary to an extent such that it is within the tuning range of the sensing device. Only in case the coherent light has the predetermined wavelength, the beam of coherent light of this predetermined wavelength is generated at the predefined beam generation location, and the coherent light diffracted by the first molecular recognition elements the diffracted light generates first constructive interference beams at the detector with the first phase. Similarly, only in case the coherent light has the predetermined wavelength, the beam of coherent light of this predetermined wavelength is generated at the predefined beam generation location, and the coherent light diffracted by the second molecular recognition elements generates second constructive interference beams at the detector with the second phase.

[0027] Due to these inverse phases of the first constructive interference beams and the second constructive interference beams, the two interdigitated recognition gratings represent an optical comparator that measures the difference in diffraction efficiency of the two interdigitated recognition gratings (i.e. the first recognition grating and the second recognition grating). The difference in diffraction efficiency is proportional to the square of the difference in scattering mass per unit area (e.g. pg / mm2, picograms per square millimeter). Thus, the signal measured in step d. may be the diffraction efficiency difference.

[0028] The scattering mass is the spatial Fourier component of the mass density distribution that fulfils the diffraction condition of the recognition gratings. All Fourier components of the mass density that do not fulfil the diffraction condition are not detectable by the sensing device. This allows for the detection or monitoring of molecular interaction, such as binding of analytes in the biological fluid sample which contains a large amount and a vast diversity of background binding partners that might interfere with the molecular interactions of the analytes in a wash-free and real-time format, i.e. it allows for wash-free and real-time immunoassays. This is because the non-specific binding of background binding partners is diluted over a large spectrum in Fourier space and is thus not detected by the sensing device.

[0029] The biological fluid sample may typically be a sample having been obtained from a subject. The biological fluid is preferably any solution or suspension derived from a human or an animal body (i.e., body fluids such as but not restricted to blood, serum, plasma, cerebrospinal fluid, interstitial fluid, saliva, lacrimal fluid, urine) or is derived from in vitro cellular or biochemical systems. It is clear however that the method according to the invention as such is typically performed in-vitro. In some embodiments, the biological fluid sample has been obtained from a subject (e.g. a human subject), which has not yet been diagnosed with a proteinopathy. The biological fluid sample may also comprise all biological fluids and biochemically processed forms of tissue, cells, or excrements derived from a human or an animal body or from in vitro cell culture systems (i.e. , tissue or cell extracts or homogenates, stool samples). A biological fluid sample may also comprise intact cells or subcellular structures (for instance but not restricted to cellular nuclei, lysosomes, exosomes, vesicles, nucleic acids containing molecules such a DNA and RNA) isolated from a human or an animal body or derived from in vitro cell culture systems. The biological fluid sample typically contains the analyte associated with proteinopathy. Such an analyte is a moiety, in particular a molecular moiety, which is known to play a role in proteinopathy. For example, it may be a moiety which is in a certain form or three dimensional structure or which occurs at different levels in a biological fluid sample obtained from a patient suffering from a proteinopathy as compared to a healthy subject or a patient with a different condition. A biological fluid is preferably any aqueous fluid derived from a human or an animal body (i.e., body fluids such as but not restricted to blood, serum, plasma, cerebrospinal fluid, interstitial fluid, saliva, lacrimal fluid, urine) or derived from in vitro cellular or biochemical systems.

[0030] It is generally understood that the biological fluid sample may comprise a single analyte associated with proteinopathy or also multiple analytes associated with proteinopathy. In the latter case, the method may be performed for only one, a portion or all of the analytes associated with proteinopathy being present in the biological fluid sample.

[0031] The term “binding” as used herein can relate to any chemical bonding or physical force of attraction event in particular on molecular level, such as but not limited to one or more of covalent bonding, hydrogen bonding, ionic binding, Van-der-Waals forces, and the like. Similarly, a molecular interaction of the first molecular recognition elements or second molecular recognition elements as used herein typically comprises a chemical bonding or physical force of attraction event between the corresponding molecular recognition element and an interaction partner, such as the analyte associated with proteinopathy or a background binding partner. As mentioned above, the plurality of first molecular recognition elements are configured to bind the analyte associated with proteinopathy. In particular, of the first and second molecular recognition elements only the first molecular recognition elements are configured to bind the analyte associated with proteinopathy. Since the signal generated at each sensing spot is generated from the interference of the first constructive interference beams and the second constructive interference beams, which have inversed phases, the signal can then be a direct measure for the analyte associated with proteinopathy. It is understood the plurality of first molecular recognition elements are typically not bound to the second unit cells and the plurality of second molecular recognition elements are typically not bound to the first unit cells.

[0032] Typically the first molecular recognition elements and / or the second molecular recognition elements comprise one or more binding sites, such as binding sites with which they can bind to a target, for example an analyte associated with proteinopathy. In particular, the first molecular recognition elements and the second molecular recognition elements may be different from each other in that, and in particular only in that, their binding sites are different from each other.

[0033] In some embodiments, step b., i.e. providing the biological fluid sample to the one or more sensing spots of the evanescent illuminator, is performed such that the biological fluid sample is provided with a flow direction. That is, the biological fluid sample is provided such that it flows across the first surface of the evanescent illuminator and / or the one or more sensing spots. This may for example be achieved by a fluidic system of the sensing device. Such a fluidic system may for example comprise one or more channels, such as microchannels.

[0034] In some embodiments, the first molecular recognition elements and the second molecular recognition elements can be or comprise antibodies, particularly nanobodies, proteins, peptides, engineered sequences of natural L- or artificial D-type amino acids, peptidic polymers derived from amino acid-like molecules, oligonucleotides, single or double stranded sense or antisense oligonucleotide sequences or structures or combinations thereof. In particular embodiments, the first molecular recognition elements and the second molecular recognition elements are or comprise antibodies. It may also be possible that the first and / or second molecular recognition elements are chemical and / or physical binders being configured to bind the analyte associated with proteinopathy in any of the chemical bonding or physical force of attraction event as mentioned above under the definition of “binding”.

[0035] In some embodiments, the first molecular recognition elements comprise first linker elements. The first linker elements may for example be bound to the first unit cells. In some embodiments, the first molecular recognition elements additionally comprise a first binder unit being configured to bind the analyte associated with proteinopathy. In particular embodiments, the first linker elements may each be bound to the first unit cell and a first binder unit. The first binder unit may be antibodies, particularly nanobodies, proteins, peptides, engineered sequences of natural L- or artificial D-type amino acids, peptidic polymers derived from amino acid-like molecules, oligonucleotides, single or double stranded sense or antisense oligonucleotide sequences or structures or combinations thereof. For example, the first binder unit and the first linker element may together form a first molecular recognition element.

[0036] Such first molecular recognition elements may be generated as follows: The first unit cells may comprise a plurality of first binding sites. Then, a first binder unit being configured to bind the analyte associated with proteinopathy and being bound to a first linker forming element is exposed to the first binding sites. The first binding sites and the first linker forming element are configured to selectively react with each other to form a first linker element. Thereby, the first linker element is bound to both the first binding site and the first binder unit and is arranged between them.

[0037] The binding sites bound to the first and second unit cells may thus serve as anchor points for anchoring, i.e. immobilizing, certain first and second molecular recognition elements, respectively binder units. By undergoing a binding reaction with a linker forming element, a linker element is formed, which can be part of the corresponding molecular recognition element.

[0038] In some embodiments, the second molecular recognition elements comprise second linker elements or a second linker forming element. A linker forming element is generally a moiety which can undergo a reaction, in particular a selective reaction, with a reaction partner, such as a binding site, in particular a second binding site being bound to the second unit cells. It may therefore be possible that a binding site is bound to the second unit cells and a linker forming element is bound to a second binder unit. The linker forming element and the second binding site may then undergo a reaction with each other forming together the second linker element. Such a second linker element is then bound to the second unit cell and the second binder unit. In general, the second binder unit may be different from the first binder unit. For example, the second binder unit may be antibodies, particularly nanobodies, proteins, peptides, engineered sequences of natural L- or artificial D-type amino acids, peptidic polymers derived from amino acid-like molecules, oligonucleotides, single or double stranded sense or antisense oligonucleotide sequences or structures or combinations thereof. For example, the second binder unit and the second linker element may together form a second molecular recognition element.

[0039] Furthermore, the first linker forming element and the second linker forming element may be different from each other. Additionally or alternatively, the first binding sites and the second binding sites may be different from each other.

[0040] In some embodiments, the first linker forming elements are configured to selectively react with the first binding sites and optionally not with the second binding sites. In some embodiments, the second linker forming elements are configured to selectively react with the second binding sites and optionally not with the first binding sites. In particular embodiments, the first binding sites and the first linker forming elements may be complementary oligonucleotide strands being particularly configured to form a double stranded DNA (e.g. as a first linker element). In particular embodiments, the second binding sites and the second linker forming elements may be complementary oligonucleotide strands being particularly configured to form a double stranded DNA. (e.g. as a second linker element).

[0041] In particular embodiments, the first molecular recognition elements may each comprise or consist of a first linker element being bound to the first unit cells and a first binder unit being configured to bind the analyte associated with proteinopathy. In addition, the second molecular recognition elements may comprise, or in some embodiments only comprise a second linker forming element, e.g. as described above.

[0042] In some embodiments, the first molecular recognition elements, respectively the first binder units, are configured to bind to a monomeric form of an analyte associated with proteinopathy. In certain embodiments, the first molecular recognition elements (respectively the first binder units) are configured to selectively bind to the monomeric form of an analyte associated with proteinopathy over its formed aggregate (i.e. its aggregated form).

[0043] In some embodiments, the first molecular recognition elements (respectively the first binder units) are configured to bind to a formed aggregate of an analyte associated with proteinopathy (i.e. its formed aggregate). In certain embodiments, the first molecular recognition elements (respectively the first binder units) are configured to selectively bind to the formed aggregate (i.e. its aggregated form) of an of an analyte associated with proteinopathy over its monomeric form.

[0044] The method according to the invention allows to directly measure the captured mass (i.e. any molecular interaction between the molecular recognition elements and the analyte) in highly complex biological samples and can almost completely suppress environmental noise because it employs the spatial affinity lock-in principle and is inherently selfreferencing. Furthermore, the method can measure the mass increase at the molecular recognition elements with quadratic sensitivity. Thus, the method according to the invention can detect the mass of an analyte associated with proteinopathy or its formed aggregate, and can enable to determine the constitution or in situ formation of a complex aggregate. A formed aggregate may be complex, however as long as it exposes at least one binding site which can interact with the first and optionally second molecular recognition elements it can be detected. This is particularly favorable for certain analytes associated with proteinopathy, because they have been found to recruit additional material, such as any residual biological material, for example cellular components and subcellular components, such as mitochondria, cell membranes, vesicular membranes, nucleic acids, proteins and fragments thereof, which results in growing aggregates of highly heterogeneous and complex nature.

[0045] In some embodiments, the first molecular recognition elements and / or the second molecular recognition elements of a given first unit cell or second unit cell may each comprise only a single binding site (i.e. a binding site for binding to a target, such as the analyte associated with proteinopathy) per molecular recognition element or they may each comprise multiple binding sites per molecular recognition element, in particular multiple different binding sites per molecular recognition element. In certain embodiments, the first molecular recognition elements of a given unit cell may comprise only a single binding site being configured to bind the analyte associated with proteinopathy per first molecular recognition element or they may each comprise multiple binding sites being configured to bind the analyte associated with proteinopathy per first molecular recognition element, in particular multiple different binding sites being configured to bind the analyte associated with proteinopathy per first molecular recognition element.

[0046] In some embodiments, the first surface of the evanescent illuminator comprises a plurality of sensing spots. In such embodiments, it is understood that in this case each sensing spot contains first and second molecular recognition elements. Further, upon generation of the beam in step c. a portion of coherent light is typically scattered at each sensing spot, i.e. at each of its first and second molecular recognition elements in the manner described in step, a. i. to iii. Therefore, each sensing spot may generate its own signal from the interference of the corresponding first constructive interference beams and second constructive interference beams. In this case step d. comprises the measurement of each of these signals. In some embodiments, the measured signal or the measured signals is / are stored after step d. in a memory unit being part of or under control of a control unit, such as a circuit or a microprocessor.

[0047] In some embodiments, the first molecular recognition elements of a sensing spot, in particular of each sensing spot, of the plurality of sensing spots are different from the first molecular recognition elements of at least some of or all of the other sensing spots. Such embodiments allow multiplexing. In particular, the first molecular recognition elements of each sensing spot of the plurality of sensing spots are different from the first molecular recognition elements of each other sensing spot. In some embodiments, the first molecular recognition elements of one or of a group of sensing spots are unique as compared to other sensing spots of the sensing device. In particular, the first molecular recognition elements of each sensing spot or of each group of sensing spots are unique as compared to other sensing spots of the sensing device.

[0048] In some embodiments, the second molecular recognition elements of a sensing spot, in particular of each sensing spot, of the plurality of sensing spots are different from the second molecular recognition elements of at least some of or all of the other sensing spots. In particular, the second molecular recognition elements of each sensing spot of the plurality of sensing spots are different from the second molecular recognition elements of each other sensing spot. In some embodiments, the second molecular recognition elements of one sensing spot or of a group of sensing spots are unique as compared to other sensing spots of the sensing device. In particular, the second molecular recognition elements of each sensing spot or of each group of sensing spots are unique as compared to other sensing spots of the sensing device. In other embodiments, the plurality of second molecular recognition elements is the same for each sensing spot.

[0049] One advantage of providing a plurality, i.e. more than one, sensing spots with different first molecular recognition elements is that it allows to break down the complexity of the analyte of proteinopathy and allows for immunosignaturing, e.g. mass-weighted immunosignaturing. For example, a first sensing spot may comprise first molecular recognition elements which are configured to bind a first binding site of the analyte associated with proteinopathy, while a second sensing spot may be configured to bind a second binding site of the analyte associated with proteinopathy. Or, the different first molecular recognition elements bind the analyte associated with proteinopathy with different binding affinities. Since analytes associated with proteinopathy can in some embodiments be not only single molecules, but be aggregates of multiple moieties, such as cellular components or fragments thereof, proteins, nucleic acids and the like, using such multiple sensing spots can provide detailed information on the analyte. Body liquids derived from patients suffering from proteinopathies might have a similar immunoreaction profile as controls, as aggregated forms contribute with quadratic intensity to the measured signal, they can be distinguished from healthy controls. By using different first molecular recognition elements, the complexity of the analyte can be broken down. Each signal generated at the detector, respectively at specific detector units of the detector, is dependent on the mass increase effect of a molecular interaction between the corresponding first molecular recognition elements and the analyte. Therefore, each signal may be used as a quantity or parameter of the analyte in the high dimensional vector space. By employing multiple sensing spots with different first molecular recognition elements, an immunosignature, e.g. mass-weighted immunosignature, of biological fluid sample and / or the analyte can be generated. This immunosignature can then be used to identify or monitor the analyte associated with proteinopathy and allows to draw conclusions on the identity, nature and / or clinical or pathologic state of the proteinopathy of a subject from which the biological fluid sample has been obtained, in particular without sample work-up. This aspect should not be underestimated as aggregated proteins are soft and complexly constituted supramolecular assemblies of proteins, lipids and biological-chemical matter. Their corresponding high molecular weight makes them well detectable over monomeric forms, and on top of molecular recognition which also happens with monomers.

[0050] In some embodiments, the difference of first molecular recognition elements of a given sensing spot from first molecular recognition elements of another sensing spot is characterized in that first molecular recognition elements or at least their binding sites, are chemically distinct from each other. Additionally or alternatively, the difference of first molecular recognition elements of a given sensing spot from first molecular recognition elements of another sensing spot is in some embodiments characterized in that they are configured to bind a different analyte associated with proteinopathy.

[0051] Additionally or alternatively, the difference of first molecular recognition elements of a given sensing spot from first molecular recognition elements of another sensing spot is in some embodiments characterized in that they are configured to bind a different epitope of the analyte associated with proteinopathy.

[0052] Additionally or alternatively, the difference of first molecular recognition elements of a given sensing spot from first molecular recognition elements of another sensing spot is in some embodiments characterized in that they are configured to bind to the same analyte or epitope with a different binding affinity.

[0053] In some embodiments, at least some or all of the plurality of sensing spots, in particular their first and / or second molecular recognition elements, compete for the analyte associated with proteinopathy and step d. is performed for a predetermined measurement time to monitor competing of the sensing spots for the analyte associated with proteinopathy. In certain embodiments, also the signal measured for the predetermined measurement time is considered a time dependent signal and is comprised in an immunosignature of the biological fluid sample.

[0054] In some embodiments, in particular in embodiments in which at least some of the plurality of sensing spots compete for the analyte associated with proteinopathy, step b. is performed such that the biological fluid sample is provided to the plurality of sensing spots such that it comes in contact with one sensing spot after the other. These embodiments may be considered as a serial competition measurement. In such a serial competition measurement, at least some or even all sensing spots are in fluidic communication with each other. That is, the sensing spots are not separated from each other by a wall structure. In a serial competition measurement, it is possible that the biological fluid sample is provided with a flow direction, i.e. it flows in a particular direction and may thus sequentially contact the sensing spots. Preferably in such embodiments, step c. and optionally step d. is performed during and optionally already prior to step b. It may also be possible that the biological fluid sample is concomitantly provided to the plurality of sensing spots, in particular such that it comes essentially simultaneously in contact with all sensing spots. Preferably also in such embodiments, step c. and optionally step d. is performed during and optionally already prior to step b. In preferred embodiments of such serial competition measurements, the sensing device comprises sensing spots which comprise different first and / or different second molecular recognition elements. That is, the first unit cell of a given sensing spot may bind first molecular recognition elements which are different and preferably unique, as compared to the first molecular sensing spots being bound to the first unit cells of another sensing spot of the sensing device.

[0055] In some embodiments, at least some of the sensing spots are separated from each other, in particular by a wall structure. Such a wall structure may be configured to prevent that the biological fluid sample flows between the separated sensing spots, im particular during or after step b.. Such a measurement may be considered as a parallel measurement. In particular, the sensing spots may be divided into multiple groups of sensing spots, wherein the different groups of sensing spots are separated from each other, for example by the wall structure(s). Preferably, each group may contain one or more sensing spots. It may further be possible that each group is characterized by different first and / or second molecular recognition elements as compared to the first and / or second molecular recognition elements of another group of sensing spots. That is for example, the first group may comprise two sensing spots which each have the same first molecular recognition elements. However, the second group may also comprise two sensing spots which each have different first molecular recognition elements as the two sensing spots of the first group.

[0056] In some embodiments, the method further comprises the step of determining the origin of at least one or all of the signals which are measured at the detector. By determining the origin of the signal measured at the detector, it may for example be possible to identify the particular sensing spot from which the signal originated. Since it is known what first and / or second molecular recognition elements are present at each sensing spot, further information on the biological fluid sample and / or the analyte associated with proteinopathy can be obtained.

[0057] In some embodiments, the method further comprises the step of determining a parameter of the analyte associated with a proteinopathy from the mass difference dependent and optionally time dependent signal measured in step d.. The determined parameter may for example be single point, such as a mass or a mass per surface area, or the mass increase or decrease at the one or more sensing spots, in particular at its first molecular recognition elements, over time, or the slope or any higher derivative or its change of the mass increase or decrease at one or more sensing spots, in particular at its first molecular recognition elements, over time.

[0058] Additionally, or alternatively, the method further comprises the step of determining a parameter of an aggregate of the analyte associated with a proteinopathy or co-aggregate with the analyte associated with a proteinopathy from the mass difference dependent and optionally time dependent signal. Also in this case, the determined parameter may for example be single point, such as a mass or a mass per surface area, or the mass increase at the one or more sensing spots, in particular at its first molecular recognition elements, over time, or the slope or any higher derivative or its change of the mass increase or decrease at one or more sensing spots, in particular at its first molecular recognition elements, over time.

[0059] It is for example possible to observe in real time if the analyte associated with proteinopathy has recruited additional material to form an aggregate or co-aggregate. Furthermore, it is also possible to observe in real time the recruiting of such additional material. The additional material may be any residual biological material, for example cellular components, mitochondria, cell membranes, nucleic acids, proteins and fragments thereof.

[0060] In some embodiments, the determined parameter is comprised in the formed immunosignature of the biological fluid sample and / or of the analyte of proteinopathy. In some embodiments, the method further comprises the step of determining the presence of the aggregate or the co-aggregate from the determined parameter. In preferred embodiments, the presence of the aggregate or the co-aggregate is determined if the determined parameter exceeds a predetermined threshold within a predefined incubation time.

[0061] In some embodiments, the determining the presence of the aggregate or the co-aggregate comprises the comparison of the determined parameter with a reference parameter. In particular embodiments, the reference parameter is obtained from different patient populations, from a healthy subject or is a previous parameter of the subject from which the biological fluid sample has been obtained. In some embodiments, the determined parameter is compared with a database comprising a plurality of reference parameters, in particular a plurality of reference parameters of different patient populations, healthy subjects and / or previous parameter of the subject from which the biological fluid sample has been obtained. For example the database may comprise reference parameters for multiple patient populations which suffer from different proteinopathies (as compared to other patient groups in the database). It may also be possible that the database comprises multiple patient groups, which suffer from the same proteinopathy but at different clinical stages (as compared to other patient groups in the database). Such embodiments provide valuable data for physicians to assess the presence, nature, stage and progression of a proteinopathy.

[0062] In some embodiments, the analyte associated with proteinopathy comprises one or more of p-amyloid, tau, a-synuclein, prion proteins (such as natural or abnormally folded forms prion protein), fused in sarcoma, wild type or mutant poly-Q huntingtin, Ubiquitin, Ataxin-3, Optineurin, TAR DNA-binding protein 43, neurofibrilary light chain light (NfL), soluble or shed Triggering Receptor expressed on myeloid cells 2 (sTREM2), Chitinase-3-like protein 1 , Glial Fibrillary Acidic Protein and truncated or otherwise post-translationally modified forms of these. Post-translational modifications can comprise but are not restricted to phosphorylation, nitration, ubiquitination, glycation and glycosylation, oxidation and dityrosine bonds due to oxidation, and methylation. In certain embodiments, the analyte associated with proteinopathy may also be an aggregate or co-aggregate which comprises one or more of the above mentioned moieties (including itself or post-translationally modified forms of itself), and additional material, such as any residual biological material, for example cellular components, mitochondria, cell membranes, nucleic acids, proteins and fragments thereof.

[0063] In some embodiments, step d. is performed for a predetermined measurement time and each signal is measured as a function of the predetermined measurement time. A signal having been measured for a predetermined measurement time may in some embodiments be further comprised in the immunosignature of the biological fluid sample and / or of the analyte associated with proteinopathy. Upon measuring the signal for a predetermined measurement time, additional insight about the nature of the analyte and the proteinopathy can be gained. For example, it is possible to monitor the behavior towards the molecular recognition elements over time, in particular towards different molecular recognition elements over time, or to monitor changes over time upon exposing the provided biological fluid sample to changed conditions, such as pH, differently composed media, soluble binding partners (natural ligands) or synthesized or engineered molecules that act as artificial ligands or interfere with the stability of the analyte, such as for example aggregate stabilizer agents or aggregate destabilizer agents as mentioned herein.

[0064] In certain embodiments, during the predetermined measurement time, changes of each signal are measured, wherein the changes are effected by aggregation, in particular by aggregation of additional material in the biological sample, such as any residual biological material, for example cellular components, mitochondria, cell membranes, nucleic acids, proteins and fragments thereof, in particular cellular components and / or proteins.

[0065] In some embodiments, step d. comprises one or more single point measurements at a specific point in time, in particular an end-point measurement. In some embodiments, the single point measurements, respectively the signals obtained from the single point measurements, is comprised in the immunosignature of the biological fluid sample and / or the analyte associated with proteinopathy. It is understood that a single point measurement, in particular an end-point measurement, does not exclude the concomitant measurement for a predetermined measurement time. While it may in some embodiments be the case that step d. only comprises one or more single point measurement or only the measurement for a predetermined measurement time, both measurements may in some embodiments

[0066] 5 also be comprised in step d.

[0067] In some embodiments, the method further comprises a rinsing step. The rinsing step may be performed before step b. and in particular before, or during step d.. The rinsing step may be performed with a rinsing solution.

[0068] Alternatively or additionally, the method comprises in some embodiments a treatment step. 0 The treatment step may preferably comprise the application of the a treatment agent configured for changing the conditions. For example, the treatment agent may be selected from one or more of detergents, ionic agents, solvents, acids, bases, aggregate stabilizer agent and aggregate destabilizer agent. An aggregate stabilizer agent is an agent being configured for stabilizing aggregates and an aggregate destabilizer agent is an agent being 5 configured to destabilize, e.g. disassemble, aggregates. For example, the aggregate stabilizer agent or the aggregate destabilizer agent may be chemical molecules, such as small molecules (which have generally a molecular mass of <1000 Da) or large molecules (which have generally a molecular mass of >1000 Da), such as antibodies, Fab fragments or nanobodies. In certain embodiments, the aggregate stabilizer agent or aggregate0 destabilizer agent may be recombinant or synthesized forms of the analyte associated with proteinopathy, natural ligands, synthesized or engineered molecules that act as artificial ligands or interfere with the stability of the aggregated analyte. Such embodiments which comprise the application of aggregate stabilizer agent or an aggregate destabilizer agent are advantageous, because early stage proteinopathies may a have low level of rigid and5 stable aggregates whereas later stage proteinopathies or multiple system atrophy may have a higher level of rigid type aggregates with less tendency to decay. Thus, the application of an aggregate stabilizer agent or an aggregate destabilizer agent may provide insights on the stage of proteinopathy. Furthermore, the signal measured may in some embodiments also be comprised in the immunosignature of the biological fluid sample and / or the analyte associated with proteinopathy.

[0069] In some embodiments step d. is performed for a predetermined measurement time during the rinsing step and / or during the treatment step. This allows to monitor the changes of the measured signal over time and the effect of the rinsing step and / or treatment step. Such a signal measured over time may in some embodiments also be comprised in the immunosignature of the biological fluid sample and / or the analyte associated with proteinopathy.

[0070] In some embodiments, step d. comprises the single point measurement at a specific point in time before the rinsing step and / or treatment step. In some embodiments, step d. comprises the single point measurement at a specific point in time after the rinsing step and / or treatment step. In some embodiments, such measurement, respectively the signal obtained from such a measurement may also be comprised in the immunosignature of the biological fluid sample and / or the analyte associated with proteinopathy.

[0071] In some embodiments after step b. incubation of the one or more sensing spots with a labeled, in particular mass-labeled or fluorescence-labeled, binding agent is performed. The mass-labeled binding agent is a binding agent preferably having a known, identifiable and optionally unique mass. In certain embodiments, the mass labeled binding agent may for example comprise nanoparticles. The nanoparticles preferably comprise a transition metal or a transition metal oxide, such as gold, TiC>2, Ta2Os and silver. If a fluorescent-labeled binding agent is used, the fluorescent signal can in some embodiments be measured with a fluorescent detector. The fluorescent detector is typically an additional and thus separate detector. In some embodiments, the detected fluorescent signal is comprised in the immunosignature of the biological fluid sample and / or of the analyte associated with proteinopathy. In some embodiments, the binding agent may comprise a predefined binding site, which may particularly be configured to bind to the analyte associated with proteinopathy. In general, the predefined binding site of the binding agent may in some embodiments be configured such that it does not bind to the first and / or second molecular recognition elements.

[0072] In some embodiments, the binding agent is configured to bind to the analyte associated with proteinopathy, in particular to bind to lysosomal markers, mitochondrial markers, nucleotides, sugars, post-translational modifications or mitochondrial DNA of the analyte associated with a proteinopathy. This may in particular be advantageous if the analyte is an aggregate or co-aggregate of additional material, such as any residual biological material, for example cellular components, mitochondria, cell membranes, nucleic acids, proteins and fragments thereof, in particular cellular components and / or proteins.

[0073] In some embodiments, the signal measured during or after incubation of the one or more sensing spots with a labeled, in particular mass-labeled or fluorescence-labeled, binding agent, is also comprised in the immunosignature of the biological fluid sample and / or of the analyte of proteinopathy.

[0074] In some embodiments, the method comprises after step b. the step of performing a seed amplification assay. This does not mean that the seed amplification assay step must necessarily be performed before step c, although this may in some embodiments be the case. It may well be performed together with step c.. Performing the seed amplification assay comprises adding a binding reagent sample, in particular a protein sample to the one or more sensing spots. It is understood that the binding reagent sample is typically added after step b. since the analyte associated with proteinopathy being present in the biological fluid sample acts as a seed to trigger aggregation, for example aggregation of other monomeric proteins, in particular recombinant or synthetic forms of the monomeric analyte. Further, step d. comprises measuring each signal over a predetermined measurement time. Preferably, the protein sample comprises monomeric proteins. The monomeric proteins may act as substrate for the co- aggregation in the seed amplification assay. Depending on the presence and nature of the analyte of proteinopathy, the added proteins of the protein sample misfold and aggregate with the analyte during the predetermined measurement time. Since the aggregation causes a mass increase, it can be readily measured during step d.. In the seed amplification assay the bound analyte associated with proteinopathy (in particular an analyte forming an aggregate) may for example act in situ as a seed that coaggregates with recombinant or synthesized forms of the analytes associated with proteinopathy or other binding reagents that were provided as a treatment agent. The seed amplification assay can in some embodiments be further comprise adding in addition to the protein sample or as an alternative to the protein sample, other binding reagents. Binding reagents which may be comprised in the binding reagent sample may comprise but are not restricted to small molecules (i.e. having a mass of <1000 Da, such as PET tracer like molecules or amyloid structure binders, peptides or oligonucleotides as described above) or large molecules (i.e., having a mass of >1000 Da, such as antibodies, nanobodies, other polypeptides).

[0075] In particular embodiments a plurality of sensing spots each with different first molecular recognition elements as described herein are used. In such embodiments, the seed amplification assay produces different signals due to different interactions at the different sensing spots. The measured signals can in some embodiments be comprised in the immunosignature, e.g. mass-weighted immunosignature, of the biological fluid sample and / or of the analyte associated with proteinopathy. Such an immunosignature can be highly specific and thus unique for a certain analyte of proteinopathy and / or the clinical stage of the patient from which the biological fluid sample has been obtained.

[0076] In some embodiments, the method further comprises a secondary characterization step for characterizing the analyte associated with proteinopathy or a state of an aggregate or coaggregate formed by the analyte associated with proteinopathy. In preferred embodiments, the secondary characterization step includes ELISA, FT-IR, Raman or fluorescence spectroscopy. The secondary characterization can be performed at any time, in particular during or after step b.. The secondary characterization step provides a secondary characterization parameter. In some embodiments, the secondary characterization parameter is also comprised in the immunosignature of the biological fluid sample and / or of the analyte associated with proteinopathy. In some embodiments, the secondary characterization step is performed directly on the biological fluid sample having been provided to the one or more sensing spots of the evanescent illuminator. Thus, in such embodiments, the secondary characterization step is performed directly when the biological fluid sample is present on the sensing device.

[0077] In some embodiments, the signal or signals measured for each sensing spot during step d. form an immunosignature, e.g. mass-weighted immunosignature, of the biological fluid sample and / or of the analyte associated with proteinopathy. Optionally also other parameters of the analyte of proteinopathy are comprised in the immunosignature. For example as mentioned herein, in some embodiments, the parameter of the analyte associated with a proteinopathy determined from the mass difference dependent and optionally time dependent signal measured in step d. , the fluorescent signal of a fluorescent- labeled binding agent, signals measured during a seed amplification assay, the determined origin of at least one or all of the signals measured at the detector, the signal obtained from application of the treatment agent and / or the secondary characterization parameter are also comprised in the immunosignature of the biological fluid sample and / or of the analyte associated with proteinopathy.

[0078] The immunosignature, e.g. mass-weighted immunosignature, may in some embodiments be regarded as a vector, in particular a time-dependent vector, whose trajectory in the high dimensional vector space is unique for the screened biological fluid sample. The term “high dimensional” refers in this context to at least 3 dimensions or more. Every parameter obtained and / or every signal measured during step d. deconvolves the complexity of the biological fluid sample. The immunosignature, e.g. mass-weighted immunosignature, can be regarded as a unique fingerprint of the screened biological fluid sample. Therefore, it is in some embodiments possible to generate multiple immunosignatures over time for biological fluid samples having been obtained within defined time intervals, such annually or half annually, from the same subject and compare the immunosignatures with each other or with reference data as outlined further below. The immunosignature is thus a direct measure of aggregated proteins and / or the pathological state of the subject from which the biological fluid sample originates from. In some embodiments, the immunosignature of the biological fluid sample is compared to a database comprising immunosignatures, e.g. mass-weighted immunosignatures, of a plurality of samples. In particular the samples may be patient samples, autologous patient samples (i.e. previously screened samples from the patient from which the presently screened biological fluid sample has been obtained) and / or reference samples. In preferred embodiments, the immunosignatures of the database are each associated with a clinical status of a proteinopathy. Thus, by comparing the immunosignature of the screened biological fluid sample with the immunosignatures comprised in the database, it is possible to characterize the nature and / or clinical status of the proteinopathy with which the analyte is associated. Furthermore, it may be possible to forecast the progression of the proteinopathy at least to a certain extent. By virtue of such a database comparison it is also possible to perform a pattern recognition analysis during which the immunosignature of the screened biological fluid sample is analyzed for specific patterns which may be found in some of the immunosignatures stored in the database. This allows for detecting a proteinopathy at a very early state, in particular even before the subject from which the biological fluid sample originates, develops recognizable clinical symptoms.

[0079] Comparison with a database can in general be computer-implemented. The database may for example be stored in a memory unit, such as a server, a hard drive or a cloud. The comparison with the database may for example be conducted by a control unit, such as a circuit or a microprocessor. The high-dimensional and multimodal data type stored in relevant databases or generated in the afore described analyses may additionally be analyzed and / or processed by machine learning and artificial intelligence-type analytical methods to identify immunosignatures.

[0080] In some embodiments, the plurality of second molecular recognition elements are configured to not selectively bind the analyte associated with proteinopathy. In contrast, the plurality of first molecular recognition elements are configured to selectively bind the analyte associated with proteinopathy. In some embodiments, the plurality of second molecular recognition elements are configured to not bind the analyte associated with proteinopathy. Thus, such second molecular recognition elements are unable to bind the analyte associated with proteinopathy.

[0081] For example, in certain embodiments, the first and second molecular recognition elements may be or comprise antibodies.

[0082] While the first molecular recognition elements may comprise a paratope being configured to bind the analyte associated with proteinopathy, in particular its epitope, the second molecular recognition element may be devoid of this paratope. In particular embodiments, the absence of this paratope may be the sole difference of the binder units of the first and second molecular recognition elements, respectively the antibodies of the first and second molecular recognition elements. For example, this may already be achieved by a point mutation, such as a point mutation in the paratope which avoids binding of the analyte associated with proteinopathy. The second molecular recognition elements may also be any other molecule but may preferably be different from the first molecular recognition elements to generate binding related mass contrast between the first and second binder unit.

[0083] In some embodiments, the first molecular recognition elements and the second molecular recognition elements of each sensing spot are both configured to interact with the same background binding partners. Background binding partners may be any chemical or biological moieties which can be bound by the first and / or second molecular recognition elements, in particular beyond the analyte associated with proteinopathy. These may be present in the biological fluid sample. These may be molecules, such as small molecules or proteins, receptors on cells or cellular components being present in the sample or binders having epitopes being able to bind to the first and / or second molecular recognition elements. Such embodiments are advantageous, because since the signal obtained in step d. is due to the inversed phases of the first and second constructive beams a measurement of the difference (i.e. the mass difference) of interaction at the first and second molecular recognition elements, interactions of background binding partners being present in the sample and not being associated with the particular analyte associated with proteinopathy are inherently not or essentially not detected. This is particularly advantageous for highly complex biological fluid samples, such as CSF or human serum.

[0084] In some embodiments, the first molecular recognition elements and the second molecular recognition elements have essentially the same affinity KD to the same background binding partners.

[0085] In some embodiments, the second molecular recognition elements have an isoelectric point being between 0.8 times to 1.2 times, in particular 0.9 times to 1.1 times, more particular 0.95 times to 1.05 times, even more particular identical to, the isoelectric point of the first molecular recognition elements. Thus, in certain embodiments, the isoelectric point of the second molecular recognition elements and the first molecular recognition elements of the sensing spot (respectively for the same sensing spot of the plurality of sensing spots) may essentially be the same, in particular the same.

[0086] The first molecular recognition elements, respectively the first binder units, may be bound to the first unit cells by first linker elements. The first linker elements may be formed by a reaction, in particular a selective reaction, between a first linker forming element and a first binding site being bound to the first unit cell. The first linker elements may for example comprise two complementary oligonucleotide strands. The two complementary oligonucleotide strands may be a chip oligonucleotide strand being bound to or part of the first unit cells and a molecular recognition element oligonucleotide strand being bound to or part of the first molecular recognition element.

[0087] The second molecular recognition elements, respectively the second binder units, may be bound to the second unit cells by second linker elements. The second linker elements may be formed by a reaction, in particular a selective reaction, between a second linker forming element and a second binding site. The second binding elements may for example comprise two complementary oligonucleotide strands. The two complementary oligonucleotide strands may be a chip oligonucleotide strand being bound to or part of the second unit cells and a molecular recognition element oligonucleotide strand being bound to or part of the second molecular recognition element.

[0088] It is understood that the first linker elements and the second linker elements are typically orthogonal linker elements. That is, the first linker forming elements and the first binding sites selectively react with each other and the second linker forming elements and second linker binding sites selectively react with each other. However, there is typically no or essentially no cross-reaction. Therefore, it is possible to immobilize the first molecular recognition elements only on the first unit cells and the second molecular recognition elements only on the second unit cells. Such methods which can for example be used are described in Current Opinion in Chemical Biology, 2014, 18:8, 8-15, ISSN 1367-5931 , DOI: 10.1016 / j.cbpa.2013.10.023.

[0089] In some embodiments, the biological fluid sample comprises one or more background binding partners being different from the analyte associated with proteinopathy. The one or more background binding partners may be configured to trigger a non-specific binding signal in step d. The one or more background binding partner may for example be configured to bind to the first molecular recognition elements and / or to the second binding sites. In such embodiments, it may for example be possible that the second molecular recognition elements of the sensing device provided in step a. comprise, or consist of, the one or more background binding partners. This is advantageous, because since the method of the present disclosure relies on the mass difference between the first and second molecular recognition elements (and any entities bound thereto), using background binding partners as second molecular recognition elements saturates the second unit cells with the background binding partners before steps b. to d.. Therefore, a stable baseline is achieved and the influence of additional background binding partners in the biological fluid sample is significantly decreased. Thereby, non-specific binding events can be avoided or at least significantly reduced, which allows to monitor the specific binding events by the first molecular recognition elements to the analyte associated with proteinopathy. The background binding partner may for example be bound to the second unit cells by a second linker element as described herein. For example, the second unit cells may comprise second binding sites forming together with second linker forming elements a second linker element, which is bound to the one or more background binding partners.

[0090] As a non-limiting example, it may be possible to use a human derived biological fluid sample, such as CSF or a blood sample and to use second molecular recognition elements which comprise, or consist of, human serum albumin. Human serum albumin has been identified as one of the major components in human derived biological fluid samples being responsible for non-specific binding. In certain embodiments, the sensing device provided in step a. therefore comprises a plurality of first molecular recognition elements being configured to bind the analyte associated with proteinopathy and being bound to the first unit cells. Furthermore, the sensing device comprises a plurality of second molecular recognition elements comprising, or consisting of, human serum albumin and being bound to the second unit cells.

[0091] In certain embodiments, the method may comprise a pre-screening step, the pre-screening step comprising identifying background binding partners in the biological fluid sample. For example, this may comprise the identification of background binding partners which bind to the first molecular recognition elements and / or which trigger a non-specific binding signal in step d.. It may also be possible to perform the pre-screening step with a sensing device as described in any of the embodiments herein, for example a sensing device in which the second molecular recognition elements consist of oligonucleotide strands. After performing steps b. to d. non-specific binding to the first and / or second molecular recognition elements may be detected and background binding partners in the biological fluid sample may be identified. After this pre-screening step, step a. (i.e. providing the sensing device) may comprise to include the identified background binding partners in the second molecular recognition elements, for example by binding them to the oligonucleotide strands. Thereafter, steps b. to d. may be performed.

[0092] In some embodiments, the first molecular recognition elements are bound to the first the first unit cells with a surface density of 0.1 to 40 fmol / mm2(femto-mol / square-millimeter) in particular of 1 and 4 fmol / mm2, in the case of molecular recognition elements being antibodies or having the size of antibodies (150 kDa). For a smaller affinity element such as nanobodies or aptamers of 15-50 kDa, the first molecular recognition elements are bound to the first units cells with a surface density of 1 and 320 fmol / mm2, in particular 16 and 64 fmol / mm2. Alternatively, or additionally, the second molecular recognition elements may be may be bound to the second unit cells with a surface density of 0.1 to 40 fmol / mm2, in particular of 1 and 4 fmol / mm2, in the case of molecular recognition elements being antibodies or having the size of antibodies (150 kDa). For a smaller affinity element such as nanobodies or aptamers of 15-50 kDa, the first molecular recognition elements are bound to the first units cells with a surface density of 1 and 320 fmol / mm2, in particular 16 and 64 fmol / mm2. In particular in cases in which the first and / or second molecular recognition elements are antibodies, such a surface density is advantageous, because on the one hand it avoid interactions of the antibodies with each other and further allows for a relatively dense occupation of the unit cells, which improves the measurement and affinity of the sensing device.

[0093] In some embodiments, the biological fluid sample may be pre-treated prior to step b.. For example, the biological fluid sample may be centrifuged and / or filtered prior to step b..

[0094] In some embodiments, it is possible to repeat steps b., c. and d. and optionally also step a. at least once, at least twice, or even more. Each repetition may be considered as a screening cycle.

[0095] It is possible that each screening cycle is different. For example, it may be possible that in a screening cycle, only the blank biological fluid sample is screened, providing a blank signal. In another screening cycle, a seed amplification as described herein may be performed on the same biological fluid sample providing a seed amplification signal. In another screening cycle, incubation with a labeled binding agent as described in some embodiments herein is performed providing a labeled binding agent signal. In another screening cycle, a treatment step and / or a rinsing step is performed as described in some of the embodiments herein providing a treatment signal and / or a rinsing signal. In another screening cycle, a serial competition measurement according to some of the herein described embodiments is performed providing several competition signals. In preferred embodiments, in another screening cycle a first serial competition measurement is performed by providing the biological fluid sample in a first flow direction to the sensing spots and in a subsequent screening cycle, a second serial competition measurement is performed by providing the biological fluid sample in a second flow direction to the sensing spots which is inverse to the first flow direction. In another screening cycle, a parallel measurement according to some of the herein described embodiments is performed providing parallel measurement signals. It is possible to perform one, two, more or all of any of these screening cycles in some embodiments of the method according to the invention.

[0096] In certain embodiments the signals and / or parameters obtained in each screening cycle a comprised in the immunosignature of the biological fluid sample and / or of the analyte associated with proteinopathy.

[0097] A second aspect of the present disclosure relates to a method for screening a biological fluid sample for an analyte associated with proteinopathy, the method comprising the steps: a. Providing a sensing device, which comprises an evanescent illuminator being configured for generating an evanescent field from a beam of coherent light with a predefined wavelength on a first surface of the evanescent illuminator, wherein the first surface of the evanescent illuminator comprises one or more sensing spots wherein each sensing spot comprises a first recognition grating with a plurality of first unit cells and a second recognition grating with a plurality of second unit cells, wherein the first and second recognition grating are interdigitated with each other such that the plurality of first unit cells are arranged in an alternating manner with the second unit cells, wherein a plurality of first binding sites is bound to the first unit cells, and wherein a plurality of second binding sites being different from the first binding sites is bound to the second unit cells, and wherein the one or more sensing spots are each configured such that i. at least a portion of coherent light of the evanescent field is scattered at the plurality of first unit cells to generate first constructive interference beams at a detector of the sensing device having a first phase; ii. at least a portion of coherent light of the evanescent field is scattered at the plurality of second unit cells to generate second constructive interference beams at the detector having a second phase being inverse to the first phase; iii. the generated first constructive interference beams interfere at the detector with the second constructive interference beams to generate a mass difference dependent and optionally time dependent signal at the detector wherein the signal is quadratically proportional to a mass difference generated by molecular interactions of the plurality of first binding sites and the plurality of second binding sites; b. Performing a screening cycle, comprising: i. Immobilizing first molecular recognition elements or first binding units to the first binding sites and not to the second binding sites, wherein the first molecular recognition elements or binding units are configured to bind to the analyte associated with proteinopathy; ii. Optionally immobilizing second molecular recognition elements or second binding units or background binding partners to the second binding sites and not to the first binding sites, wherein the second molecular recognition elements or background binding partners are different from the first molecular recognition elements (respectively wherein the second binding units are different from the first binding units); iii. Providing a biological fluid sample to the one or more sensing spots of the evanescent illuminator; iv. Generating a beam of coherent light with the predetermined wavelength at a predefined beam generation location; v. Measuring the signal for each of the sensing spots at the detector, each signal being quadratically proportional to the mass difference generated by molecular interactions of the plurality of first molecular recognition elements (or first binding units) and the plurality of second molecular recognition elements (or second binding units or background binding partners); vi. Reactivating the sensing device by detaching the immobilized first molecular recognition elements or first binding units from the first binding sites and optionally by detaching the immobilized second molecular recognition elements (or second binding units or background binding partners) from the second binding sites, and optionally by washing; c. Repeating step b. once or several times with different first molecular recognition elements (or first binding units) and optionally with different or the same second molecular recognition elements (or different or the same second binding units or different or the same background binding partners).

[0098] This method is a sequential screening approach. Initially, a first type of first molecular recognition elements is immobilized to the first binding sites. Then, the behavior of this biological fluid sample to this first type of first molecular recognition elements is determined and a signal is measured. Thereafter, the immobilized first type of first molecular recognition elements is detached, e.g. cleaved, from the first binding sites. Then, the sequence is repeated with another, different type of first molecular recognition elements, These are again immobilized to the first binding sites and the behavior of this biological fluid sample to this different type of first molecular recognition elements is determined and a signal is measured.

[0099] In step b.vi. the plurality of second molecular recognition elements or second binding units or background binding partners may in some embodiments not be detached. This may for example be advantageous if the same second molecular recognition elements or second binding units or background binding partners are used for each repetition of step b. Then, step b.ii. may only be conducted when performing step b. the first time.

[0100] It is understood that the sensing device can be a sensing device as described in any of the embodiments herein, for example as with respect to the first aspect.

[0101] Repeating step b. as described above (step c.) may be done with different first molecular recognition elements or first binding units. In particular, such different first molecular recognition elements or first binding units may have a different affinity or different binding behavior to the analyte associated with proteinopathy. The second molecular recognition elements or second binding units or background binding partners may in each repetition of step b. be the same or it may be different.

[0102] For example, it may be possible that the first molecular recognition elements or first binding units used when performing step b. the first time may be configured to selectively bind to a first epitope (and optionally not to other epitopes) of the analyte associated with proteinopathy, while the first molecular recognition elements or first binding units used when performing step b. the second time (or any additional time) may be configured to selectively bind to a different epitope (and optionally not to other epitopes) of the analyte associated with proteinopathy.

[0103] It may also be possible that the first molecular recognition elements or first binding units used when performing step b. the first time may be configured to selectively bind to a monomeric form of the analyte associated with proteinopathy (and optionally not to aggregated forms), while the first molecular recognition elements or first binding units used when performing step b. the second time (or any additional time) may be configured to selectively bind to an aggregated form (and optionally not to monomeric forms) of the analyte associated with proteinopathy.

[0104] In some embodiments, the first molecular recognition elements or first binding units used when performing step b. the first time may show a different binding behavior, such as different selectivity or different affinity, to the analyte associated with proteinopathy, or certain forms thereof, as compared to the first molecular recognition elements or first binding units used when performing step b. the second time (or any additional time). In particular, the different first molecular recognition elements or first binding units may be selective for certain forms of the analyte associated with proteinopathy, such as its monomer, its oligomer, aggregate, or a specific structure (such as for example a secondary, tertiary or quaternary protein structure).

[0105] In some embodiments, the biological fluid sample comprises one or more background binding partners being different from the analyte associated with proteinopathy. The one or more background binding partners may be configured to trigger a non-specific binding signal in step b.v.. The one or more background binding partner may for example be configured to bind to the first molecular recognition elements or first binding units and / or to the second binding sites. In such embodiments, it may for example be possible that the second molecular recognition elements of the sensing device provided in step a. comprise or consist of the one or more background binding partners. This is advantageous, because since the method of the present disclosure relies on the mass difference between the first and second molecular recognition elements (and any entities bound thereto), using background binding partners as second molecular recognition elements saturates the second unit cells with the background binding partners before steps b.ii. to b.v.. Therefore, a stable baseline is achieved and the influence of additional background binding partners in the biological fluid sample is significantly decreased. Thereby, non-specific binding events can be avoided or at least significantly reduced, which allows to monitor the specific binding events by the first molecular recognition elements or first binding units to the analyte associated with proteinopathy. The background binding partner may for example be bound to the second unit cells by a second linker element as described herein. For example, the second unit cells may comprise second binding sites forming together with second linker forming elements a second linker element, which is bound to the one or more background binding partners.

[0106] As a non-limiting example, it may be possible to use a human derived biological fluid sample, such as CSF or a blood sample and to use second molecular recognition elements which comprise or consist of human serum albumin. Human serum albumin has been identified as one of the major components in human derived biological fluid samples being responsible for non-specific binding. In certain embodiments, the sensing device provided in step a. therefore comprises a plurality of first molecular recognition elements or first binding units being configured to bind the analyte associated with proteinopathy and being bound to the first unit cells. Furthermore, the sensing device comprises a plurality of second molecular recognition elements comprising or consisting of human serum albumin and being bound to the second unit cells.

[0107] In certain embodiments, the method may therefore comprise a pre-screening step, the prescreening step comprising identifying background binding partners in the biological fluid sample. For example, this may comprise the identification of background binding partners which bind to the first molecular recognition elements and / or which trigger a non-specific binding signal in step d.. It may also be possible to perform the pre-screening step with a sensing device as described in any of the embodiments herein, for example a sensing device in which the second molecular recognition elements consist of oligonucleotide strands. After performing steps b. ii. to b.iv. non-specific binding to the first and / or second molecular recognition elements may be detected and background binding partners in the biological fluid sample may be identified. After this pre-screening step, step b.ii.. (i.e. immobilizing second molecular recognition elements) may comprise to include the identified background binding partners in the second molecular recognition elements, for example by binding them to the second binding units (e.g. oligonucleotide strands) being bound to the second unit cells. Thereafter, steps b.iii. to b.vi. may be performed. Step b. may for example be repeated 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x or 10x, or even more. However, during every repetition, a different type of first molecular recognition elements may preferably be used.

[0108] Step b.vi. (i.e. reactivating the sensing device) may comprise detaching the immobilized first molecular recognition elements or first binding units from the first binding sites and optionally detaching the immobilized second molecular recognition elements (or second binding units or background binding partners) from the second binding sites. Detaching may for example be done chemically or by physically. For example, a reagent may be added which cleaves off the immobilized first or second molecular recognition elements from the first or second binding sites. As an example, in embodiments in which the first and second binding sites are oligonucleotide strands and in which the first and second molecular recognition elements comprise oligonucleotide strands being complementary to the oligonucleotide strands of the first and second binding sites, a thus formed double strand (e.g. a DNA double strand) may be cleaved by aqueous alkaline treatment, e.g. by aqueous hydroxide (such as NaOH). Alternatively, the cleavage may be done physically, for example by irradiation or heating to a cleavage temperature at which the first or second molecular recognition elements are cleaved from the first or second binding sites.

[0109] The second molecular recognition elements or second binding units or background binding partners optionally used in step b.ii. may be different from the first molecular recognition elements or first binding units, in particular such that they do not selectively bind to the analyte associated with proteinopathy.

[0110] A third aspect of the present disclosure relates to a method for screening a biological fluid sample for an analyte associated with proteinopathy, the method comprising the steps: a. Providing a sensing device, which comprises an evanescent illuminator being configured for generating an evanescent field from a beam of coherent light with a predefined wavelength on a first surface of the evanescent illuminator, wherein the first surface of the evanescent illuminator comprises a plurality of sensing spots wherein each sensing spot comprises a first recognition grating with a plurality of first unit cells and a second recognition grating with a plurality of second unit cells, wherein the first and second recognition grating are interdigitated with each other such that the plurality of first unit cells are arranged in an alternating manner with the second unit cells, wherein a plurality of first molecular recognition elements being configured to bind an analyte associated with proteinopathy, is bound to the first unit cells and wherein a plurality of second molecular recognition elements being different from the first molecular recognition elements is bound to the second unit cells, wherein each sensing spot is unique in that every sensing spot comprises different first molecular recognition elements and optionally different second molecular recognition elements as compared to the remaining sensing spots, and wherein the each sensing spot are each configured such that i. at least a portion of coherent light of the evanescent field is scattered at the plurality of first unit cells to generate first constructive interference beams at a detector of the sensing device having a first phase; ii. at least a portion of coherent light of the evanescent field is scattered at the plurality of second unit cells to generate second constructive interference beams at the detector having a second phase being inverse to the first phase; iii. the generated first constructive interference beams interfere at the detector with the second constructive interference beams to generate a mass difference dependent and optionally time dependent signal at the detector wherein the signal is quadratically proportional to a mass difference generated by molecular interactions of the plurality of first molecular recognition elements and the plurality of second molecular recognition elements; b. Providing a biological fluid sample to the plurality of sensing spots of the evanescent illuminator; c. Generating a beam of coherent light with the predetermined wavelength at a predefined beam generation location; d. Measuring the signal for each of the sensing spots at the detector, each signal being quadratically proportional to the mass difference generated by molecular interactions of the plurality of first molecular recognition elements and the plurality of second molecular recognition elements.

[0111] It is understood that the sensing device can be a sensing device as described in any of the embodiments herein, for example as with respect to the first aspect.

[0112] In some embodiments, the sensing device provided in step a. comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, sensing spots. Each sensing spot is unique in that it has unique first molecular recognition elements. Therefore the behavior of the biological fluid sample to a variety of unique first molecular recognition elements is measured in a single experiment, which allows multiplexing.

[0113] In some embodiments, the sensing spots may be unique in that the first molecular recognition elements of the corresponding sensing spots show different binding behavior, such as different selectivity or different affinity, to the analyte associated with proteinopathy, or certain forms thereof. In particular, the different first molecular recognition elements may be selective for certain forms of the analyte associated with proteinopathy, such as its monomer, its oligomer, aggregate, or a specific structure (such as for example a secondary, tertiary or quaternary protein structure).

[0114] In some embodiments, the plurality of second molecular recognition elements may be the same for each sensing spot.

[0115] In some embodiments, the first molecular recognition elements being bound to the first unit cells of at least one sensing spot are configured to selectively bind to a first epitope (and optionally not to other epitopes) of the analyte associated with proteinopathy. Furthermore, the first molecular recognition elements being bound to the first unit cells of at least another and thus different sensing spot may be configured to selectively bind to a different epitope (and optionally not to other epitopes) of the analyte associated with proteinopathy. In some embodiments, the first molecular recognition elements being bound to the first unit cells of at least one sensing spot are configured to selectively bind to an aggregated form (and optionally not to monomeric forms) of the analyte associated with proteinopathy. Furthermore the first molecular recognition elements being bound to the first unit cells of at least another sensing spot are configured to selectively bind to a monomeric form (and optionally not to aggregated forms) of the analyte associated with proteinopathy.

[0116] In some embodiments, the biological fluid sample comprises one or more background binding partners being different from the analyte associated with proteinopathy. The one or more background binding partners may be configured to trigger a non-specific binding signal in step d. The one or more background binding partner may for example be configured to bind to the first molecular recognition elements and / or to the second binding sites. In such embodiments, it may for example be possible that the second molecular recognition elements of one, more, or each of the sensing spots of the sensing device provided in step a. comprises or consists of the one or more background binding partners. This is advantageous, because since the method of the present disclosure relies on the mass difference between the first and second molecular recognition elements (and any entities bound thereto), using background binding partners as second molecular recognition elements saturates the second unit cells with the background binding partners before steps b. to d.. Therefore, a stable baseline is achieved and the influence of additional background binding partners in the biological fluid sample is significantly decreased. Thereby, nonspecific binding events can be avoided or at least significantly reduced, which allows to monitor the specific binding events by the first molecular recognition elements to the analyte associated with proteinopathy. The background binding partner may for example be bound to the second unit cells of one, more, or each of the sensing spots by a second linker element as described herein. For example, the second unit cells may comprise second binding sites forming together with second linker forming elements a second linker element, which is bound to the one or more background binding partners.

[0117] As a non-limiting example, it may be possible to use a human derived biological fluid sample, such as CSF or a blood sample and to use second molecular recognition elements which comprise or consist of human serum albumin. Human serum albumin has been identified as one of the major components in human derived biological fluid samples being responsible for non-specific binding. In certain embodiments, the sensing spots of the sensing device provided in step a. therefore comprise a plurality of first molecular recognition elements being configured to bind the analyte associated with proteinopathy and being bound to the first unit cells. Furthermore, the sensing spots of the sensing device may comprise a plurality of second molecular recognition elements comprising or consisting of human serum albumin and being bound to the corresponding second unit cells.

[0118] In certain embodiments, the method may therefore comprise a pre-screening step, the prescreening step comprising identifying background binding partners in the biological fluid sample. For example, this may comprise the identification of background binding partners which bind to the first molecular recognition elements and / or which trigger a non-specific binding signal in step d.. It may also be possible to perform the pre-screening step with a sensing device as described in any of the embodiments herein, for example a sensing device in which the second molecular recognition elements consist of oligonucleotide strands. After performing steps b. to d. non-specific binding to the first and / or second molecular recognition elements may be detected and background binding partners in the biological fluid sample may be identified. After this pre-screening step, step a. (i.e. providing the sensing device) may comprise to include the identified background binding partners in the second molecular recognition elements, for example by binding them to the oligonucleotide strands. Thereafter, steps b. to d. may be performed.

[0119] Brief description of the figures

[0120] The herein described invention will be more fully understood from the detailed description given herein below and the accompanying drawings which should not be considered limiting to the invention described in the appended claims. The drawings are showing:

[0121] Fig. 1 a schematic representation of a sensing device according to an embodiment of the invention; Fig. 2 a schematic detailed view of a sensing spot according to an embodiment of the invention;

[0122] Fig. 3a a schematic detailed view of a section of a sensing spot in use in a method according to the invention screening for an analyte associated with proteinopathy forming an aggregate;

[0123] Fig. 3b a schematic detailed view of a section of a sensing spot in use in a method according to the invention screening for an analyte associated with proteinopathy being present as a monomer;

[0124] Fig. 4 a schematic detailed view of a section of a sensing spot in use in a method according to the invention after washing and with an additional mass-labeled binding agent;

[0125] Fig. 5 a schematic detailed view of a section of a sensing spot in use in a method according to the invention where after a predetermined time interval, a treatment step including the application of a detergent is performed;

[0126] Fig. 6 a schematic detailed view of a section of a sensing spot in use in a method according to the invention where a seed amplification assay is performed;

[0127] Fig. 7 a schematic representation of a method according to an embodiment of the invention in which the measured signals form an immunosignature;

[0128] Fig. 8a, b a schematic representation of a portion of a sensing device where a serial competition measurement is performed;

[0129] Fig. 9 a schematic representation of a portion of a sensing device where a parallel measurement is performed; Fig. 10 a schematic detailed view of a section of a sensing spot in use in a method according to the invention, where a marker protein expressed on the surface of mitochondria is exposed and used to bind to first molecular recognition elements;

[0130] Fig. 11 a schematic detailed view of a section of a sensing spot in use in a method according to the invention, where a marker protein expressed on the surface of a lysosome is exposed and used to bind to first molecular recognition elements;

[0131] Fig. 12 a schematic detailed view of a section of a sensing spot in use in a method according to the invention, where a protein co-aggregates are employed as analyte associates with proteinopathy;

[0132] Fig. 13 a matrix representation of different multiplex screenings of different samples (analytes associated with proteinopathy in different forms in PBST (phosphate-buffered saline with detergent [polysorbate 20]) and blank);

[0133] Fig. 14 a matrix representation of different multiplex screenings of different samples (analytes associated with proteinopathy in different forms in cerebrospinal fluid (CSF) and blank);

[0134] Fig. 15 a matrix representation of different multiplex screenings of different samples (analytes associated with proteinopathy in different forms in human serum (HS) and blank);

[0135] Fig. 16 a schematic representation of immobilizing first and second molecular recognition elements to first and second unit cells as it may be done in certain embodiments of the disclosure.

[0136] Exemplary embodiments Figure 1 shows an embodiment of a sensing device 1 as it can be used in some embodiments of the method according to the invention. Sensing device 1 comprises an evanescent illuminator 3 which is configured to generate an evanescent field from a beam of coherent light with a predefined wavelength on a first surface of the evanescent illuminator. Evanescent illuminator 3 may in this or any other embodiment described herein be arranged on a carrier 2, e.g. a transparent carrier. Evanescent illuminator 3, respectively its first surface (which is exposed to the viewer), includes sensing spot 5. While in this embodiment the evanescent illuminator, respectively its first surface, comprises only one sensing spot, it may well be possible that the evanescent illuminator, respectively its first surface, comprises a plurality of such sensing spots. This is for example shown in Fig. 8 or 9. Sensing device 1 further comprises light source 6 and grating 4. Upon generation of a beam of coherent light with predefined wavelength by light source 6, grating 4 launches a guided mode of the waveguide which guides the light towards sensing spot 5 as indicated by the arrow.

[0137] Figure 2 shows a detailed view of a sensing spot 5. As can be seen, sensing spot 5 comprises or consists of, a first recognition grating with a plurality of first unit cells 8 and a second recognition grating with a plurality of second unit cells 9. The first and second recognition grating are interdigitated with each other such that the plurality of first unit cells 8 are arranged in an alternating manner with second unit cells 9. All first unit cells 8, which in this embodiment are in the form of curved elongated lines or bars, form together the first recognition grating. Vice versa, all second unit cells, which in this embodiment are also in the form of curved elongated lines or bars, form together the second recognition grating of sensing spot 5. A plurality of first molecular recognition elements (not shown, see for example Fig. 3a), which are configured to bind an analyte associated with proteinopathy, is bound to first unit cells 8. In addition, a plurality of second molecular recognition elements (not shown, see for example Fig. 3a) being different from the first molecular recognition elements is bound to second unit cells 9.

[0138] Referring to both Fig. 1 and 2, sensing spot 5 is configured such that at least a portion of the coherent light of the evanescent field generated by the evanescent illuminator is scattered at the plurality of first unit cells 8 to generate first constructive interference beams at a detector 7 of the sensing device, which have a first phase. In addition, sensing spot 5 is configured such that at least a portion of coherent light of the evanescent field generated by the evanescent illuminator is scattered at the plurality of second unit cells 9 to generate second constructive interference beams at the detector 7. The second constructive interference beams have a second phase which is inverse to the first phase of the first constructive interference beams. In addition, sensing spot 5 is configured such that the generated first constructive interference beams interfere at the detector with the second constructive interference beams to generate a mass difference dependent and optionally time dependent signal at detector 7. This signal is quadratically proportional to a mass difference generated by molecular interactions of the plurality of first molecular recognition elements and the plurality of second molecular recognition elements.

[0139] Fig. 3a and 3b show a comparison of the method performed on an analyte associated with proteinopathy forming an aggregate (Fig. 3a) and on an analyte being a monomer (Fig. 3b). Both figures show a detailed cross-sectional view of a first unit cell 8 and second unit cell 9 of a sensing spot (such as the sensing spot 5 shown in Fig. 1 and 2). As can be seen, a plurality of first molecular recognition elements 10 is bound to first unit cell 8 and a plurality of different second molecular recognition elements 11 is bound to second unit cell 9. The analyte of proteinopathy 13 forms an aggregate which exposes several binding sites denoted by a, b, c, d and e. In general as used herein, different letters refer to different binding sites. That is, in this example, analyte 13 associated with proteinopathy has four different types of binding sites a, b, c, d and e. As can be seen, first molecular recognition elements comprise a binding site A, while second molecular recognition elements are devoid of such a binding site A. In general as used herein, a binding site denoted by a capital letter, such as binding site “A” is generally configured to bind a binding site denoted with the same lower case letter, such as binding site “a” but preferably not a binding site with another lower case letter. It can be seen in Fig, 3a that after providing a biological fluid sample to sensing the sensing spot and thus to its first and second unit cells, the analyte 13 associated with proteinopathy binds with its binding site a to a binding site A of a first molecular recognition element. In the graph below, the signal measured at the detector is shown. Since non-binding background material being present in the biological fluid sample does not interact with first or second molecular recognition elements, or since these do so with essentially equal probability, the obtained signal is inherently self-referencing and it is not necessary to wash away background material to obtain a measurement and a reliable signal. It can be seen that the measurement of the signal obtained from the sensing spot commences during time interval 1. Binding of the analyte 13 associated with proteinopathy occurs during time interval 2 which leads to a mass increase being clearly visible in the obtained time dependent signal. Since the first molecular recognition elements 10 can be selected such that they are highly selective binders for binding sites a of analyte 13, already such a measurement can provide detailed information on the nature or clinical picture of a proteinopathy. Further, because the system is essentially only dependent on the mass of the binding partner with which the molecular recognition elements interact, it is irrelevant how complex and how heterogeneous the aggregate formed by analyte 13 is. Fig. 3b shows the signal obtained if only monomers are detected. As the measured signal is quadratically dependent on the mass difference interacting with the molecular recognition elements, aggregates formed by the analyte associated with proteinopathy have a significantly more intense signal.

[0140] Similarly to Fig. 3a, Fig. 4 also shows the screening for an analyte associated with proteinopathy which forms an aggregate. Again, as it can be seen from the measured signal, measurement commences during time interval 1. In time interval 2, the biological fluid sample comprising amongst others the aggregated analyte 13 associated with proteinopathy is added. Thereafter, in time interval 5, a mass-labeled binding agent 14 is added. For example, such a mass-labeled binding agent may comprise a gold nanoparticle as mass label.

[0141] Furthermore, the mass-labeled binding agent may in this or any other embodiment as described herein, comprise a predefined binding site, which may particularly be configured to bind to the analyte associated with proteinopathy. In general, the predefined binding site may for example be configured such that it does not bind to the first and / or second molecular recognition elements. As can be seen in Fig. 4, upon addition of the mass-labeled binding agent, the signal increases in time interval 5, because the binding agent binds to the analyte 13 which is already bound to first molecular recognition elements 10. Such embodiments are advantageous as these further can help to deconvolve and thus characterize the proteinopathy from which the patient as the source of the biological fluid sample being screened suffers. For example, if one knows that in specific types of diseases show an analyte which exposes binding site b, the mass increase shows the presence of such a binding site b and may thus help to exclude other proteinopathies. It is understood that the mass label has a defined and known mass.

[0142] Fig. 5 shows a method according to an embodiment of the invention in which a treatment step is performed. In this embodiment, the treatment step comprises the application of a detergent. As can be seen in the upper part, an aggregated analyte is bound to the first molecular recognition element. The corresponding signal increase is seen in time intervals 2 to 4. Then, in time interval 5 a detergent is applied which results in removal of parts of or the complete analyte (see middle part). Due to the loss of mass interacting with the first molecular recognition elements, the signal decreases. The measured signal can generally further be comprised in the immunosignature of the analyte of proteinopathy and / or the biological fluid sample. If it is known for example that certain analytes are sensitive towards different treatments, such as detergents, pH, etc., then such treatment steps can be used to provide further information on the analyte associated with proteinopathy.

[0143] Fig. 6 depicts an embodiment of the method according to the invention in which after step b. a seed amplification assay is performed. As can be seen in the upper part of Fig. 6, the biological fluid sample contains analytes 13 associated with proteinopathy. After providing the biological fluid sample, a protein sample comprising monomeric proteins 15 is added to the corresponding sensing spot comprising first unit cell 8 and second unit cell 9. Since the analyte associated with proteinopathy shows for example prion-like behavior, it effects misfolding and aggregation of added monomeric proteins. Therefore, as seen in the middle part of Fig. 6. The aggregation increases and additional aggregates form. Since the mass increase due to advancing aggregation can be directly measured, the seed amplification can be observed in real time. This is shown in the lower part of Fig. 6 showing the measured signal over time. As can be seen, the measured signal continuously increases during time interval 2, which directly indicates that seed amplification occurs.

[0144] Fig. 7 shows an embodiment of the method according to the invention in which the sensing device (not shown) comprises a plurality of sensing spots 5’a, 5”a, 5’b, 5”b, 5’c, 5”c, 5’d

[0145] 5 and 5”d. Each sensing spot comprises first unit cells with first molecular recognition elements bound thereto and second unit cells with second molecular recognition elements bound thereto. Preferably, at least some or even all of the sensing spots can in this or any other embodiment described herein comprise unique first molecular recognition elements and / or unique second molecular recognition elements. The term “unique” means in this0 context that the first (or second) molecular recognition elements of a particular sensing spot are different from the first (or second) molecular recognition elements of the other sensing spots of the sensing device, however typically the first molecular recognition elements within each sensing spot are typically the same. As can be seen from the graph depicting the time dependent measured signal, one signal per sensing spot is obtained once the biological 5 fluid sample S is provided to the sensing spots. These signals can then all represent a parameter in a high dimensional vector space as indicated by the matrix a11 - a43. This matrix of parameters may in this or any other embodiments described herein be an immunosignature of the biological fluid sample and / or of the analyte associated with proteinopathy having been screened with the method according to the invention. This0 immunosignature can then by a control unit being for example part of a computer, be compared with a database being stored in a memory unit of this computer. If this database then contains data associated with a clinical status of a proteinopathy of patient samples, autologous patient samples (i.e. previously screened samples from the patient from which the presently screened biological fluid sample has been obtained) and / or reference5 samples, it is possible to characterize the nature and / or clinical status of the proteinopathy with which the analyte is associated. Furthermore, it is possible to forecast the progression of the proteinopathy. Additionally, also other parameters can be included into the immunosignature, such as the signal(s) obtained from an seed amplification assay as described herein or the signal obtained from the addition of a mass-labeled binding agent0 as described herein. Fig. 8a and b show embodiments of the method according to the invention in which the plurality of sensing spots of the sensing device compete for the analyte associated with proteinopathy. Both Fig. 8a and Fig. 8b show serial competition measurements and the arrow indicates the direction of flow with which the biological fluid sample is provided. That is, in Fig. 8a, the biological fluid sample is provided such that it first contacts the two sensing spots being denoted by “A”, then the two sensing spots being denoted by “B” and then the two sensing spots being denoted by “C”. If for example the first two sensing spots comprise first molecular recognition elements having binding site A, the central two sensing spots comprise different first molecular recognition elements having binding site B and the last sensing spots comprise yet other, different first molecular recognition elements having binding site C, the behavior of the analyte associated with proteinopathy to these different first molecular recognition elements can provide valuable information on the analyte and the proteinopathy. Figure 8b shows a serial competition measurement with the same sensing device as shown in Fig. 8a, however, the direction of flow is inversed. Performing such serial competition measurements, i.e. a first one with a first direction of flow and a second one with the inversed direction of flow provides additional information, which allows to further characterize the analyte and the proteinopathy.

[0146] Fig. 9 shows a parallel measurement. As can be seen the sensing device comprises three groups of sensing spots, each comprising two sensing spots. The first group comprises sensing spots with first molecular recognition elements having binding site A. The second group comprises sensing spots with first molecular recognition elements having binding site B. The third group comprises sensing spots with first molecular recognition elements having binding site C. The sensing device comprises wall structures 12 which separate the groups of sensing spots from each other. The wall structures may in this or any other embodiment form channels, such as microfluidic channels, which are part of a fluidic system. As can be seen, the biological fluid sample can be provided to the sensing device upstream of the sensing spots. It then flows in parallel over the sensing spots. Due to the presence of wall structures 12, the biological fluid sample cannot flow from one channel into another. Fig. 10 shows another embodiment of the method according to the invention. As can be seen, the sensing spot of the employed sensing device comprises first molecular recognition elements 10 bound to first unit cell 8, which each comprise two different binding sites A and B. This is beneficial as it allows for a more accurate characterization of analyte 13 which expresses on its surface complementary binding sites I epitopes a and b. It is known for certain proteinopathies that cell residues, such as mitochondria or parts thereof are recruited and form an aggregate. Further, it is known that such mitochondria or its parts may comprise a specific marker protein such as protein b, e.g. VDAC. Thus, exploiting this knowledge by equipping the first molecular recognition elements with binding sites A binding to a certain binding site I epitope a on the marker and also with binding sites B binding specifically to the mitochondrial protein b, can further characterize the nature / composition of the analyte.

[0147] Fig. 11 shows a similar principle as Fig. 10, but in this case a lysosome is part of the analyte which specifically exposes surface protein c, such as LAMP1. Therefore, if the first molecular recognition elements comprise binding site C being configured to bind to protein c, further details on the composition of the analyte and the nature of the associated proteinopathy can be provided.

[0148] Fig. 12 shows another variant being similar to the examples shown in Fig. 10 and 11. In this case, a protein co-aggregate is screened as analyte 13 associated with proteinopathy. In this variant one may for example assume that the patient suffers from a specific proteinopathy which involves a protein aggregate that has a specific post-translational modification d. By employing a sensing device having a sensing spots with first recognition elements which comprise each two binding sites A and D, wherein the latter is specific for the post-translational modification d, it is possible to generate information on the nature of the proteinopathy, respectively the analyte associated therewith.

[0149] Fig. 13 shows a matrix of representation of different multiplex screenings of different samples (analytes associated with proteinopathy in different forms in PBST (phosphate- buffered saline with detergent [polysorbate 20]) and blank). Samples S are as follows: SO is a blank sample (i.e. PBST); S1 is wild-type monomeric a-syn in PBST; S2 is wild-type aggregated a-syn in PBST; S3 is phosphorylated (pSer129) a-syn in PBST; S4 is aggregated phosphorylated (pSer129) a-syn in PBST; S4 are fibrils of a-syn in PBST; Abeta is beta-amyloid in PBST. A series of multiplexed screening experiments has been performed. Every measurement shows the mass dependent binding signal (x-axis: time in s, y-axis: coherent mass density in pg / mm2) for the sample indicated in response to different first and second molecular recognition elements. As indicated by the designation Ab1 , Ab2, Ab3, Ab4, Ab5 and Ab6, each row represents a series of experiments in which specific antibodies Ab1 , Ab2, Ab3, Ab4, Ab5 and Ab6 were used as part of the first molecular recognition elements being configured to bind an analyte associated with proteinopathy, e.g. and epitope thereof. The roman column designations I to X serve to identify different probed second molecular recognition elements. The sensing devices have been provided according to step a. as described herein. The first unit cells contained first binding sites, being oligonucleotides. They were exposed to the indicated antibody samples Ab1 to Ab6 which comprised a complementary oligonucleotide strand being bound to an antibody (i.e. a binding unit), employing DNA directed immobilization as it is for example employed in peptide arrays. Thereby, Ab1 to Ab6 were bound to the first unit cells. The second unit cells contained second binding sites, which are differently constituted oligonucleotide strands (but different than the oligonucleotide strands of the first binding sites). In columns I, II and III aFLAG being connected to an oligonucleotide complementary to the oligonucleotide of the second binding sites has been immobilized to the second unit cells. In columns IV to X human serum albumin being connected to an oligonucleotide complementary to the oligonucleotide of the second binding sites has been immobilized to the second unit cells. For the measurement, the different provided sensing devices have been exposed to the biological fluid samples SO to S6 as indicated (10 pM for S1 to S4 and 2 pM for S5 and S6).

[0150] Fig. 13 shows that monomeric a-syn (S1) having a much lower molecular weight than its aggregated forms, is well recognized by Ab1 to Ab5 to different extents with the lowest on rate for Ab5. This illustrates the methods benefit of providing a very specific immunosignature, e.g. mas-weighted immunosignature, as different behaviors to different antibodies can be readily determined in a series of a few experiments. Additionally, the presence of aggregated a-syn (such as S2) shows a different behavior to Ab1 to Ab6. For example, it can be seen that a response is observed for Ab2 and Ab4, which is similar to the response observed for the monomeric form. In contrast, Ab1 and Ab3 show an even increased response as compared to the monomeric form. This shows that Ab1 and Ab3 can be used to identify aggregated and monomeric a-syn. Ab1 and Ab3 used are C-terminal specific antibodies and Ab2 and Ab4 are N-terminal specific antibodies. Without wishing to be bound to a theory, it may be hypothesized that C-terminal epitopes may be accessible in the aggregates formed, while N-terminal epitopes may be inaccessible. It should be mentioned that monomer-free analytes cannot be provided and that the signals are mass-weighted responses of monomers which contain aggregates on top of the monomer background. From Western Blots Ab5 and Ab6 are antibodies were supposed not to bind to aggregated forms (S2), but surprisingly showed an unexpected off- target activity. Such an activity cannot readily be determined by prior art methods.

[0151] Regarding the phosphorylated series S3 and S4, it can be seen that Ab5 shows good high affinity in regard of S2, since Ab5 has been designed to be specific for this target (cf. response of Ab5 towards S1). Ab1 , Ab2, Ab3 and Ab4 show a comparable behavior towards the phosphorylated a-syn as compared to the wild-type monomeric form. No response is observed for Ab6 in regard to S3. For the aggregated phosphorylated a-syn a response similar to the monomers (S2) is observed for Ab2 and Ab4, which are both N-terminal specific, which may be shielded or otherwise not accessible in the aggregated form, while an increased response is observed for Ab1 and Ab3. The latter two are both C-terminal specific and the corresponding epitope may therefore be accessible also in the aggregated form. Ab5 shows an increased response for the aggregated form (S4) and thus, the corresponding epitope seems to be available even in the aggregate form (the higher molecular weight of the aggregated species increases the mass detected by this recognition). Strikingly, Ab6 which does not show any response for the monomeric form S3 shows a pronounced response for the aggregated form S4 and is thus able to identify an aggregate in view of a monomer and thus with high selectivity. Fig. 14 shows a matrix of representation of different multiplex screenings of different samples (analytes associated with proteinopathy in different forms in cerebrospinal fluid (CSF) and blank). Samples S are as follows: SO is a blank sample (i.e. CSF); S1 is wildtype monomeric a-syn in CSF; S2 is wild-type aggregated a-syn in CSF; S3 is phosphorylated (pSer129) a-syn in CSF; S4 is aggregated phosphorylated (pSer129) a-syn in CSF; S4 are fibrils of a-syn in CSF; Abeta is beta-amyloid in CSF. A series of screening experiments has been performed. Every measurement shows the signal measured (x-axis: time in s, y-axis: coherent mass density in pg / mm2) for the sample indicated in response to different first and second molecular recognition elements. As indicated by the designation Ab1 , Ab2, Ab3, Ab4, Ab5 and Ab6, each row represents a series of experiments in which specific antibodies Ab1 , Ab2, Ab3, Ab4, Ab5 and Ab6 were used as part of the first molecular recognition elements being configured to bind an analyte associated with proteinopathy. Ab1 to Ab6 are the same as used in Fig. 13. The roman column designations I to X serve to identify different second molecular recognition elements. The sensing devices have been provided according to step a. as described herein. The first unit cells contained first binding sites, being oligonucleotides. They were exposed to the indicated antibody samples Ab1 to Ab6 which comprised a complementary oligonucleotide strand being bound to an antibody (i.e. a binding unit). Thereby, Ab1 to Ab6 were bound to the first unit cells. The second unit cells contained second binding sites, which are also oligonucleotide strands (but different than the oligonucleotide strands of the first binding sites). In columns I, II and III no additional component has been immobilized and thus the oligonucleotide strands of the second unit cells form the second molecular recognition elements. In columns IV to X human serum albumin being connected to an oligonucleotide complementary to the oligonucleotide of the second binding sites has been immobilized to the second unit cells. For the measurement, the different provided sensing devices have been exposed to the biological fluid samples SO to S6 as indicated (10 pM for S1 to S4 and 2 pM for S5 and S6).

[0152] The results shown in CSF are in line with the results obtained for PBST and show in general that no matrix amplification in CSF is observed as compared to PBST, which supports that it can be distinguished between monomeric and aggregated forms of the same protein in mixtures, as well as in mixtures with CSF. Entries of columns III and VI show an important detail. Both columns show the behavior of S2 (wild-type aggregated a-syn) to AB1 to Ab6. For column III no additional component has been immobilized and thus the oligonucleotide strands of the second unit cells form the second molecular recognition elements. In contrast, for column VI human serum albumin (HSA), which has been identified as one of sources of non-specific binding, is immobilized to the second unit cells and is thus part of the second molecular recognition elements. It can be seen that the signals in column II for example for Ab1 , Ab2, Ab3 and Ab5 are relatively complex. For Ab3 a very strong signal is measured in column III as compared to column VI. It is hypothesized that this may be due to non-specific binding events of components in CSF which overlap the specific binding signal to be measured. When the second unit cells are bound immobilized with HAS as shown in column VI, these non-specific responses are eliminated and a clear signal is obtained (of. Ab3 in column III and VI).

[0153] Fig. 15 shows a matrix of representation of different multiplex screenings of different samples (analytes associated with proteinopathy in different forms in human serum (HS) and blank). Samples S are as follows: SO is a blank sample (i.e. HS); S1 is wild-type monomeric a-syn in HS; S2 is wild-type aggregated a-syn in HS; S3 is phosphorylated (pSer129) a-syn in HS; S4 is aggregated phosphorylated (pSer129) a-syn in HS; S4 are fibrils of a-syn in HS; Abeta is oligomeric beta-amyloid in HS. A series of screening experiments has been performed. Every measurement shows the signal measured (x-axis: time in s, y-axis: coherent mass density in pg / mm2) for the sample indicated in response to different first and second molecular recognition elements. As indicated by the designation Ab1 , Ab2, Ab3, Ab4, Ab5 and Ab6, each row represents a series of experiments in which specific antibodies Ab1 , Ab2, Ab3, Ab4, Ab5 and Ab6 were used as part of the first molecular recognition elements being configured to bind an analyte associated with proteinopathy. Ab1 to Ab6 are the same as used in Fig. 13 and 14. The roman column designations I to X serve to identify different second molecular recognition elements. The sensing devices have been provided according to step a. as described herein. The first unit cells contained first binding sites, being oligonucleotides. They were exposed to the indicated antibody samples Ab1 to Ab6 which comprised a complementary oligonucleotide strand being bound to an antibody (i.e. a binding unit). Thereby, Ab1 to Ab6 were bound to the first unit cells. The second unit cells contained second binding sites, which are also oligonucleotide strands (but different than the oligonucleotide strands of the first binding sites). In columns I, II and III no additional component has been immobilized and thus the oligonucleotide strands of the second unit cells form the second molecular recognition elements. In columns IV to X human serum albumin being connected to an oligonucleotide complementary to the oligonucleotide of the second binding sites has been immobilized to the second unit cells. For the measurement, the different provided sensing devices have been exposed to the biological fluid samples SO to S6 as indicated (10 pM for S1 to S4 and 2 pM for S5 and S6).

[0154] Fig. 15 demonstrates that human serum (HS) has an extremely strong matrix induced response (i.e. shows a strong signal being triggered by non-specific binding of components in HS. For columns I to III in which no additional component has been immobilized and thus the oligonucleotide strands of the second unit cells form the second molecular recognition elements, this non-specific binding signal is highly complex and overlaps any specific binding signal of the antibodies to the analyte associated with proteinopathy. This nonspecific binding is reduced in columns IV to X, in which human serum albumin (HSA), which has been identified as one of sources of non-specific binding, is immobilized to the second unit cells and thus part of the second molecular recognition elements. Additionally Ab3 and Ab6 show a clear response in the presence of aggregated a-syn (S2, column VI).

[0155] Fig. 16 shows the immobilization process as it can be used in some embodiments of the invention. A sensing device may comprise first unit cell 8 and second unit cell 9. Before immobilization, a first binding site 20 (for example an oligonucleotide strand) is bound to first unit cell 8 and an orthogonal second binding site 21 (for example a different oligonucleotide strand) is bound to second unit cell 9. Then, first binder unit 18 (e.g. an antibody) being bound to a linker forming element 22 (e.g. an oligonucleotide being complementary to binding site 20) is provided and linker forming element 22 and binding site 20 form together first linker element 16. First linker element 16 does then form together with binder unit 18 first molecular recognition element 10. Vice versa, a second molecular recognition element 11 can be formed from second linker element 17 and second binder unit 19 by selective reaction between second binding site 21 and second linker forming element 23.

[0156] List of designations

[0157] 1 sensing device

[0158] 2 carrier

[0159] 3 evanescent illuminator

[0160] 4 grating

[0161] 5 sensing spot

[0162] 5’a, 5”a sensing spot

[0163] 5’b, 5”b sensing spot

[0164] 5’c, 5”c sensing spot

[0165] 5’d, 5”d sensing spot

[0166] 6 light source

[0167] 7 detector

[0168] 8 first unit cell

[0169] 9 second unit cell

[0170] 10 first molecular recognition elements

[0171] 11 second molecular recognition elements

[0172] 12 wall structure

[0173] 13 analyte of proteinopathy

[0174] 14 mass labeled binding agent

[0175] 15 monomeric proteins

[0176] 16 first linker element

[0177] 17 second linker element

[0178] 18 first binder unit

[0179] 19 second binder unit

[0180] 20 first binding site

[0181] 21 second binding site

[0182] 22 first linker forming element 23 second linker forming element

Claims

Claims1. A method for screening a biological fluid sample for an analyte associated with proteinopathy, the method comprising the steps: a. Providing a sensing device (1), which comprises an evanescent illuminator (3) being configured for generating an evanescent field from a beam of coherent light with a predefined wavelength on a first surface of the evanescent illuminator (3), wherein the first surface of the evanescent illuminator (3) comprises one or more sensing spots (5) wherein each sensing spot (5) comprises a first recognition grating with a plurality of first unit cells (8) and a second recognition grating with a plurality of second unit cells (9), wherein the first and second recognition grating are interdigitated with each other such that the plurality of first unit cells (8) are arranged in an alternating manner with the second unit cells (9), wherein a plurality of first molecular recognition elements (10) being configured to bind an analyte (13) associated with proteinopathy, is bound to the first unit cells (8), and wherein a plurality of second molecular recognition elements (11) being different from the first molecular recognition elements (10) is bound to the second unit cells (9), and wherein the one or more sensing spots (5) are each configured such that i. at least a portion of coherent light of the evanescent field is scattered at the plurality of first unit cells (8) to generate first constructive interference beams at a detector (7) of the sensing device (1) having a first phase; ii. at least a portion of coherent light of the evanescent field is scattered at the plurality of second unit cells (9) to generate second constructive interference beams at the detector (7) having a second phase being inverse to the first phase;iii. the generated first constructive interference beams interfere at the detector with the second constructive interference beams to generate a mass difference dependent and optionally time dependent signal at the detector (7) wherein the signal is quadratically proportional to a mass difference generated by molecular interactions of the plurality of first molecular recognition elements (10) and the plurality of second molecular recognition elements (11); b. Providing a biological fluid sample to the one or more sensing spots (5) of the evanescent illuminator (3); c. Generating a beam of coherent light with the predetermined wavelength at a predefined beam generation location; d. Measuring the signal for each of the sensing spots at the detector (7), each signal being quadratically proportional to the mass difference generated by molecular interactions of the plurality of first molecular recognition elements (10) and the plurality of second molecular recognition elements (11).

2. The method according to claim 1 , wherein the first surface of the evanescent illuminator (3) comprises a plurality of sensing spots (5), wherein the first molecular recognition elements (10) of a sensing spot (5), in particular of each sensing spot (5), of the plurality of sensing spots (5) are different from the first molecular recognition elements (10) of at least some of or all of the other sensing spots (5).

3. The method according to claim 2, wherein the first molecular recognition elements (10) of the sensing spot (5) are different from the first molecular recognition elements (10) of at least some of or all of the other sensing spots (5) in that they are chemically distinct from each other and / or in that they are configured to bind a different analyte (13) associated with proteinopathy and / or in that they are configured to bind a different epitope of the analyte (13) associated with proteinopathy, and / or in that they areconfigured to bind to the same analyte (13) associated with proteinopathy or epitope of the analyte (13) associated with proteinopathy with a different binding affinity.

4. The method according to claim 2 or 3, wherein at least some of the plurality of sensing spots (5) compete for the analyte (13) associated with proteinopathy and wherein step d. is performed for a predetermined measurement time to monitor competing of the sensing spots (5) for the analyte (13) associated with proteinopathy.

5. The method according to any of claims 2 to 4, further comprising the step of determining the origin of at least one or all of the signals measured at the detector (7).

6. The method according to any of the previous claims, the method further comprising: determining a parameter of the analyte (13) associated with a proteinopathy from the mass difference dependent and optionally time dependent signal; and / or: determining a parameter of an aggregate of the analyte (13) associated with a proteinopathy or co-aggregate with the analyte (13) associated with a proteinopathy from the mass difference dependent and optionally time dependent signal.

7. The method according to claim 6, the method further comprising the step: determining the presence of the aggregate or the co-aggregate from the determined parameter, wherein preferably the presence of the aggregate or the co-aggregate is determined if the determined parameter exceeds a predetermined threshold within a predefined incubation time.

8. The method according to claim 7, wherein determining the presence of the aggregate or the co-aggregate comprises the comparison of the determined parameter with a reference parameter, in particular a reference parameter being obtained from different patient populations or from a healthy subject.

9. The method according to any of the previous claims, wherein the analyte (13) associated with proteinopathy comprises one or more of p-amyloid, tau, a-synuclein,prion proteins, fused in sarcoma, wild type or mutant poly-Q huntingtin, Ubiquitin, Ataxin-3, Optineurin, TAR DNA-binding protein 43, neurofibrilary light chain light (NfL), soluble or shed Triggering Receptor expressed on myeloid cells 2 (sTREM2), Chitinase-3-like protein 1 , Glial Fibrillary Acidic Protein and truncated or otherwise post-translationally modified forms of these.

10. The method according to any of the previous claims, wherein step d. is performed for a predetermined measurement time and wherein each signal is measured as a function of the measurement time, wherein preferably during the predetermined measurement time changes of each signal are measured, wherein the changes are effected by aggregation, in particular by aggregation of additional material in the biological fluid sample, such as any residual biological material, for example cellular components, mitochondria, cell membranes, nucleic acids, proteins and fragments thereof.

11. The method according to any of the previous claims, wherein step d. comprises a single point measurement at a specific point in time, in particular an end-point measurement.

12. The method according to any of the previous claims, wherein a rinsing step is performed after step b. and in particular before or during step d, and / or wherein a treatment step is performed after step b, wherein the treatment step preferably comprises the application of a treatment agent, such as a detergent, ionic agent, solvent, acid, base, aggregate stabilizer agent and / or aggregate destabilizer agent.

13. The method according to any of the previous claims, wherein after step b. incubation of the one or more sensing spots with a labeled, in particular mass-labeled or fluorescence-labeled, binding agent is performed, wherein preferably the labeled binding agent is configured to bind to the analyte (13) associated with a proteinopathy, in particular to bind to lysosomal markers, mitochondrial markers, nucleotides, sugars,post-translational modifications or mitochondrial DNA of the analyte associated with a proteinopathy.

14. The method according to any of the previous claims, wherein after step b. a seed amplification assay is performed by adding a binding reagent sample, in particular a protein sample, to the one or more sensing spots, and measuring each signal over a predetermined measurement time.

15. The method according to any of the previous claims, wherein the method further comprises a secondary characterization step for characterizing the analyte (13) associated with proteinopathy or a state of an aggregate or co-aggregate formed by the analyte (13) associated with proteinopathy, wherein the secondary characterization step preferably includes ELISA, FT-IR, Raman or fluorescence spectroscopy.

16. The method according to claim 15, wherein the secondary characterization step is performed directly on the biological fluid sample having been provided to the one or more sensing spots (5) of the evanescent illuminator (3).

17. The method according to any of the previous claims, wherein the signals measured in step d. form an immunosignature of the biological fluid sample.

18. The method according to claim 17, wherein the immunosignature of the biological fluid sample is compared to a database comprising immunosignatures of a plurality of samples, in particular patient samples, autologous patient samples and / or reference samples, wherein the immunosignatures of the database are preferably each associated with a clinical status of a proteinopathy.

19. The method according to any of the previous claims, wherein the plurality of second molecular recognition elements (11) are configured to not bind the analyte (13) associated with proteinopathy.

20. The method according to claim 19 wherein the first molecular recognition elements(10) and the second molecular recognition elements (11) are both configured to interact with the same background binding partners, wherein preferably the first molecular recognition elements (10) and the second molecular recognition elements(11) have essentially the same affinity KD to the same background binding partners.

21. The method according to any of the previous claims, wherein the biological fluid sample comprises a background binding partner and wherein the second molecular recognition elements of the sensing device (1) provided in step a. comprise or consist of the background binding partner.

22. The method according to claim 21 , wherein the background binding partner is human serum albumin.

23. The method according to claim 21 or 22, further comprising a pre-screening step before step a., which comprises the identification of the background binding partner in the biological fluid.

24. A method for screening a biological fluid sample for an analyte associated with proteinopathy, the method comprising the steps: a. Providing a sensing device (1), which comprises an evanescent illuminator (3) being configured for generating an evanescent field from a beam of coherent light with a predefined wavelength on a first surface of the evanescent illuminator (3), wherein the first surface of the evanescent illuminator (3) comprises one or more sensing spots (5) wherein each sensing spot (5) comprises a first recognition grating with a plurality of first unit cells (8) and a second recognition grating with a plurality of second unit cells (9), wherein the first and second recognition grating are interdigitated with each other such that the plurality of first unit cells (8) are arranged in an alternating manner with the second unit cells (9), wherein a plurality of firstbinding sites is bound to the first unit cells (8), and wherein a plurality of second binding sites being different from the first binding sites is bound to the second unit cells, and wherein the one or more sensing spots (5) are each configured such that i. at least a portion of coherent light of the evanescent field is scattered at the plurality of first unit cells (8) to generate first constructive interference beams at a detector (7) of the sensing device (1) having a first phase; ii. at least a portion of coherent light of the evanescent field is scattered at the plurality of second unit cells (9) to generate second constructive interference beams at the detector (7) having a second phase being inverse to the first phase; iii. the generated first constructive interference beams interfere at the detector with the second constructive interference beams to generate a mass difference dependent and optionally time dependent signal at the detector (7) wherein the signal is quadratically proportional to a mass difference generated by molecular interactions of the plurality of first binding sites and the plurality of second binding sites; b. Performing a screening cycle, comprising: i. Immobilizing first molecular recognition elements or first binding units to the first binding sites and not to the second binding sites, wherein the first molecular recognition elements or first binding units are configured to bind to the analyte associated with proteinopathy; ii. Optionally immobilizing second molecular recognition elements or second binding units or background binding partners to the secondbinding sites and not to the first binding sites, wherein the second molecular recognition elements or second binding units or background binding partners are different from the first molecular recognition elements or first binding units; iii. Providing a biological fluid sample to the one or more sensing spots (5) of the evanescent illuminator (3); iv. Generating a beam of coherent light with the predetermined wavelength at a predefined beam generation location; v. Measuring the signal for each of the sensing spots at the detector (7), each signal being quadratically proportional to the mass difference generated by molecular interactions of the plurality of first molecular recognition elements (10) or first binding units and the plurality of second molecular recognition elements (11) or second binding units or background binding partners; vi. Reactivating the sensing device by detaching the immobilized first molecular recognition elements or first binding units from the first binding sites and optionally by detaching the immobilized second molecular recognition elements or second binding units or background binding partners from the second binding sites, and optionally by washing; c. Repeating step b. once or several times with different first molecular recognition elements or first binding units and optionally with different or the same second molecular recognition elements or second binding units or background binding partners.

25. The method according to claim 24, wherein each signal measured form an immunosignature of the biological fluid sample.

26. The method according to claim 25, wherein the immunosignature of the biological fluid sample is compared to a database comprising immunosignatures of a plurality of samples, in particular patient samples, autologous patient samples and / or reference samples, wherein the immunosignatures of the database are preferably each associated with a clinical status of a proteinopathy.

27. A method for screening a biological fluid sample for an analyte associated with proteinopathy, the method comprising the steps: a. Providing a sensing device (1), which comprises an evanescent illuminator (3) being configured for generating an evanescent field from a beam of coherent light with a predefined wavelength on a first surface of the evanescent illuminator (3), wherein the first surface of the evanescent illuminator (3) comprises a plurality of sensing spots (5) wherein each sensing spot (5) comprises a first recognition grating with a plurality of first unit cells (8) and a second recognition grating with a plurality of second unit cells (9), wherein the first and second recognition grating are interdigitated with each other such that the plurality of first unit cells (8) are arranged in an alternating manner with the second unit cells (9), wherein a plurality of first molecular recognition elements (10) being configured to bind an analyte (13) associated with proteinopathy, is bound to the first unit cells (8) and wherein a plurality of second molecular recognition elements (11) being different from the first molecular recognition elements (10) is bound to the second unit cells (9), wherein each sensing spot is unique in that every sensing spot comprises different first molecular recognition elements and optionally different second molecular recognition elements as compared to the remaining sensing spots (5), and wherein the each sensing spot (5) are each configured such thati. at least a portion of coherent light of the evanescent field is scattered at the plurality of first unit cells (8) to generate first constructive interference beams at a detector (7) of the sensing device (1) having a first phase; ii. at least a portion of coherent light of the evanescent field is scattered at the plurality of second unit cells (9) to generate second constructive interference beams at the detector (7) having a second phase being inverse to the first phase; iii. the generated first constructive interference beams interfere at the detector with the second constructive interference beams to generate a mass difference dependent and optionally time dependent signal at the detector (7) wherein the signal is quadratically proportional to a mass difference generated by molecular interactions of the plurality of first molecular recognition elements (10) and the plurality of second molecular recognition elements (11); b. Providing a biological fluid sample to the plurality of sensing spots (5) of the evanescent illuminator (3); c. Generating a beam of coherent light with the predetermined wavelength at a predefined beam generation location; d. Measuring the signal for each of the sensing spots at the detector (7), each signal being quadratically proportional to the mass difference generated by molecular interactions of the plurality of first molecular recognition elements (10) and the plurality of second molecular recognition elements (11).

28. The method according to claims 27, wherein the first molecular recognition elements(10) being bound to the first unit cells (8) of at least one sensing spot are configuredto selectively bind to a first epitope of the analyte associated with proteinopathy; and wherein the first molecular recognition elements (10) being bound to the first unit cells (8) of at least another sensing spot are configured to selectively bind to a different epitope of the analyte associated with proteinopathy.

29. The method according to claim 27 or 28, wherein the first molecular recognition elements (10) being bound to the first unit cells (8) of at least one sensing spot are configured to selectively bind to an aggregated form of the analyte associated with proteinopathy; and wherein the first molecular recognition elements (10) being bound to the first unit cells (8) of at least another sensing spot are configured to selectively bind to a monomeric form of the analyte associated with proteinopathy.

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