Method for screening biological fluid samples for analytes related to protein disorders

Abstract

This specification discloses a method for screening biological fluid samples for proteinosis-related analytes, which depends on a signal proportional to the square of the mass difference resulting from molecular interactions between a plurality of first molecular recognition elements (10) and a plurality of second molecular recognition elements.

Description

[Technical Field]

[0001] Areas of disclosure The present invention relates to the field of analysis of biological fluid samples, particularly in the field of protein disorders. The present invention relates to a method for screening biological fluid samples for analytes related to protein disorders. [Background technology]

[0002] Background, prior art Proteinopathy is a group of disorders that can affect the entire body. In the brain or peripheral nervous system, they are characterized by the appearance of pathological extracellular or intracellular protein accumulations. Pathology can be analyzed postmortem brain or peripheral tissue biopsy, and the spatiotemporal distribution and abundance of proteinopathy are used for postmortem confirmation of disease staging and clinical diagnosis. In some neurodegenerative disorders, the order of proteinopathy appearance extends throughout the nervous system along neuroanatomical connections. This observation has led to the hypothesis that specific pathogenic species of the proteins involved (sometimes called "pathogenic seeds") may spread between neurons in a time-dependent and region-specific manner, leading to interneuronal propagation and pathogenesis. Some of the proteins produced during the pathogenic process and their different proteoforms may reach bodily fluids such as cerebrospinal fluid or blood. Pathogenic seeds are also hypothesized to be present in different bodily fluids. Neuropathological features are associated with synaptic and overall neuronal dysfunction, and ultimately neuronal loss. Neuropathology is also accompanied by abnormally regulated focal congenital immune cells, a process that may sometimes manifest as neuroinflammation. The accompanying and ongoing destruction of brain structure and function can occur over decades and is reflected in non-motor function (i.e., cognitive and overall social behavior) and, in some disabilities, in the uncontrollable deterioration of motor function. The early stages of neurodegeneration and the onset of proteinopathy often unnoticed for several years. Often, the initial symptoms are slight or unrecognizable, not initially specific to a single disorder, and so-called "basic symptoms" may not be observed; therefore, the disease stage before diagnosis is considered "preclinical" or prodromal when a specific higher probability of developing a disorder is achieved based on preclinical symptoms. Clear detection of the preclinical stage and early differentiation of potential disease trajectories will help identify patients and improve diagnosis for future novel therapies for neurodegenerative disorders. Furthermore, it would be desirable to enable better classification and stratification of patients into clinical subtypes, as well as monitoring of disease progression and treatment effectiveness.

[0003] Abnormal proteinaceous cellular inclusions are scattered throughout the brains of patients with Alzheimer's disease, Parkinson's disease, Huntington's disease, or other related protein disorders of the nervous system. In some disorders, similar inclusions are also found in the peripheral nervous system, where some proteins or their peptide fragments accumulate in the extracellular space of the brain to form so-called plaques (for example, in Alzheimer's disease, these are called senile plaques or amyloid plaques). Analytical and therapeutically, such pathogenesis proteins have been drug targets and diagnostic markers for decades. Analysis of brain tissue and exploration of biochemical disease mechanisms in cell culture and in vivo model systems suggest that disease-related proteins undergo abnormal metabolism and posttranslational modifications, accumulating in poorly soluble portions, which can be complex biochemical materials, reaching micrometer dimensions and often crowded with recalcitrant materials containing oligomers, protofibrils, fibrils, or aggregated protein assemblies. These proteinaceous inclusions may contain disintegrated organelles, lipid vesicles, and other unspecified structures and materials of cellular origin. Therefore, these pathological features, which may be analytes associated with proteinopathy, are highly diverse and heterogeneous. Analysis of the composition of brain cell inclusions and plaques is only possible postmortem. All data from human tissues and model systems suggest multifactorial contributions to the development of proteinopathy, including prolonged maturation based on specific endogenous factors (i.e., genetic susceptibility, aging processes) or exogenous triggers (i.e., viral or bacterial infection, toxins), and accumulation of specific proteins due to overproduction or dysfunctional clearance mechanisms or lipid turnover. Abundant proteins can reach high local or intracellular concentrations and are therefore more likely to accumulate and become part of the pathological features of different proteinopathy. The multifactorial causes, the development of multiple proteinopathy within the same disease range, and the inability to obtain biological samples closest to the neurodegenerative process (i.e., directly from living human brains) exacerbate the selection of appropriate diagnostic and treatment options at the individual patient level.Brain imaging using specific radiotrace (i.e., positron emission tomography, PET) during the course of disease has so far allowed for the detection of only one type of protein inclusion (i.e., tau tangles) and extracellular amyloid-β plaques. Some proteins or their proteoforms that cause certain proteinopathy can be specifically detected and quantified by immunoassays or mass spectrometry in bodily fluids such as blood or cerebrospinal fluid. For only a very small number of specific monomeric β-amyloid peptides and tau proteoforms, this has so far served as biomarker-based support for clinical diagnosis. Detection of individual aggregate forms of proteins by immunoassay has so far not been robust enough for proper validation and recognition, or lacked sufficient sensitivity and specificity to function as diagnostic markers. Another method proposes detecting aggregated proteins in biological fluids by using so-called seed amplification assays. In this method, the biological fluid is suspected to contain protein aggregates that are associated with certain proteinopathy and can act as seeds that trigger the aggregation of monomeric recombinant forms of the same protein that are added as substrates to the amplification process. In vitro amplified aggregates are typically detectable by amyloid-specific fluorescent dyes. To date, this assay has shown low interlaboratory reproducibility, its performance allows only qualitative readouts, and it requires several days of handling and incubation. Combined, this makes it difficult to develop current seed amplifications into diagnostic assays.

[0004] From a technical standpoint, the prerequisite for currently known methods for screening proteinopathy is a molecular entity that selectively stains or labels rich characteristic proteins. Detection of aggregated proteins requires molecules selective for such substances. Because aggregates underlie proteinopathy, they can be unpredictably complex and may harbor other proteins, lipids, and the aforementioned disintegrated organelles. This fact makes it difficult to achieve selectivity for aggregates over monomers in biological fluids, particularly in Parkinson's disease. The failure of known PET tracer programs and the lack of biomarkers support the view that known methods cannot adequately distinguish between aggregated and non-aggregated substances.

[0005] The dissociation constants of antibodies targeting inherently disordered proteins typically have an affinity of nM (nanomole). Structured epitopes and / or targets with multiple binding sites allow for achieving pM (picomole) affinity, with well-structured oligomers and coherently assembled fibrils being examples of such. All antibodies tested in Parkinson's disease (PD) targeting aSYN aggregates have failed to distinguish between patients and controls in the biofluid, at least with established detection principles. This may be related to 1) unfavorable concentration ratios of aggregates and monomers in the investigated groups, 2) nonspecific binding and lack of detection sensitivity in the biofluid due to the applied physical detection principle, 3) generally low abundance of targeted epitopes, and / or 4) broader spatial structure and constitutive complexity of the targeted aggregates. Published data suggest that the concentration range, meaning the concentration or amount of the targeted epitope here, is consistent with the affinity selectivity limits of all recognition elements established to date. Seed amplification assays are the only method that allows for the distinction between PD patients and healthy controls. Interestingly, this assay measures the nucleation ability of large fluid volumes, and it is known that it does not require antibodies or other recognition elements in the first place; a small amount of seed may be sufficient to initiate the amplification process.

[0006] Ideally, however, aggregates and co-aggregates associated with proteinopathy should be detected as analytes in easily accessible biological fluids without requiring complex tissue sampling (particularly in human brain tissue, which is mostly sampled postmortem), and methods for investigating and identifying analytes in undisturbed biological fluids should be applied. [Overview of the project]

[0007] Summary of Disclosure A general objective of the present invention is to advance the latest technology in the field of proteinopathy screening methods, preferably by completely or partially overcoming the shortcomings of the prior art.

[0008] In a favorable embodiment, a method is provided that enables screening for protein disorders in bodily fluid samples obtained from patients. In a particularly favorable embodiment, a method is provided that enables early detection of protein disorders. In a favorable embodiment, a method is provided that can be carried out on relatively readily obtainable biological fluid samples such as blood or urine.

[0009] In other advantageous embodiments, methods are provided that enable the identification of patients at high risk of developing the disease or those with specific subtypes of the disease, thereby enabling more personalized treatment and monitoring. In some preferred embodiments, methods are provided that enable improved classification and stratification of patients into clinical subtypes, as well as monitoring of disease progression and treatment effectiveness.

[0010] In a further advantageous embodiment, a method is provided that enables monitoring of disease progression and treatment response, particularly during clinical trials.

[0011] In a further advantageous embodiment, a method having a high signal-to-noise ratio is provided.

[0012] This general objective is achieved by the subject matter of the independent claims. Further advantageous embodiments are derived from the dependent claims and the overall disclosure.

[0013] According to aspects of the present invention, the present invention relates to a method for screening biological fluid samples for analytes related to protein disorders. In the first embodiment, the method is as follows: a. A step of providing a sensing device, the sensing device comprising an evanescent illuminator, the evanescent illuminator configured to generate an evanescent field on a first surface of the evanescent illuminator from a beam of coherent light having a predetermined wavelength. On the first surface of the evanescent illuminator, each evanescent illuminator comprises one or more sensing spots. Each sensing spot comprises a first recognition grid having a plurality of first unit cells and a second recognition grid having a plurality of second unit cells. The first and second recognition grids are interlocked with each other such that the plurality of first unit cells are arranged alternately with the second unit cells and vice versa. A plurality of first molecular recognition elements configured to bind to analytes associated with proteinosis are bound to the first unit cells. Furthermore, a plurality of second molecular recognition elements, different from the first molecular recognition elements, are bound to the second unit cells. In addition, each of the one or more sensing spots, i) At least a portion of the coherent light of the evanescent field is scattered in a plurality of first unit cells to generate a first constructive interference beam in a detector of a sensing device, and the first constructive interference beam is configured to have a first phase, ii) At least a portion of the coherent light of the evanescent field is scattered in a plurality of second unit cells to generate a second constructive interference beam in the detector, and the second constructive interference beam is configured to have a second phase which is the opposite of the first phase of the first constructive interference beam. iii) A step of providing a sensing device in which a first constructive interference beam generated interferes with a second constructive interference beam in a detector to generate a mass difference-dependent and optionally time-dependent signal in the detector, wherein the signal is configured to be proportional to the square of the mass difference generated by molecular interactions between a plurality of first molecular recognition elements and a plurality of second molecular recognition elements. b. A step of providing a biological fluid sample to one or more sensing spots of an evanescent illuminator. c. A step of generating a beam of coherent light having a predetermined wavelength at a predefined beam generation position. d. A step of measuring a signal for each of one or more sensing spots in a detector, wherein each signal is proportional to the square of the mass difference generated by molecular interactions between a plurality of first molecular recognition elements and a plurality of second molecular recognition elements of the corresponding sensing spot.

[0014] The method according to the present invention uses a recognition element such as an antibody or other elements described in the embodiments herein, but avoids the most central limitations of established detection principles. Similarly, since the basic detection principle converts the higher mass associated with the captured aggregates with a squared signal intensity, the sensing device can easily achieve selectivity for aggregates rather than monomers. A broader result is that the signal intensity is no longer proportional to or limited by the amount of the epitope, which may be rare on the surface of the analyte associated with proteinosis, i.e., difficult to access. This is especially true when the analyte forms aggregates. The total mass of the analyte is converted independently of the secondary surrounding or general chemical composition of the analyte. The spatial lock-in detection principle of the sensing device is to suppress ambient noise and exclude nonspecific binding. In summary, the method according to the present invention preferably represents a technique for measuring the mass of an analyte, particularly aggregates, associated with a target epitope, even in the presence of a monomer, and even when the analyte concentration is below the affinity limit of the molecular recognition element. The detection principle applicable to specific areas of proteinopathy, where it is necessary to distinguish aggregated high molecular weight analytes from monomers exposed to similarly configured epitopes, goes beyond the concept of established immunosensors. Even if the same amount of binding events are actually counted, the sensitivity to aggregates can be far higher than the sensitivity to monomers. Therefore, the method of the present invention can essentially weigh and distinguish aggregates in proteinopathy.

[0015] The reference of steps with letters such as a., b., c., d., and i., ii., iii. does not imply a specific order of steps, but rather these letters function as reference letters to identify specific steps of the claimed method. The method may be carried out stepwise from step a. to step d., but the claimed invention also includes cases where the steps are carried out in a different order, or where at least some steps are carried out simultaneously. For example, the invention also includes cases where step c., i.e., the generation of a coherent light beam, is carried out before, after, and / or during step b., i.e., the provision of a biological fluid sample.

[0016] In this specification, the term “comprising” is generally understood to mean that the features following it are included, but the existence of other features is not excluded insofar as it renders the claims unenforceable. On the other hand, when the expression “consisting of” is used, there are no further features other than those following it.

[0017] "A predetermined wavelength" refers to a wavelength of coherent light that is known in advance and is typically a single wavelength (meaning that coherent light is monochromatic).

[0018] The term "predefined beam generation position" refers to the position where a beam of coherent light is generated, and is also known in advance. Of course, if the sensor is adjustable (for example, within a very small range with respect to the precise position of the light source, the collision direction of the coherent light beam, a given wavelength of coherent light, or the precise position of the detector), the predefined beam generation position can be varied to such an extent that it falls within the adjustment range of the sensing device.

[0019] The term "unit cell" is not limited to any particular dimensions and merely serves to identify the position or region to which either the first or the second molecular recognition element is attached. This does not mean that the unit cell must necessarily be three-dimensional, although it may be so in some embodiments. Further, the unit cell can have any desired shape, which can be regular or irregular. In other words, the first unit cell is also referred to as the "first region" and the second unit cell is also referred to as the "second region". Further, a plurality of first unit cells together form a first recognition lattice, and a plurality of second unit cells together form a second recognition lattice.

[0020] Furthermore, it will be understood by those skilled in the art that the coherent light beam generated in step c. and the first unit cell are typically configured such that a portion of the coherent light scattered in the plurality of first unit cells generates a first constructive interference beam in the detector of the sensing device having a first phase. Similarly, it will be understood by those skilled in the art that the coherent light beam generated in step c. and the second unit cell are typically configured such that a portion of the coherent light scattered in the plurality of second unit cells generates a second constructive interference beam in the detector of the sensing device having a second phase opposite to the first phase.

[0021] It is further understood that the sensing device comprises a detector. The detector can comprise one or more different detector subunits in some embodiments. Preferably, the detector can comprise one detector subunit per sensing spot of the evanescent illuminator. In certain embodiments, each detector subunit can be associated with a single sensing spot. The detector is typically a photodetector.

[0022] It is understood by those skilled in the art that the detector is arranged at a predefined detection position, and each detector subunit is arranged at a predefined detection position. The term "predefined detection position" refers to the position where the first constructive interference beam interferes with the second constructive interference beam, and the position where the signal described in step d. is measured. The predefined detection position is known in advance. Also, if the sensing device is adjustable, the predefined detection position can vary to such an extent that it is within the adjustment range of the sensing device. Only when the coherent light has a predetermined wavelength, a beam of coherent light of this predetermined wavelength is generated at a predefined beam generation position, and the coherent light diffracted by the first molecular recognition element generates a first constructive interference beam at the detector in a first phase. Similarly, only when the coherent light has a predetermined wavelength, a beam of coherent light of this predetermined wavelength is generated at a predefined beam generation position, and the coherent light diffracted by the second molecular recognition element generates a second constructive interference beam at the detector in a second phase.

[0023] Due to these opposite phases of the first constructive interference beam and the second constructive interference beam, the two meshing recognition gratings represent an optical comparator that measures the difference in diffraction efficiency between the two meshing 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 / mm 2 , picograms per square millimeter). Therefore, the signal measured in step d. can be the difference in diffraction efficiency.

[0024] The scattered mass is the spatial Fourier component of the mass density distribution that satisfies the diffraction conditions of the recognition grating. All Fourier components of the mass density that do not satisfy the diffraction conditions are undetectable by the sensing device. This enables the detection or monitoring of molecular interactions, such as the binding of an analyte in a wash-free and real-time format, in biological fluid samples containing a large and vast diversity of background binding partners that can interfere with the molecular interactions of the analyte, i.e., enabling wash-free and real-time immunoassays. This is because the nonspecific binding of background binding partners is diluted over a large spectrum in Fourier space and is therefore undetectable by the sensing device.

[0025] A biological fluid sample is typically a sample obtained from a subject. The biological fluid is preferably any solution or suspension derived from a human or animal body (i.e., body fluids such as blood, serum, plasma, cerebrospinal fluid, interstitial fluid, saliva, tears, urine, etc.), or derived from an in vitro cell system or biochemical system. However, it is clear that the method according to the present invention is typically carried out in vitro. In some embodiments, the biological fluid sample is obtained from a subject (e.g., a human subject) that has not yet been diagnosed with a proteinopathy. The biological fluid sample may also include all biological fluids and biochemically processed forms of tissues, cells, or excretions derived from a human or animal body or an in vitro cell culture system (i.e., tissue or cell extracts or homogenates, fecal samples). The biological fluid sample may also include intact cells or intracellular structures (e.g., nuclei, lysosomes, exosomes, vesicles, nucleic acids containing molecules such as DNA and RNA, etc.) isolated from a human or animal body or derived from an in vitro cell culture system. Biological fluid samples typically contain analytes associated with proteinopathy. Such analytes are parts, particularly molecular parts, known to play a role in proteinopathy. For example, it may be a specific form or three-dimensional structure, or a part that occurs at different levels in biological fluid samples obtained from patients with proteinopathy compared to healthy subjects or patients with different symptoms. The biological fluid is preferably any aqueous fluid derived from the human or animal body (i.e., body fluids such as blood, serum, plasma, cerebrospinal fluid, interstitial fluid, saliva, tears, urine, etc., but not limited to), or derived from an in vitro cell system or biochemical system.

[0026] It is generally understood that biological fluid samples may contain a single analyte related to proteinosis or multiple analytes related to proteinosis. In the latter case, the method may be performed on only one, some, or all of the proteinosis-related analytes present in the biological fluid sample.

[0027] As used herein, the term “bonding” can refer to any chemical bond or physical attractive event at the molecular level, particularly one or more such as, but not limited to, covalent bonds, hydrogen bonds, ionic bonds, van der Waals forces, etc. Similarly, the molecular interactions of the first or second molecular recognition element as used herein typically involve chemical bonds or physical attractive events between the corresponding molecular recognition element and an interaction partner or background binding partner, such as an analyte associated with proteinosis.

[0028] As described above, multiple first molecular recognition elements are configured to bind to analytes associated with proteinosis. In particular, of the first and second molecular recognition elements, only the first molecular recognition elements are configured to bind to analytes associated with proteinosis. Since the signal generated at each sensing spot is produced from the interference of the first and second constructive interference beams having opposite phases, the signal can serve as a direct measure of the analytes associated with proteinosis. It is understood that multiple first molecular recognition elements are typically not bound to the second unit cell, and multiple second molecular recognition elements are typically not bound to the first unit cell.

[0029] Typically, the first molecular recognition element and / or the second molecular recognition element include one or more binding sites, such as binding sites, on which they can bind to a target, for example, an analyte associated with a proteinosis. In particular, the first molecular recognition element and the second molecular recognition element may differ from each other in that their binding sites are different, and only in that respect.

[0030] In some embodiments, step b, i.e., supplying a biological fluid sample to one or more sensing spots of the evanescent illuminator, is performed such that a flow direction is provided to the biological fluid sample. That is, the biological fluid sample is supplied so as to flow across the first surface and / or one or more sensing spots of the evanescent illuminator. This can be achieved, for example, by a fluid system of the sensing device. Such a fluid system may include, for example, one or more channels, such as microchannels.

[0031] In some embodiments, the first and second molecular recognition elements may be antibodies, particularly nanobodies, proteins, peptides, manipulated sequences of native L-type or artificial D-type amino acids, peptide polymers derived from amino acid-like molecules, oligonucleotides, single- or double-stranded sense or antisense oligonucleotide sequences or structures, or combinations thereof, or include them. In certain embodiments, the first and second molecular recognition elements are antibodies or include antibodies. The first and / or second molecular recognition elements may also be chemical and / or physical binders configured to bind to proteinosis-related analytes in either chemical bonding or physical attraction events as described above under the definition of “binding”.

[0032] In some embodiments, the first molecular recognition element includes a first linker element. The first linker element may be bound to, for example, a first unit cell. In some embodiments, the first molecular recognition element further includes a first binder unit configured to bind to an analyte associated with proteinosis. In certain embodiments, each of the first linker elements may be bound to a first unit cell and a first binder unit. The first binder unit may be an antibody, particularly a nanobody, a protein, a peptide, an engineered sequence of natural L-type or artificial D-type amino acids, a peptide polymer derived from an amino acid-like molecule, an oligonucleotide, a single-stranded or double-stranded sense or antisense oligonucleotide sequence or structure or a combination thereof. For example, the first binder unit and the first linker element may together form the first molecular recognition element.

[0033] Such a first molecular recognition element may be generated as follows: A first unit cell may contain a plurality of first binding sites. A first binder unit, configured to bind to an analyte associated with proteinosis and bound to a first linker-forming element, is then exposed to the first binding site. The first binding site and the first linker-forming element are configured to react selectively with each other to form a first linker element. Thus, the first linker element is bound to both the first binding site and the first binder unit and positioned between them.

[0034] Therefore, the binding sites attached to the first and second unit cells can function as anchor points for fixing, i.e., immobilizing, specific first and second molecular recognition elements to the binder unit, respectively. Linker elements, which may be part of the corresponding molecular recognition elements, are formed through binding reactions with linker-forming elements.

[0035] In some embodiments, the second molecular recognition element includes a second linker element or a second linker-forming element. The linker-forming element is generally a portion that can react, particularly selectively, with a reaction partner, such as a binding site, especially a second binding site bound to a second unit cell. Thus, it may be possible to bind the binding site to the second unit cell and the linker-forming element to the second binder unit. The linker-forming element and the second binding site can then react with each other to form a second linker element together. Such a second linker element is then bound to the second unit cell and the second binder unit. Generally, the second binder unit may be different from the first binder unit. For example, the second binder unit may be an antibody, especially a nanobody, a protein, a peptide, an engineered sequence of natural L-type or artificial D-type amino acids, a peptide polymer derived from an amino acid-like molecule, an oligonucleotide, a single-stranded or double-stranded sense or antisense oligonucleotide sequence or structure, or a combination thereof. For example, a second binder unit and a second linker element may together form a second molecular recognition element.

[0036] Furthermore, the first linker-forming element and the second linker-forming element may be different from each other. Additionally or alternatively, the first bonding site and the second bonding site may be different from each other.

[0037] In some embodiments, the first linker-forming element is configured to selectively react with the first binding site and optionally not with the second binding site. In some embodiments, the second linker-forming element is configured to selectively react with the second binding site and optionally not with the first binding site. In certain embodiments, the first binding site and the first linker-forming element may be complementary oligonucleotide chains specifically configured to form double-stranded DNA (e.g., as the first linker element). In certain embodiments, the second binding site and the second linker-forming element may be complementary oligonucleotide chains specifically configured to form double-stranded DNA (e.g., as the second linker element).

[0038] In certain embodiments, each first molecular recognition element may include, or consist of, a first linker element bound to a first unit cell and a first binder unit configured to bind to an analyte associated with proteinosis. In addition, the second molecular recognition element may include, for example, a second linker-forming element as described above, or, in some embodiments, may consist solely of that.

[0039] In some embodiments, the first molecular recognition element, each first binder unit, is configured to bind to the monomeric form of the proteinosis-related analyte. In certain embodiments, the first molecular recognition element (each first binder unit) is configured to selectively bind to the monomeric form of the proteinosis-related analyte, to its formed aggregates (i.e., its aggregated form).

[0040] In some embodiments, the first molecular recognition element (each a first binder unit) is configured to bind to formed aggregates of the proteinosis-related analyte (i.e., the formed aggregates). In certain embodiments, the first molecular recognition element (each a first binder unit) is configured to selectively bind to the formed aggregates of the proteinosis-related analyte (i.e., the aggregate form) relative to its monomer form.

[0041] The method according to the present invention enables the direct measurement of capture mass (i.e., any molecular interaction between molecular recognition elements and the analyte) in highly complex biological samples, and since it uses the spatial affinity lock-in principle and is inherently self-referential, it can almost completely suppress environmental noise. Furthermore, the method can measure the mass increase at molecular recognition elements with square sensitivity. Thus, the method according to the present invention can detect the mass of analytes associated with proteinosis or the aggregates formed thereon, making it possible to determine the composition or in situ formation of complex aggregates. The formed aggregates may be complex, but they can be detected as long as they expose at least one binding site that can interact with a first and optionally second molecular recognition element that can be detected. This is particularly advantageous for certain analytes associated with proteinosis, because they have been found to recruit additional material such as any residual biological material, e.g., cellular and intracellular components such as mitochondria, cell membranes, vesicle membranes, nucleic acids, proteins and their fragments, resulting in the growth of aggregates of a highly heterogeneous and complex nature.

[0042] In some embodiments, the first molecular recognition elements and / or second molecular recognition elements of a given first or second unit cell may each contain only a single binding site (i.e., a binding site for binding to a target such as an analyte associated with a proteinosis) or each molecular recognition element may contain multiple binding sites, in particular multiple different binding sites. In certain embodiments, the first molecular recognition elements of a given unit cell may each contain only a single binding site configured to bind to an analyte associated with a proteinosis, or they may each contain multiple binding sites, in particular multiple different binding sites, configured to bind to an analyte associated with a proteinosis.

[0043] In some embodiments, the first surface of the evanescent illuminator includes a plurality of sensing spots. In such embodiments, it is understood that each sensing spot includes first and second molecular recognition elements. Also, when generating the beam in step c, a portion of the coherent light is scattered typically at each sensing spot, i.e., at each of its first and second molecular recognition elements in the manner described in steps ai to iii. Thus, each sensing spot may generate its own signal from the interference of the corresponding first and second constructive interference beams. In this case, step d includes measuring each of these signals.

[0044] In some embodiments, the measured signal(s) are stored after step d. in a memory unit that is part of a control unit such as a circuit or microprocessor, or under the control of the control unit.

[0045] In some embodiments, a sensing spot among a plurality of sensing spots, particularly the first molecular recognition element of each sensing spot, is different from at least some or all of the first molecular recognition elements of other sensing spots. Such embodiments enable multiplexing. In particular, the first molecular recognition element of each sensing spot among a plurality of sensing spots is different from the first molecular recognition elements of other sensing spots. In some embodiments, the first molecular recognition element of one of the sensing spots or a group of sensing spots is unique compared to other sensing spots in the sensing device. In particular, the first molecular recognition element of each sensing spot or each group of sensing spots is unique compared to other sensing spots in the sensing device.

[0046] In some embodiments, a sensing spot among a plurality of sensing spots, particularly the second molecular recognition element of each sensing spot, is different from at least some or all of the second molecular recognition elements of other sensing spots. In particular, the second molecular recognition element of each sensing spot among a plurality of sensing spots is different from the second molecular recognition elements of other sensing spots. In some embodiments, the second molecular recognition element of one sensing spot or group of sensing spots is unique compared to other sensing spots in the sensing device. In particular, the second molecular recognition element of each sensing spot or group of sensing spots is unique compared to other sensing spots in the sensing device. In other embodiments, a plurality of second molecular recognition elements are the same for each sensing spot.

[0047] One advantage of providing multiple, i.e., two or more, sensing spots having different first molecular recognition elements is that it allows for the decomposition of the complexity of proteinosis analytes and enables immunosignatures, such as mass-weighted immunosignatures. For example, a first sensing spot may include a first molecular recognition element configured to bind to a first binding site of a proteinosis-related analyte, and a second sensing spot may be configured to bind to a second binding site of a proteinosis-related analyte. Alternatively, different first molecular recognition elements may bind to proteinosis-related analytes with different binding affinities. Since proteinosis-related analytes can be not only a single molecule in some embodiments but also aggregates of multiple parts such as cellular components or their fragments, proteins, nucleic acids, etc., using multiple such sensing spots can provide detailed information about the analyte. Body fluids from patients with proteinosis may have a similar immunoreaction profile to controls and can be distinguished from healthy controls, since the aggregate morphology contributes to the measured signal with squared intensity. By using different first molecular recognition elements, the complexity of the analyte can be decomposed. Each signal generated in the detector depends on the mass-increasing effect of the molecular interaction between the corresponding first molecular recognition element and the analyte in a specific detector unit of the detector. Therefore, each signal can be used as a quantity or parameter of the analyte in a high-dimensional vector space. By using multiple sensing spots with different first molecular recognition elements, an immunosignature of a biological fluid sample and / or analyte, such as a mass-weighted immunosignature, can be generated. This immunosignature can then be used to identify or monitor analytes associated with proteinopathy, and in particular, without post-processing of the sample, conclusions can be drawn regarding the identity, nature, and / or clinical or pathological state of the proteinopathy of the subject obtained from the biological fluid sample. This aspect should not be underestimated, as aggregated proteins are soft and constitute complex supramolecular assemblies of proteins, lipids, and biochemicals.These corresponding high molecular weights make them sufficiently detectable in molecular recognition, both in monomeric form and in monomeric form.

[0048] In some embodiments, the difference between a first molecular recognition element of a given sensing spot and a first molecular recognition element of another sensing spot is that the first molecular recognition elements, or at least their binding sites, are chemically distinct from each other.

[0049] Additionally or alternatively, the difference between a first molecular recognition element at a given sensing spot and a first molecular recognition element at another sensing spot is, in some embodiments, characterized in that they are configured to bind to different analytes associated with proteinosis.

[0050] Additionally or alternatively, the difference between a first molecular recognition element at a given sensing spot and a first molecular recognition element at another sensing spot is, in some embodiments, characterized in that they are configured to bind to different epitopes of analytes associated with proteinosis.

[0051] Additionally or alternatively, the difference between a first molecular recognition element at a given sensing spot and a first molecular recognition element at another sensing spot is, in some embodiments, characterized in that they are configured to bind to the same analyte or epitope with different binding affinities.

[0052] In some embodiments, multiple sensing spots, particularly at least some or all of their first and / or second molecular recognition elements, compete for an analyte associated with proteinosis, and step d. is performed over a predetermined measurement time to monitor the competition of sensing spots for the analyte associated with proteinosis. In certain embodiments, signals measured over a predetermined measurement time are also considered time-dependent signals and are included in the immunosignature of the biological fluid sample.

[0053] In some embodiments, particularly in embodiments where at least some of the multiple sensing spots compete for an analyte related to a proteinosis, step b. is performed such that a biological fluid sample is provided to the multiple sensing spots and sequentially contacts one sensing spot at a time. These embodiments can be considered as a continuous competitive measurement. In such a continuous competitive measurement, at least some or even all of the sensing spots are in fluid communication with each other; that is, the sensing spots are not separated from each other by a wall structure. In a continuous competitive measurement, it is possible to provide a flow direction to the biological fluid sample, i.e., to flow in a specific direction and thus sequentially contact the sensing spots. Preferably, in such embodiments, steps c. and optionally d. are performed during step b. and optionally before step b. The biological fluid sample may also be provided to multiple sensing spots simultaneously, in particular, so as to contact all sensing spots essentially at the same time. Preferably, in such embodiments as well, steps c. and optionally d. are performed during step b. and optionally before step b. In a preferred embodiment of such continuous competitive measurement, the sensing device includes sensing spots comprising different first and / or different second molecular recognition elements. That is, a first unit cell of a given sensing spot may be coupled to a first molecular recognition element that is different, and preferably unique, to a first molecular sensing spot coupled to a first unit cell of another sensing spot of the sensing device.

[0054] In some embodiments, at least a portion of the sensing spots are separated from each other, particularly by wall structures. Such wall structures may be configured to prevent the biological fluid sample from flowing between the separated sensing spots, particularly during or after step b. Such measurements can be considered parallel measurements. In particular, the sensing spots can be divided into multiple sensing spot groups, and different sensing spot groups are separated from each other, for example, by wall structures(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 compared to the first and / or second molecular recognition elements of another sensing spot group. That is, for example, the first group may include two sensing spots, each having the same first molecular recognition element. However, the second group may also include two sensing spots, each having a different first molecular recognition element, as two sensing spots of the first group.

[0055] In some embodiments, the method further includes the step of determining the source of at least one or all of the signals measured by the detector. By determining the source of the signals measured by the detector, it may be possible to identify, for example, a specific sensing spot from which the signal was emitted. Since it is known which first and / or second molecular recognition elements are present at each sensing spot, further information can be obtained regarding the biological fluid sample and / or analytes associated with proteinosis.

[0056] In some embodiments, the method further includes a step of determining parameters of an analyte associated with proteinosis from the mass difference-dependent and optionally time-dependent signals measured in step d. The determined parameters may be, for example, a single point, e.g., mass or mass per unit surface area, or an increase or decrease in mass over time at one or more sensing spots, particularly its first molecular recognition element, or the gradient or any higher derivative or change thereof of the increase or decrease in mass over time at one or more sensing spots, particularly its first molecular recognition element.

[0057] Additionally or alternatively, the method further includes the step of determining parameters of aggregates of proteinosis-related analytes or co-aggregates with proteinosis-related analytes from mass difference-dependent and optionally time-dependent signals. In this case, the determined parameters may be, for example, a single point, such as mass or mass per unit surface area, or one or more sensing spots, particularly the increase in mass over time at their first molecular recognition element, or the gradient or any higher-order derivative or change thereof of the increase or decrease in mass over time at one or more sensing spots, particularly at their first molecular recognition element.

[0058] For example, it is possible to observe in real time whether an analyte associated with proteinosis has recruited additional material to form aggregates or coagulates. Furthermore, it is also possible to observe such recruitment of additional material in real time. The additional material may be any residual biological material, such as cellular components, mitochondria, cell membranes, nucleic acids, proteins, and fragments thereof.

[0059] In some embodiments, the determined parameters are included in the formed immunosignature of the biological fluid sample and / or proteinosis analyte.

[0060] In some embodiments, the method further includes a step of determining the presence of aggregates or coagulants from the determined parameters. In a preferred embodiment, the presence of aggregates or coagulants is determined if the determined parameters exceed a predetermined threshold within a predetermined incubation time.

[0061] In some embodiments, determining the presence of aggregates or coagulants involves comparing the determined parameter with a reference parameter. In certain embodiments, the reference parameter is obtained from a different patient population, from a healthy subject, or from the subject from which the biofluid sample was obtained. In some embodiments, the determined parameter is compared to a database containing multiple reference parameters, particularly multiple reference parameters from different patient populations, from healthy subjects, and / or from the subject from which the biofluid sample was obtained. For example, the database may contain reference parameters for multiple patient populations suffering from different proteinopathy (compared to other patient populations in the database). The database may also contain multiple patient populations suffering from the same proteinopathy but at different clinical stages (compared to other patient populations in the database). Such embodiments provide useful data for physicians to assess the presence, nature, stage, and progression of proteinopathy.

[0062] In some embodiments, the proteinopathy-related analytes include β-amyloid, tau, α-synuclein, prion proteins (such as native or abnormally folded forms of prion proteins), fusion sarcomas, wild-type or mutant polyQ huntingtin, ubiquitin, ataxin-3, optinurin, TAR DNA-binding protein 43, neurofibrillary light chain (NfL), soluble or detachable trigger receptor 2 (sTREM2) expressed on myeloid cells, chitinase-3-like protein 1, glial fibrillary acidic proteins, and one or more truncated or other post-translational modified forms thereof. Post-translational modifications may include, but are not limited to, phosphorylation, nitration, ubiquitination, glycation and glycosylation, dityrosine binding by oxidation and oxidation, and methylation. In certain embodiments, the analytes associated with proteinosis may also be aggregates or coagulants comprising one or more of the aforementioned parts (including themselves or their post-translational modified forms) and additional materials such as any residual biological material, e.g., cellular components, mitochondria, cell membranes, nucleic acids, proteins and their fragments.

[0063] In some embodiments, step d. is performed over a predetermined measurement time, and each signal is measured as a function of the predetermined measurement time. The signals measured over the predetermined measurement time may, in some embodiments, further be included in the immunosignature of the biological fluid sample and / or analytes associated with proteinosis. Measuring the signals over a predetermined measurement time can provide further insights into the nature of the analytes and proteinosis. For example, it is possible to monitor the behavior over time with respect to molecular recognition elements, in particular the behavior over time with respect to different molecular recognition elements, or to monitor the changes over time when the provided biological fluid sample is exposed to altered conditions, such as pH, a medium of different composition, a soluble binding partner (natural ligand), or a synthetic or manipulated molecule that acts as an artificial ligand or interferes with the stability of the analyte, such as agglomeration stabilizers or agglomeration destabilizers as referred herein.

[0064] In certain embodiments, changes in each signal are measured during a predetermined measurement time, and these changes are brought about by aggregation, in particular by aggregation of any residual biological material, such as cellular components, mitochondria, cell membranes, nucleic acids, proteins and their fragments, especially additional material in the biological sample such as cellular components and / or proteins.

[0065] In some embodiments, step d. includes one or more single-point measurements at a specific time point, in particular an endpoint measurement. In some embodiments, the signals obtained from each single-point measurement are included in the immunosignature of the biological fluid sample and / or analyte associated with proteinosis. It is understood that single-point measurements, in particular an endpoint measurement, do not exclude simultaneous measurements over a predetermined measurement time. In some embodiments, step d. may include only one or more single-point measurements or only measurements over a predetermined measurement time, while in some embodiments, both measurements may be included in step d.

[0066] In some embodiments, the method further includes a rinsing step. The rinsing step may be performed before step b, in particular before or during step d. The rinsing step may be performed using a rinsing solution.

[0067] Alternatively or additionally, the method, in some embodiments, includes a processing step. The processing step may preferably include the application of a treatment agent configured to alter conditions. For example, the treatment agent may be selected from one or more of the following: detergents, ionic agents, solvents, acids, bases, flocculation stabilizers, and flocculation destabilizers. A flocculation stabilizer is an agent configured to stabilize aggregates, while a flocculation destabilizer is an agent configured to destabilize, for example, decompose, aggregates. For example, a flocculation stabilizer or flocculation destabilizer may be a chemical molecule such as a small molecule (generally having a molecular mass of ≤1000 Da) or a large molecule (generally having a molecular mass of >1000 Da), such as an antibody, Fab fragment, or nanobody. In certain embodiments, the flocculation stabilizer or flocculation destabilizer may be a recombinant or synthetic form of a synthetic or manipulated molecule that acts as a proteinosis-related analyte, a natural ligand, an artificial ligand, or interferes with the stability of the aggregate analyte. Such embodiments, including the application of aggregation stabilizers or aggregation destabilizers, are advantageous because early proteinosis may have low levels of rigid, stable aggregates, while later proteinosis or multiple system atrophy may have high levels of rigid-type aggregates that are less prone to collapse. Therefore, the application of aggregation stabilizers or aggregation destabilizers may provide insight into the stage of proteinosis. Furthermore, in some embodiments, the measured signals may also be included in the immunosignature of biological fluid samples and / or analytes associated with proteinosis.

[0068] In some embodiments, step d is performed over a predetermined measurement time during the rinsing and / or processing steps. This makes it possible to monitor changes in the measurement signal over time as well as the effects of the rinsing and / or processing steps. In some embodiments, such signals measured over time may also be included in the immunosignature of the biological fluid sample and / or analytes associated with proteinosis.

[0069] In some embodiments, step d. includes a single-point measurement at a specific point in time prior to the rinsing and / or processing steps. In some embodiments, step d. includes a single-point measurement at a specific point in time after the rinsing and / or processing steps. In some embodiments, each such measurement, and the signals obtained from such measurements, may also be included in the immunosignature of the biological fluid sample and / or analytes associated with proteinosis.

[0070] In some embodiments, after step b, incubation is performed of one or more sensing spots with a labeled, particularly mass-labeled or fluorescently labeled, binder. The mass-labeled binder is preferably a binder having a known, identifiable, and optionally unique mass. In certain embodiments, the mass-labeled binder may include, for example, nanoparticles. The nanoparticles preferably include transition metals or transition metal oxides such as gold, TiO2, Ta2O5, and silver. When a fluorescently labeled binder is used, in some embodiments, the fluorescence signal can be measured with a fluorescence detector. The fluorescence detector is typically an additional, and therefore separate, detector. In some embodiments, the detected fluorescence signal is included in the immunosignature of the biological fluid sample and / or the analyte associated with the proteinosis. In some embodiments, the binder may include a predefined binding site that can be specifically configured to bind to the analyte associated with the proteinosis. Generally, the predefined binding site of the binder may, in some embodiments, be configured not to bind to the first and / or second molecular recognition element.

[0071] In some embodiments, the binder is configured to bind to analytes associated with proteinosis, particularly to lysosomal markers, mitochondrial markers, nucleotides, sugars, post-translational modifications, or mitochondrial DNA of the analytes associated with proteinosis. This may be particularly advantageous when the analyte is aggregates or coagulations of any residual biological material, such as cellular components, mitochondria, cell membranes, nucleic acids, proteins and their fragments, and especially additional materials such as cellular components and / or proteins.

[0072] In some embodiments, the signals measured during or after incubation of one or more sensing spots with a labeled, particularly mass-labeled or fluorescently labeled, binder are also included in the immunosignature of the biological fluid sample and / or proteinosis analyte.

[0073] In some embodiments, the method includes a step of performing a seed amplification assay after step b. This does not mean that the seed amplification assay step must necessarily be performed before step c, but it may be in some embodiments. It may be performed together with step c. Performing a seed amplification assay involves adding a binding reagent sample, in particular a protein sample, to one or more sensing spots. It is understood that the binding reagent sample is typically added after step b. This is because the proteinosis-related analytes present in the biological fluid sample act as seeds that trigger aggregation (e.g., aggregation of other monomeric proteins, in particular recombinant or synthetic forms of the monomeric analytes). Furthermore, step d. includes measuring each signal over a predetermined measurement time. Preferably, the protein sample contains monomeric proteins. Monomeric proteins can act as substrates for co-aggregation in the seed amplification assay. Depending on the presence and nature of the proteinosis analytes, the added proteins in the protein sample will misfold with the analytes and aggregate during the predetermined measurement time. Aggregation causes a mass increase and can therefore be easily measured during step d. In seed amplification assays, the conjugated analytes associated with proteinopathy (particularly those that form aggregates) may act in situ as seeds to co-aggregate with recombinant or synthetic forms of other conjugating reagents provided, for example, as proteinopathy-related analytes or processing agents. In some embodiments, seed amplification assays may further include the addition of other conjugating reagents in addition to or as a substitute for the protein sample. The conjugating reagents that may be included in the sample may include, but are not limited to, small molecules (i.e., having a mass of ≤1000 Da, e.g., the aforementioned PET tracer-like molecules or amyloid structure binders, peptides or oligonucleotides) or large molecules (i.e., having a mass of >1000 Da, e.g., antibodies, nanobodies, or other polypeptides).

[0074] In certain embodiments, multiple sensing spots are used, each having a different first molecular recognition element described herein. In such embodiments, the seed amplification assay produces different signals due to different interactions at different sensing spots. In some embodiments, the measured signals may be included in the immunosignature of a biological fluid sample and / or an analyte associated with a proteinopathy, e.g., a mass-weighted immunosignature. Such immunosignatures may be highly specific and therefore unique to the clinical stage of the patient from which the particular analyte and / or biological fluid sample of the proteinopathy was obtained.

[0075] In some embodiments, the method further includes a secondary characterization step for characterizing the analyte associated with the proteinosis, or the state of aggregates or coagulations formed by the analyte associated with the proteinosis. In preferred embodiments, the secondary characterization step includes ELISA, FT-IR, Raman, or fluorescence spectroscopy. The secondary characterization can be performed at any time, particularly during or after step b. The secondary characterization step provides secondary characterization parameters. In some embodiments, the secondary characterization parameters are also included in the immunosignature of the biological fluid sample and / or the analyte associated with the proteinosis.

[0076] In some embodiments, the secondary characterization process is performed directly on the biological fluid sample provided to one or more sensing spots of the evanescent illuminator. Thus, in such embodiments, the secondary characterization process is performed directly while the biological fluid sample is present on the sensing device.

[0077] In some embodiments, the signals (one or more) measured for each sensing spot during step d. form an immunosignature (e.g., a mass-weighted immunosignature) of the biological fluid sample and / or proteinosis-related analyte. Optionally, other parameters of the proteinosis analyte are also included in the immunosignature. For example, as referred to herein, in some embodiments, the parameters of the proteinosis-related analyte determined from the mass-difference-dependent and optionally time-dependent signals measured in step d., the fluorescence signal of the fluorescent labeling binder, the signals measured during the seed amplification assay, the determined source of at least one or all of the signals measured in the detector, the signals obtained from the application of the treatment agent, and / or secondary characterization parameters are also included in the immunosignature of the biological fluid sample and / or proteinosis-related analyte.

[0078] An immunosignature, such as a mass-weighted immunosignature, can in some embodiments be considered a vector, particularly a time-dependent vector, whose trajectory in a high-dimensional vector space is unique to the screened biological fluid sample. In this context, the term “high-dimensional” refers to at least three dimensions or more. All parameters and / or signals obtained during step d. deconvolve the complexity of the biological fluid sample. An immunosignature, such as a mass-weighted immunosignature, can be considered a unique fingerprint of the screened biological fluid sample. Thus, in some embodiments, it is possible to generate multiple immunosignatures over time for biological fluid samples obtained from the same subject within a defined time interval, such as annually or semi-annually, and to compare the immunosignatures with each other or with reference data further outlined below. Thus, an immunosignature is a direct measure of the pathological state of the subject from which the aggregated proteins and / or biological fluid sample originate.

[0079] In some embodiments, the immunosignature of a biological fluid sample is compared to a database containing immunosignatures of multiple samples, such as mass-weighted immunosignatures. In particular, the samples may be patient samples, autologous patient samples (i.e., previously screened samples from patients from whom the currently screened biological fluid sample was obtained), and / or reference samples. In preferred embodiments, each immunosignature in the database is associated with the clinical state of proteinopathy. Thus, by comparing the immunosignature of a screened biological fluid sample with the immunosignatures contained in the database, it is possible to characterize the nature and / or clinical state of the proteinopathy to which the analyte is associated. Furthermore, it may be possible to predict, at least to some extent, the progression of proteinopathy. Such database comparisons also make it possible to perform pattern recognition analysis in which the immunosignature of a screened biological fluid sample is analyzed for certain patterns that may be found in some of the immunosignatures stored in the database. This makes it possible to detect proteinopathy in very early stages, even before the subject from which the biological fluid sample originates develops recognizable clinical symptoms.

[0080] The comparison with the database can generally be implemented on a computer. The database can be stored in a memory unit such as a server, hard drive, or cloud. The comparison with the database may also be performed by a control unit such as a circuit or microprocessor. The high-dimensional and multimodal data types stored in the relevant database or generated in the aforementioned analysis can be further analyzed and / or processed by machine learning and artificial intelligence analytical methods for identifying immunosignatures.

[0081] In some embodiments, multiple second molecular recognition elements are configured not to selectively bind to analytes associated with proteinosis. In contrast, multiple first molecular recognition elements are configured to selectively bind to analytes associated with proteinosis.

[0082] In some embodiments, multiple second molecular recognition elements are configured not to bind to analytes associated with proteinosis. Therefore, such second molecular recognition elements cannot bind to analytes associated with proteinosis.

[0083] For example, in certain embodiments, the first and second molecular recognition elements may be antibodies or may include antibodies.

[0084] The first molecular recognition element may include a paratope configured to bind to an analyte associated with proteinosis, particularly its epitope, while the second molecular recognition element may lack this paratope. In certain embodiments, the absence of this paratope may be the only difference between the binder units of the first and second molecular recognition elements, and between the antibodies of the first and second molecular recognition elements, respectively. For example, this can already be achieved by point mutations, such as point mutations within the paratope that avoid binding to the analyte associated with proteinosis. The second molecular recognition element may also be any other molecule, but preferably it may be different from the first molecular recognition element in order to generate a binding-associated mass contrast between the first and second binder units.

[0085] In some embodiments, both the first and second molecular recognition elements of each sensing spot are configured to interact with the same background binding partner. The background binding partner can be any chemical or biological part that can be bound by the first and / or second molecular recognition elements, particularly beyond the analytes associated with proteinosis. 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 present in the sample, or binders having epitopes that can bind to the first and / or second molecular recognition elements. Such embodiments have the advantage that, because the signals obtained in step d. are due to the out-of-phase first and second constructive beams, interactions of background binding partners present in the sample but unrelated to specific analytes associated with proteinosis are not inherently detected or essentially undetectable when measuring the difference in interactions (i.e., mass difference) between the first and second molecular recognition elements. This is particularly advantageous for highly complex biological fluid samples such as CSF or human serum.

[0086] In some embodiments, the first molecular recognition element and the second molecular recognition element have essentially the same affinity K for the same background binding partner. D It holds.

[0087] In some embodiments, the second molecular recognition element has an isoelectric point 0.8 to 1.2 times, particularly 0.9 to 1.1 times, more specifically 0.95 to 1.05 times, or even more specifically, the same as, that of the first molecular recognition element. Thus, in certain embodiments, the isoelectric points of the second molecular recognition element and the first molecular recognition element at a sensing spot (for the same sensing spot of multiple sensing spots, respectively) may be essentially the same, particularly identical.

[0088] Each first molecular recognition element, each first binder unit, can be bound to a first unit cell by a first linker element. The first linker element can be formed by a reaction, particularly a selective reaction, between a first linker-forming element and a first binding site bound to the first unit cell. The first linker element may include, for example, two complementary oligonucleotide chains. The two complementary oligonucleotide chains may be a tip oligonucleotide chain bound to the first unit cell or a portion thereof, and a molecular recognition element oligonucleotide chain bound to the first molecular recognition element or a portion thereof.

[0089] Each second molecular recognition element, each second binder unit, may be bound to a second unit cell by a second linker element. The second linker element may be formed by a reaction, particularly a selective reaction, between a second linker-forming element and a second binding site. The second binding element may include, for example, two complementary oligonucleotide chains. The two complementary oligonucleotide chains may be a tip oligonucleotide chain bound to the second unit cell or a portion thereof, and a molecular recognition element oligonucleotide chain bound to the second molecular recognition element or a portion thereof.

[0090] It is understood that the first and second linker elements are typically orthogonal linker elements; that is, the first linker-forming element and the first binding site selectively react with each other, and the second linker-forming element and the second linker binding site selectively react with each other. However, typically, cross-reactions are absent or essentially absent. Therefore, it is possible to immobilize the first molecular recognition element only on the first unit cell and the second molecular recognition element only on the second unit cell. For example, such a method can be used, as described in Current Opinion in Chemical Biology, 2014, 18:8, 8-15, ISSN 1367-5931, DOI:10.1016 / j.cbpa.2013.10.023.

[0091] In some embodiments, the biological fluid sample includes one or more background binding partners distinct from the analyte associated with the proteinosis. One or more background binding partners may be configured to trigger a nonspecific binding signal in step d. One or more background binding partners may be configured to bind, for example, to a first molecular recognition element and / or a second binding site. In such embodiments, for example, the second molecular recognition element of the sensing device provided in step a. may include or consist of 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 to them), using background binding partners as the second molecular recognition element ensures that the second unit cell is saturated with background binding partners before steps b. through d. Thus, a stable baseline is achieved, and the influence of further background binding partners in the biological fluid sample is significantly reduced. This makes it possible to avoid or at least significantly reduce nonspecific binding events, thereby enabling monitoring of specific binding events by the first molecular recognition element to the analyte associated with the proteinosis. A background bonding partner may be bonded to a second unit cell, for example, by a second linker element as described herein. For example, the second unit cell may include a second bonding site that forms a second linker element, which is bonded to one or more background bonding partners, together with a second linker-forming element.

[0092] As a non-limiting example, it may be possible to use a human-derived biological fluid sample, such as CSF or blood sample, and to use a second molecular recognition element containing or consisting of human serum albumin. Human serum albumin has been identified as one of the major components in human-derived biological fluid samples responsible for nonspecific binding. Therefore, in certain embodiments, the sensing device provided in step a. includes a plurality of first molecular recognition elements configured to bind to an analyte associated with proteinosis and to bind to a first unit cell. Furthermore, the sensing device includes a plurality of second molecular recognition elements containing or consisting of human serum albumin and bound to a second unit cell.

[0093] In certain embodiments, the method may include a pre-screening step, which includes identifying background binding partners in a biological fluid sample. For example, this may include, in step d, the identification of background binding partners that bind to a first molecular recognition element and / or trigger a nonspecific binding signal. The pre-screening step may also be carried out using a sensing device as described in any embodiment of this specification, for example, a sensing device in which the second molecular recognition element consists of an oligonucleotide chain. After performing steps b. to d., nonspecific 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. Following this pre-screening step, step a. (i.e., providing the sensing device) may include incorporating the identified background binding partners into the second molecular recognition element, for example, by attaching them to an oligonucleotide chain. Steps b. to d. may then be carried out.

[0094] In some embodiments, the first molecular recognition element is an antibody or has an antibody size (150 kDa), with a molecular recognition capacity of 0.1 to 40 fmol / mm³. 2 (Femtomoles / square millimeter), especially 1 and 4 fmol / mm² 2is bound to the first unit cell at a surface density of. For smaller affinity elements such as nanobodies or aptamers of 15 - 50 kDa, the first molecular recognition element binds to the first unit cell at a surface density of 1 and 320 fmol / mm 2 , particularly 16 and 64 fmol / mm 2 . Alternatively or additionally, if the molecular recognition element is an antibody or has the size of an antibody (150 kDa), the second molecular recognition element can be bound to the second unit cell at a surface density of 0.1 - 40 fmol / mm 2 , particularly 1 and 4 fmol / mm 2 . For smaller affinity elements such as nanobodies or aptamers of 15 - 50 kDa, the first molecular recognition element binds to the first unit cell at a surface density of 1 and 320 fmol / mm 2 , particularly 16 and 64 fmol / mm 2 . Particularly, when the first and / or the second molecular recognition element is an antibody, such a surface density is advantageous as it avoids the interaction between antibodies on the one hand and further enables a relatively high density occupation of the unit cell, thereby improving the measurement and affinity of the sensing device.

[0095] In some embodiments, the biological fluid sample can be pretreated before step b. For example, before step b, the biological fluid sample may be centrifuged and / or filtered through a filter.

[0096] In some embodiments, steps b., c., d., and optionally step a. can be repeated at least once, at least twice, or even more times. Each repetition can be considered a screening cycle.

[0097] Each screening cycle may differ. For example, in one screening cycle, only a blank biological fluid sample may be screened, providing a blank signal. In another screening cycle, seed amplification as described herein may be performed on the same biological fluid sample, providing a seed amplification signal. In yet another screening cycle, incubation with a labeling binder as described in some embodiments herein may be performed, providing a labeling binder signal. In yet another screening cycle, processing and / or rinsing steps as described in some embodiments herein may be performed, providing a processing signal and / or rinsing signal. In yet another screening cycle, a series of competitive measurements as described in some embodiments herein may be performed, providing several competitive signals. In a preferred embodiment, in yet another screening cycle, a first series of competitive measurements is performed by providing a biological fluid sample to a sensing spot in a first flow direction, and in a subsequent screening cycle, a second series of competitive measurements is performed by providing a biological fluid sample to a sensing spot in a second flow direction opposite to the first flow direction. In yet another screening cycle, parallel measurements as described in some embodiments herein may be performed, providing parallel measurement signals. In some embodiments of the method according to the present invention, it is possible to perform one, two, more, or all of these screening cycles.

[0098] In certain embodiments, the signals and / or parameters obtained in each screening cycle are included in the immunosignature of the biological fluid sample and / or the analyte associated with the proteinosis.

[0099] A second aspect of this disclosure relates to a method for screening biological fluid samples for analytes related to proteinosis, wherein the method is a. A step of providing a sensing device, the sensing device comprising an evanescent illuminator, the evanescent illuminator configured to generate an evanescent field on a first surface of the evanescent illuminator from a beam of coherent light having a predetermined wavelength, the first surface of the evanescent illuminator comprising one or more sensing spots, each sensing spot comprising a first recognition grid having a plurality of first unit cells and a second recognition grid having a plurality of second unit cells, the first and second recognition grids interlock with each other such that the plurality of first unit cells are alternately arranged with the second unit cells, a plurality of first coupling sites are coupled to the first unit cells, a plurality of second coupling sites different from the first coupling sites are coupled to the second unit cells, and each of the one or more sensing spots is i. At least a portion of the coherent light of the evanescent field is scattered in a plurality of first unit cells to generate a first constructive interference beam having a first phase in the detector of the sensing device, ii. At least a portion of the coherent light of the evanescent field is scattered in a plurality of second unit cells to generate a second constructive interference beam in the detector having a second phase opposite to the first phase, iii. A sensing device comprising the steps of providing a sensing device in which a first constructive interference beam generated interferes with a second constructive interference beam in a detector to generate a mass difference-dependent and optionally time-dependent signal in the detector, wherein the signal is configured to be proportional to the square of the mass difference generated by molecular interactions between a plurality of first binding sites and a plurality of second binding sites. b. A step in which a screening cycle is carried out, i. Immobilizing a first molecular recognition element or first binding unit to a first binding site rather than a second binding site, wherein the first molecular recognition element or binding unit is configured to bind to an analyte associated with proteinosis. ii. Optionally, immobilizing a second molecular recognition element, a second binding unit, or a background binding partner at the second binding site instead of the first binding site, wherein the second molecular recognition element or background binding partner is different from the first molecular recognition element (in each case, the second binding unit is different from the first binding unit). iii. Providing a biological fluid sample to one or more sensing spots of an evanescent illuminator. iv. To generate a beam of coherent light having a predetermined wavelength at a predefined beam generation position. v. Measuring a signal for each sensing spot in the detector, wherein each signal is proportional to the square of the mass difference generated by molecular interactions between a plurality of first molecular recognition elements (or first binding units) and a plurality of second molecular recognition elements (or second binding units or background binding partners). vi. A step of performing a screening cycle, which includes reactivating the sensing device by optionally cleaning, by removing the immobilized first molecular recognition element or first binding unit from the first binding site, and optionally removing the immobilized second molecular recognition element (or second binding unit or background binding partner) from the second binding site. c. The process includes repeating step b. once or several times, using different first molecular recognition elements (or first binding units), and optionally using different or the same second molecular recognition elements (or different or the same second binding units or different or the same background binding partners).

[0100] This method is a sequential screening technique. First, a first molecular recognition element of a first type is immobilized at a first binding site. Next, the behavior of the biological fluid sample with respect to this first molecular recognition element of the first type is determined and the signal is measured. Subsequently, the immobilized first molecular recognition element of the first type is detached from the first binding site, for example, by cleavage. Then, the sequence is repeated with a different first molecular recognition element, which is again immobilized at the first binding site, and the behavior of the biological fluid sample with respect to this different first molecular recognition element is determined and the signal is measured.

[0101] In step b.vi, the multiple second molecular recognition elements or second binding units or background binding partners do not need to be separated in some embodiments. This may be advantageous, for example, when the same second molecular recognition elements or second binding units or background binding partners are used in each iteration of step b. Next, step b.ii. may be performed only when step b. is performed for the first time.

[0102] It is understood that the sensing device may be, for example, a sensing device described in any of the embodiments herein with respect to a first aspect.

[0103] As described above, step b. can be repeated (step c.) using different first molecular recognition elements or first binding units. In particular, such different first molecular recognition elements or first binding units may have different affinities or different binding behaviors to the proteinosis-related analytes. The second molecular recognition element or second binding unit or background binding partner may be the same or different in each iteration of step b.

[0104] For example, the first molecular recognition element or first binding unit used when step b. is performed for the first time may be configured to selectively bind to a first epitope of the proteinosis-related analyte (and optionally not to other epitopes), while the first molecular recognition element or first binding unit used when step b. is performed a second time (or any additional time) may be configured to selectively bind to a different epitope of the proteinosis-related analyte (and optionally not to other epitopes).

[0105] Furthermore, the first molecular recognition element or first binding unit used when step b. is performed for the first time may be configured to selectively bind to the monomeric form of the proteinosis-related analyte (and optionally not to the aggregated form), while the first molecular recognition element or first binding unit used when step b. is performed for the second time (or any additional time) may be configured to selectively bind to the aggregated form of the proteinosis-related analyte (and optionally not to the monomeric form).

[0106] In some embodiments, the first molecular recognition element or first binding unit used when step b. is performed for the first time may exhibit different binding behavior, such as different selectivity or different affinity, to the proteinosis-related analyte or a particular form thereof, compared to the first molecular recognition element or first binding unit used when step b. is performed a second time (or any additional time). In particular, different first molecular recognition elements or first binding units may be selective to a particular form of the proteinosis-related analyte, such as its monomer, its oligomer, aggregate, or a particular structure (e.g., secondary, tertiary, or quaternary protein structure).

[0107] In some embodiments, the biological fluid sample includes one or more background binding partners distinct from the analytes associated with the proteinosis. One or more background binding partners may be configured to trigger a nonspecific binding signal in step bv. One or more background binding partners may be configured to bind, for example, to a first molecular recognition element or a first binding unit and / or a second binding site. In such embodiments, for example, the second molecular recognition element of the sensing device provided in step a. may include or consist of 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 to them), using background binding partners as the second molecular recognition element ensures that the second unit cell is saturated with background binding partners from step b.ii. to bv. Thus, a stable baseline is achieved, and the influence of further background binding partners in the biological fluid sample is significantly reduced. This makes it possible to avoid or at least significantly reduce nonspecific binding events, thereby enabling monitoring of specific binding events by the first molecular recognition element or first binding unit to analytes associated with proteinosis. Background binding partners may be bound to a second unit cell by, for example, a second linker element as described herein. For example, the second unit cell may include a second binding site that forms a second linker element together with a second linker-forming element, which is bound to one or more background binding partners.

[0108] As a non-limiting example, it may be possible to use a human-derived biological fluid sample, such as CSF or blood sample, and to use a second molecular recognition element containing or consisting of human serum albumin. Human serum albumin has been identified as one of the major components in human-derived biological fluid samples responsible for nonspecific binding. Therefore, in certain embodiments, the sensing device provided in step a. comprises a plurality of first molecular recognition elements or first binding units configured to bind to an analyte associated with proteinosis and bound to a first unit cell. Furthermore, the sensing device comprises a plurality of second molecular recognition elements containing or consisting of human serum albumin and bound to a second unit cell.

[0109] Accordingly, in certain embodiments, the method may include a pre-screening step, which includes identifying background binding partners in a biological fluid sample. For example, this may include, in step d, the identification of background binding partners that bind to the first molecular recognition element and / or trigger a nonspecific binding signal. The pre-screening step may also be carried out using a sensing device as described in any embodiment of this specification, for example, a sensing device in which the second molecular recognition element consists of an oligonucleotide chain. After performing steps b.ii. to b.iv., nonspecific 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. Following this pre-screening step, step b.ii. (i.e., immobilizing the second molecular recognition element) may include incorporating the identified background binding partners into the second molecular recognition element, for example, by binding to a second binding unit (e.g., an oligonucleotide chain) bound to a second unit cell. Subsequently, steps b.iii. to b.vi. may be carried out.

[0110] Step b. may be repeated, for example, once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times, or more. However, a different type of first molecular recognition element may preferably be used with each iteration.

[0111] Step b.vi. (i.e., reactivating the sensing device) may include removing the immobilized first molecular recognition element or first binding unit from the first binding site, and optionally removing the immobilized second molecular recognition element (or second binding unit or background binding partner) from the second binding site. Removal may be performed, for example, chemically or physically. For example, a reagent may be added to cleave the immobilized first or second molecular recognition element from the first or second binding site. In an embodiment in which the first and second binding sites are oligonucleotide chains and the first and second molecular recognition elements include oligonucleotide chains complementary to the oligonucleotide chains of the first and second binding sites, the thus formed double helix (e.g., DNA double helix) may be cleaved by aqueous alkali treatment, for example, by aqueous hydroxide (e.g., NaOH). Alternatively, cleavage may be performed physically, for example, by irradiation or heating to a cleavage temperature at which the first or second molecular recognition element is cleaved from the first or second binding site.

[0112] The second molecular recognition element or second binding unit or background binding partner used in step b.ii may optionally be different from the first molecular recognition element or first binding unit so as not to selectively bind to analytes associated with proteinosis in particular.

[0113] A third aspect of this disclosure relates to a method for screening biological fluid samples for analytes related to proteinosis, the method being: a. A step of providing a sensing device, the sensing device comprising an evanescent illuminator, the evanescent illuminator configured to generate an evanescent field on a first surface of the evanescent illuminator from a beam of coherent light having a predetermined wavelength, the first surface of the evanescent illuminator comprising a plurality of sensing spots, each sensing spot comprising a first recognition grid having a plurality of first unit cells and a second recognition grid having a plurality of second unit cells, the first and second recognition grids interlocking with each other such that the plurality of first unit cells are alternately arranged with the second unit cells, a plurality of first molecular recognition elements configured to bind analytes associated with proteinosis being coupled to the first unit cells, a plurality of second molecular recognition elements different from the first molecular recognition elements being coupled to the second unit cells, each sensing spot being unique in that all sensing spots contain different first molecular recognition elements and optionally different second molecular recognition elements compared to the remaining sensing spots, each sensing spot is i. At least a portion of the coherent light of the evanescent field is scattered in a plurality of first unit cells to generate a first constructive interference beam having a first phase in the detector of the sensing device, ii. At least a portion of the coherent light of the evanescent field is scattered in a plurality of second unit cells to generate a second constructive interference beam in the detector having a second phase opposite to the first phase, iii. A sensing device comprising the steps of providing a sensing device in which a first constructive interference beam generated interferes with a second constructive interference beam in a detector to generate a mass difference-dependent and optionally time-dependent signal in the detector, wherein the signal is configured to be proportional to the square of the mass difference generated by molecular interactions between a plurality of first molecular recognition elements and a plurality of second molecular recognition elements. b. A process of providing a biological fluid sample to multiple sensing spots of an evanescent illuminator. c. A step of generating a beam of coherent light having a predetermined wavelength at a predefined beam generation position. d. A step of measuring a signal for each sensing spot in a detector, wherein each signal is proportional to the square of the difference in mass generated by molecular interactions between a plurality of first molecular recognition elements and a plurality of second molecular recognition elements.

[0114] It is understood that the sensing device may be, for example, a sensing device described in any of the embodiments herein with respect to a first aspect.

[0115] In some embodiments, the sensing device provided in step a. includes 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 a unique first molecular recognition element. Thus, the behavior of a biological fluid sample against various unique first molecular recognition elements can be measured in a single experiment, thereby enabling multiplexing.

[0116] In some embodiments, the sensing spots may be unique in that the first molecular recognition element of the corresponding sensing spot exhibits different binding behaviors, such as different selectivity or different affinity, to the proteinosis-related analyte or a particular form thereof. In particular, different first molecular recognition elements may be selective to a particular form of the proteinosis-related analyte, such as its monomer, oligomer, aggregate, or a specific structure (e.g., a secondary, tertiary, or quaternary protein structure).

[0117] In some embodiments, multiple second molecular recognition elements may be the same for each sensing spot.

[0118] In some embodiments, a first molecular recognition element bound to a first unit cell of at least one sensing spot is configured to selectively bind to a first epitope of the proteinosis-related analyte (and optionally not to other epitopes). Furthermore, a first molecular recognition element bound to a first unit cell of at least another, and therefore different, sensing spot may be configured to selectively bind to a different epitope of the proteinosis-related analyte (and optionally not to other epitopes).

[0119] In some embodiments, a first molecular recognition element bound to a first unit cell of at least one sensing spot is configured to selectively bind to the aggregated form of the proteinosis-related analyte (and optionally not to the monomeric form). Furthermore, a first molecular recognition element bound to a first unit cell of at least another sensing spot is configured to selectively bind to the monomeric form of the proteinosis-related analyte (and optionally not to the aggregated form).

[0120] In some embodiments, the biological fluid sample includes one or more background binding partners distinct from the analytes associated with the proteinosis. One or more background binding partners may be configured to trigger a nonspecific binding signal in step d. One or more background binding partners may be configured to bind, for example, to a first molecular recognition element and / or a second binding site. In such embodiments, for example, one, multiple, or each of the second molecular recognition elements among the sensing spots of the sensing device provided in step a. may include or consist of 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 to them), using background binding partners as second molecular recognition elements allows the second unit cell to be saturated with background binding partners before steps b. through d. Thus, a stable baseline is achieved, and the influence of further background binding partners in the biological fluid sample is significantly reduced. This makes it possible to avoid or at least significantly reduce nonspecific binding events, thereby enabling monitoring of specific binding events by the first molecular recognition element to analytes associated with proteinosis. Background binding partners may be bound to one, more, or each of the second unit cells of the sensing spots by, for example, the second linker element described herein. For example, the second unit cell may include a second binding site that forms a second linker element together with a second linker-forming element, with the second linker element bound to one or more background binding partners.

[0121] As a non-limiting example, it may be possible to use a human-derived biological fluid sample, such as CSF or blood sample, and to use a second molecular recognition element containing or consisting of human serum albumin. Human serum albumin has been identified as one of the major components in human-derived biological fluid samples responsible for nonspecific binding. Therefore, in certain embodiments, the sensing spot of the sensing device provided in step a. is configured to bind to an analyte associated with proteinosis and includes a plurality of first molecular recognition elements bound to a first unit cell. Furthermore, the sensing spot of the sensing device may include a plurality of second molecular recognition elements containing or consisting of human serum albumin and bound to a corresponding second unit cell.

[0122] Accordingly, in certain embodiments, the method may include a pre-screening step, which includes identifying background binding partners in a biological fluid sample. For example, this may include, in step d, the identification of background binding partners that bind to the first molecular recognition element and / or trigger a nonspecific binding signal. The pre-screening step may also be carried out using a sensing device as described in any embodiment of this specification, for example, a sensing device in which the second molecular recognition element consists of an oligonucleotide chain. After performing steps b. to d., nonspecific 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 include incorporating the identified background binding partners into the second molecular recognition element, for example, by attaching them to an oligonucleotide chain. Steps b. to d. may then be carried out.

[0123] The inventions described herein should be better understood from the detailed description and accompanying drawings provided below, and the accompanying drawings should not be considered to be limited to the inventions described in the accompanying claims. The drawings are as follows: [Brief explanation of the drawing]

[0124] [Figure 1] A schematic diagram of a sensing device according to one embodiment of the present invention. [Figure 2] A schematic detail diagram of a sensing spot according to one embodiment of the present invention. [Figure 3a] A schematic detail diagram of a cross-section of a sensing spot used in the method according to the present invention for screening analytes associated with aggregate-forming protein disorders. [Figure 3b] A schematic detail diagram of a cross-section of a sensing spot used in the method according to the present invention for screening analytes associated with proteinosis that exist as monomers. [Figure 4] A schematic detail diagram of a cross-section of a sensing spot used in the method according to the present invention after washing and with an additional mass labeling binder. [Figure 5] A schematic detail diagram of a cross-section of a sensing spot used in a method according to the present invention, in which a processing step including the application of detergent is carried out after a predetermined time interval. [Figure 6] A schematic detail diagram of a cross-section of a sensing spot used in the method according to the present invention in which a seed amplification assay is performed. [Figure 7] A schematic diagram of a method according to one embodiment of the present invention, in which the measured signal forms an immune signature. [Figure 8] A schematic diagram of some of the sensing devices in which continuous competitive measurement is performed. [Figure 9] A schematic diagram of some of the sensing devices in which parallel measurements are performed. [Figure 10] A schematic detail of a cross-section of a sensing spot used in the method according to the present invention, in which a marker protein expressed on the surface of a mitochondria is exposed and used to bind to a first molecular recognition element. [Figure 11] A schematic detail diagram of a cross-section of a sensing spot used in the method according to the present invention, in which a marker protein expressed on the surface of a lysosome is exposed and used to bind to a first molecular recognition element. [Figure 12]A schematic detail of a cross-section of a sensing spot used in the method according to the present invention, in which protein co-aggregates are used as analytes associated with proteinosis. [Figure 13] Matrix display of different multiplex screenings of different samples (analytes and blanks related to different forms of proteinosis in PBST (phosphate-buffered saline containing detergent [polysorbate 20])). [Figure 14] Matrix representation of different multiplex screenings of different samples (analytes and blanks related to different forms of proteinosis in cerebrospinal fluid (CSF)). [Figure 15] Matrix representation of different multiplex screenings of different samples (analytes and blanks related to different forms of proteinosis in human serum (HS)). [Figure 16] A schematic diagram of immobilizing the first and second molecular recognition elements on the first and second unit cells, as may be done in certain embodiments of this disclosure. [Modes for carrying out the invention]

[0125] Exemplary Embodiments Figure 1 shows an embodiment of a sensing device 1 that can be used in several embodiments of the method according to the present invention. Sensing device 1 comprises an evanescent illuminator 3 configured to generate an evanescent field on a first surface of the evanescent illuminator from a beam of coherent light having a predetermined wavelength. The evanescent illuminator 3 may be placed on a carrier 2, for example, a transparent carrier, in this embodiment or any other embodiment described herein. Each evanescent illuminator 3 has a first surface (exposed to the observer) that includes a sensing spot 5. In this embodiment, each evanescent illuminator, each having a first surface, includes only one sensing spot, but it is quite possible that each evanescent illuminator, each having a first surface, includes multiple such sensing spots. This is shown, for example, in Figure 8 or Figure 9. Sensing device 1 further comprises a light source 6 and a grid 4. When the light source 6 generates a beam of coherent light having a predetermined wavelength, the grid 4 activates an induction mode of a waveguide that guides the light toward the sensing spot 5, as indicated by the arrows.

[0126] Figure 2 shows a detailed view of the sensing spot 5. As can be seen from the figure, the sensing spot 5 includes, or consists of, a first recognition grid having a plurality of first unit cells 8 and a second recognition grid having a plurality of second unit cells 9. The first and second recognition grids interlock with each other such that the plurality of first unit cells 8 are arranged alternately with the second unit cells 9. In this embodiment, all the first unit cells 8, which are in the form of curved elongated lines or bars, together form the first recognition grid. Conversely, in this embodiment, all the second unit cells, which are in the form of curved elongated lines or bars, together form the second recognition grid of the sensing spot 5. A plurality of first molecular recognition elements (not shown, see, e.g., Figure 3a) configured to bind to analytes associated with proteinosis are bound to the first unit cells 8. In addition, a plurality of second molecular recognition elements (not shown, see, e.g., Figure 3a) distinct from the first molecular recognition elements are bound to the second unit cells 9.

[0127] Referring to both Figures 1 and 2, the 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 in a plurality of first unit cells 8 to generate a first constructive interference beam having a first phase in the detector 7 of the sensing device. In addition, the 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 in a plurality of second unit cells 9 to generate a second constructive interference beam in the detector 7. The second constructive interference beam has a second phase opposite to the first phase of the first constructive interference beam. Furthermore, the sensing spot 5 is configured such that the generated first constructive interference beam interferes with the second constructive interference beam in the detector to generate a mass difference-dependent and optionally time-dependent signal in the detector 7. This signal is proportional to the square of the mass difference generated by the molecular interactions between the plurality of first molecular recognition elements and the plurality of second molecular recognition elements.

[0128] Figures 3a and 3b show a comparison of methods performed on an analyte associated with proteinosis forming aggregates (Figure 3a) and an analyte that is a monomer (Figure 3b). Both figures show detailed cross-sectional views of the first unit cell 8 and the second unit cell 9 of the sensing spot (e.g., sensing spot 5 shown in Figures 1 and 2). As can be seen from the figures, multiple first molecular recognition elements 10 are bound to the first unit cell 8, and multiple different second molecular recognition elements 11 are bound to the second unit cell 9. The proteinosis analyte 13 forms aggregates that expose several binding sites indicated by a, b, c, d, and e. Generally, as used herein, different letters refer to different binding sites. That is, in this example, the proteinosis analyte 13 has four different types of binding sites a, b, c, d, and e. As can be seen from the figures, the first molecular recognition elements include binding site A, while the second molecular recognition elements lack such binding site A. Generally, as used herein, binding sites indicated by uppercase letters such as binding site "A" generally bind to binding sites indicated by the same lowercase letter such as binding site "a", but preferably not to binding to other lowercase binding sites. In Figure 3a, after providing a biological fluid sample to sense the sensing spot, and thus its first and second unit cells, it can be seen that the proteinosis-related analyte 13 binds to binding site A of the first molecular recognition element at its binding site a. The following graph shows the signals measured by the detector. The obtained signals are inherently self-referential because the unbound background material present in the biological fluid sample does not interact with the first or second molecular recognition element, or because they interact with essentially equal probability, and there is no need to wash away the background material to obtain measured and reliable signals. It can be seen that the measurement of the signal obtained from the sensing spot begins during time interval 1. Binding of the proteinosis-related analyte 13 occurs during time interval 2, which results in a mass increase clearly visible in the obtained time-dependent signal.Since the first molecular recognition elements 10 can be selected to be highly selective binders for the binding site a of the analyte 13, such measurements can already provide detailed information about the nature or clinical picture of proteinopathy. Furthermore, since the system is essentially dependent only on the mass of the binding partner with which the molecular recognition elements interact, the complexity and heterogeneity of the aggregates formed by the analyte 13 are irrelevant. Figure 3b shows the signal obtained when only monomers are detected. Since the measured signal is square-dependent on the mass difference interacting with the molecular recognition elements, aggregates formed by analytes associated with proteinopathy have a significantly stronger signal.

[0129] Similar to Figure 3a, Figure 4 also shows the screening of analytes associated with proteinosis that form aggregates. Here again, as can be seen from the measured signals, the measurement begins during time interval 1. During time interval 2, a biological fluid sample containing aggregate analytes 13, particularly those associated with proteinosis, is added. Subsequently, during time interval 5, a mass-labeled binder 14 is added. For example, such a mass-labeled binder may contain gold nanoparticles as a mass label.

[0130] Furthermore, the mass-labeled binder may include a predefined binding site that can be specifically configured to bind to an analyte associated with a proteinosis in this embodiment or any other embodiment described herein. Generally, the predefined binding site may be configured not to bind to, for example, the first and / or second molecular recognition element. As seen in Figure 4, upon addition of the mass-labeled binder, the signal increases at time intervals of 5, as the binder binds to the analyte 13, which is already bound to the first molecular recognition element 10. Such embodiments are advantageous because they can further deconvolute and thus help characterize the proteinosis that the patient suffers from as a source of biological fluid samples to be screened. For example, if it is known that an analyte exposes binding site b in a particular type of disease, the mass increase can indicate the presence of such binding site b and thus help to rule out other proteinosis. It is understood that the mass label has a defined known mass.

[0131] Figure 5 shows a method according to one embodiment of the present invention in which a processing step is carried out. In this embodiment, the processing step includes the application of a detergent. As seen at the top, the aggregated analyte is bound to the first molecular recognition element. The corresponding signal increase is observed at time intervals 2–4. Then, at time interval 5, the detergent is applied, resulting in the removal of some or all of the analyte (see the middle portion). Due to the loss of mass interacting with the first molecular recognition element, the signal decreases. The measured signal may generally be further included in the immunosignature of proteinosis analytes and / or biological fluid samples. For example, if a particular analyte is known to be sensitive to different treatments such as detergent, pH, etc., such processing steps can be used to provide further information about the analyte associated with proteinosis.

[0132] Figure 6 shows one embodiment of the method according to the present invention, in which a seed amplification assay is performed after step b. As seen in the upper part of Figure 6, the biological fluid sample contains an analyte 13 related to proteinosis. After providing the biological fluid sample, a protein sample containing monomeric protein 15 is added to the corresponding sensing spots, which include a first unit cell 8 and a second unit cell 9. Since the analyte related to proteinosis exhibits, for example, prion-like behavior, it leads to misfolding and aggregation of the added monomeric protein. Thus, as seen in the central part of Figure 6, aggregation increases and further aggregates are formed. Seed amplification can be observed in real time because the mass increase due to the progression of aggregation can be measured directly. This is shown in the lower part of Figure 6, which shows the measured signal over time. As can be seen from the figure, the measured signal increases continuously over time interval 2, which directly indicates that seed amplification is occurring.

[0133] Figure 7 shows one embodiment of the method according to the present invention, wherein a sensing device (not shown) comprises a plurality of sensing spots 5'a, 5''a, 5'b, 5''b, 5'c, 5''c, 5'd, and 5''d. Each sensing spot includes a first unit cell to which a first molecular recognition element is coupled, and a second unit cell to which a second molecular recognition element is coupled. Preferably, in this embodiment or any other embodiment described herein, at least some, or even all, sensing spots may include a unique first molecular recognition element and / or a unique second molecular recognition element. The term “unique” in this context means that the first (or second) molecular recognition element of a particular sensing spot is different from the first (or second) molecular recognition elements of other sensing spots of the sensing device, although typically the first molecular recognition elements within each sensing spot are typically the same. As can be seen from the graph showing the time-dependent measurement signals, when a biological fluid sample S is supplied to the sensing spots, one signal is obtained for each sensing spot. All of these signals can represent parameters in a high-dimensional vector space, as shown by matrices a11-a43. This matrix of parameters may, in this embodiment or any other embodiment described herein, be an immunosignature of a biological fluid sample and / or analyte associated with a proteinosis screened by the method according to the present invention. This immunosignature can then be compared with a database stored in the memory unit of a computer, for example, by a control unit which is part of the computer. If this database contains data relating to the clinical status of proteinosis in patient samples, self-patient samples (i.e., previously screened samples from patients from whom the biological fluid sample currently being screened was obtained) and / or reference samples, it is possible to characterize the nature and / or clinical status of the proteinosis associated with the analyte. It is also possible to predict the progression of the proteinosis.In addition, other parameters, such as signals obtained from the seed amplification assay described herein or signals obtained from the addition of the mass labeling binder described herein, may also be included in the immunosignature.

[0134] Figures 8 a) and b) illustrate embodiments of the method according to the present invention, in which multiple sensing spots of a sensing device compete for an analyte associated with proteinosis. Both a) and b) show a continuous competitive measurement, and arrows indicate the direction of flow in which the biological fluid sample is provided. That is, in a), the biological fluid sample is provided so as to first contact two sensing spots indicated by "A", then two sensing spots indicated by "B", and then two sensing spots indicated by "C". For example, if the first two sensing spots include a first molecular recognition element having a binding site A, the two middle sensing spots include a different first molecular recognition element having a binding site B, and the last sensing spot includes yet another different first molecular recognition element having a binding site C, and the behavior of the analyte associated with proteinosis with respect to these different first molecular recognition elements can provide valuable information about the analyte and proteinosis. b) shows a continuous competitive measurement using the same sensing device as shown in a), but with the flow direction reversed. By performing such a series of competitive measurements, namely a first competitive measurement with a first flow direction and a second competitive measurement with the opposite flow direction, additional information is provided that enables further characterization of the analyte and proteinosis.

[0135] Figure 9 shows parallel measurement. As can be seen from the figure, the sensing device includes three groups of sensing spots, each containing two sensing spots. The first group includes a sensing spot having a first molecular recognition element having a binding site A. The second group includes a sensing spot having a first molecular recognition element having a binding site B. The third group includes a sensing spot having a first molecular recognition element having a binding site C. The sensing device includes wall structures 12 that separate the groups of sensing spots from each other. In this embodiment or any other embodiment, the wall structures can form channels such as microfluidic channels that are part of a fluid system. As can be seen from the figure, a biological fluid sample can be supplied 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 to the other.

[0136] Figure 10 shows another embodiment of the method according to the present invention. As can be seen from the figure, the sensing spot of the sensing device used comprises a first molecular recognition element 10 bound to a first unit cell 8, each containing two different binding sites A and B. This is beneficial as it allows for more precise characterization of the analyte 13 expressing surface complementary binding sites / epitopes a and b. Certain proteinosis is known in which cellular residues, such as mitochondria or parts thereof, are recruited to form aggregates. Furthermore, it is known that such mitochondria or parts thereof may contain certain marker proteins, such as protein b, e.g., VDAC. Therefore, by utilizing this knowledge by providing the first molecular recognition element with binding site A that binds to a specific binding site / epitope a on a marker and binding site B that specifically binds to mitochondrial protein b, the properties / composition of the analyte can be further characterized.

[0137] Figure 11 illustrates a similar principle to Figure 10, but in this case, the lysosome is part of the analyte that specifically exposes surface proteins c, such as LAMP1. Therefore, if the first molecular recognition element includes a binding site C configured to bind to protein c, further details regarding the composition of the analyte and the properties of the associated proteinosis can be provided.

[0138] Figure 12 shows another modification similar to the examples shown in Figures 10 and 11. In this case, protein coagulants are screened as analytes 13 associated with proteinopathy. In this modification, for example, it can be assumed that the patient has a specific proteinopathy involving protein aggregates with a specific post-translational modification d. By using a sensing device having a sensing spot having a first recognition element containing two binding sites A and D, the latter being specific to post-translational modification d, it is possible to generate information about the nature of the proteinopathy and the analytes associated with it, respectively.

[0139] Figure 13 shows a matrix of different multiplex screenings of different samples (analytes and blanks related to different forms of proteinosis in PBST (phosphate-buffered saline containing detergent [Polysorbate 20])). Sample S is as follows: S0 is the blank sample (i.e., PBST). S1 is wild-type monomer α-syn in PBST. S2 is wild-type aggregated α-syn in PBST. S3 is phosphorylated (pSer129) α-syn in PBST. S4 is aggregated phosphorylated (pSer129) α-syn in PBST. S4 is α-syn fibrils in PBST. Abeta is beta-amyloid in PBST. A series of multiplex screening experiments were performed. All measurements show the mass-dependent binding signals of the samples in response to different first and second molecular recognition elements (x-axis: time (s), y-axis: coherent mass density (pg / mm³)). 2The notation Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 indicates that each row represents a series of experiments in which specific antibodies Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 were used as part of a first molecular recognition element configured to bind to an analyte associated with proteinosis and, for example, its epitope. The Roman alphabet notation I-X helps to identify different probed second molecular recognition elements. The sensing device is provided according to step a. described herein. The first unit cells contained a first binding site which was an oligonucleotide. They were exposed to the indicated antibody samples Ab1-Ab6 containing complementary oligonucleotide chains (i.e., binding units) bound to the antibody, using DNA-directed immobilization as used, for example, in peptide arrays. This bound Ab1-Ab6 to the first unit cells. The second unit cells contained a second binding site which was an oligonucleotide chain of a different configuration (but different from the oligonucleotide chain of the first binding site). In rows I, II, and III, aFLAG is immobilized in the second unit cell, with a complementary oligonucleotide linked to the oligonucleotide of the second binding site. In rows IV–X, human serum albumin is immobilized in the second unit cell, with a complementary oligonucleotide linked to the oligonucleotide of the second binding site. For measurement, different provided sensing devices were exposed to biological fluid samples S0–S6 as shown (10 μM for S1–S4, and 2 μM for S5 and S6).

[0140] Figure 13 shows that monomer α-syn(S1), which has a much lower molecular weight than its aggregated form, is sufficiently recognized to varying degrees by Ab1-Ab5, with Ab5 exhibiting the lowest on-rate. This demonstrates the advantage of a method that provides highly specific immunosignatures, such as mass-weighted immunosignatures, as different behaviors for different antibodies can be easily determined in a series of several experiments.

[0141] Furthermore, the presence of aggregated α-syn (such as S2) exhibits different behavior for Ab1-Ab6. For example, responses were observed for Ab2 and Ab4, which are similar to the responses observed for monomeric forms. In contrast, Ab1 and Ab3 show an even greater response compared to monomeric forms. This indicates that aggregated and monomeric α-syn can be identified using Ab1 and Ab3. Ab1 and Ab3 used are C-terminal specific antibodies, while Ab2 and Ab4 are N-terminal specific antibodies. While we do not wish to be constrained by theory, it can be assumed that the C-terminal epitope may be accessible in the formed aggregates, while the N-terminal epitope may be inaccessible. It should be noted that we cannot provide monomer-free analytes, and the signals are the monomer mass-weighted responses containing aggregates on top of a monomer background. From Western blotting, antibodies Ab5 and Ab6 were thought not to bind to the aggregated form (S2), but surprisingly, they showed unexpected off-target activity. Such activity cannot be easily determined by prior art methods.

[0142] Regarding phosphorylation series S3 and S4, Ab5 is designed to be specific to this target, and therefore Ab5 exhibits good high affinity for S2 (see Ab5's response to S1). Ab1, Ab2, Ab3, and Ab4 behave similarly to phosphorylated α-syn compared to their wild-type monomer forms. No response is observed for Ab6 with respect to S3. For aggregated phosphorylated α-syn, similar responses are observed for Ab2 and Ab4 to the monomer (S2), both of which are N-terminal specific and may be shielded or otherwise inaccessible in the aggregated form, while increased responses are observed for Ab1 and Ab3. The latter two are both C-terminal specific, and therefore the corresponding epitopes may be accessible even in the aggregated form. Ab5 shows an increased response to the aggregated form (S4), and therefore the corresponding epitope appears to be available even in the aggregated form (higher molecular weight of the aggregated species increases the mass detected by this recognition). Surprisingly, Ab6, which does not respond to monomeric form S3, shows a significant response to aggregated form S4, and therefore, aggregates can be identified from the monomer perspective, and thus with high selectivity.

[0143] Figure 14 shows a matrix of different multiplex screenings of different samples (analytes and blanks related to different forms of proteinosis in cerebrospinal fluid (CSF)). Sample S is as follows: S0 is the blank sample (i.e., CSF). S1 is wild-type monomer α-syn in CSF. S2 is wild-type aggregated α-syn in CSF. S3 is phosphorylated (pSer129) α-syn in CSF. S4 is aggregated phosphorylated (pSer129) α-syn in CSF. S4 is α-syn fibrils in CSF. Abeta is beta-amyloid in CSF. A series of screening experiments were performed. All measurements show the measured signals of the samples in response to different first and second molecular recognition elements (x axis: time (s), y axis: coherent mass density (pg / mm³) 2The diagram shows the following. As indicated by the notations 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 a first molecular recognition element configured to bind to analytes associated with proteinosis. Ab1-Ab6 are similar to those in Figure 13. The Roman alphabet notations I-X help to identify different second molecular recognition elements. The sensing devices are provided according to step a. described herein. The first unit cells contained a first binding site which was an oligonucleotide. They were exposed to the indicated antibody samples Ab1-Ab6 which contained complementary oligonucleotide chains (i.e., binding units) bound to the antibodies. This resulted in Ab1-Ab6 binding to the first unit cells. The second unit cells contained a second binding site which was similarly an oligonucleotide chain (but different from the oligonucleotide chain of the first binding site). In columns I, II, and III, no additional components are immobilized; therefore, the oligonucleotide chain of the second unit cell forms the second molecular recognition element. In columns IV–X, human serum albumin, linked to an oligonucleotide complementary to the oligonucleotide of the second binding site, is immobilized on the second unit cell. For measurement, different provided sensing devices were exposed to the biological fluid samples S0–S6 as shown (10 μM for S1–S4, and 2 μM for S5 and S6).

[0144] The results shown for CSF are consistent with those obtained for PBST, generally indicating that matrix amplification in CSF is not observed compared to PBST, which supports the distinction between monomeric and aggregated forms of the same protein in the mixture and in the mixture with CSF. Entries in columns III and VI provide important details. Both columns show the behavior of S2 (wild-type aggregated α-syn) for AB1-Ab6. For column III, no additional components are immobilized, and therefore the oligonucleotide chain of the second unit cell forms the second molecular recognition element. In contrast, for column VI, human serum albumin (HSA), identified as one of the nonspecific binding sources, is immobilized in the second unit cell and is therefore part of the second molecular recognition element. For example, the signals in column II for Ab1, Ab2, Ab3, and Ab5 are relatively complex. For Ab3, a much stronger signal is measured in column III compared to column VI. This is presumably due to nonspecific binding events of components in the CSF that overlap with the specific binding signal being measured. When the second unit cell is bound and immobilized with the HAS as shown in column VI, these nonspecific responses are eliminated and a clear signal is obtained (see columns III and VI, Ab3).

[0145] Figure 15 shows a matrix of different multiplex screenings of different samples (analytes and blanks related to different forms of proteinopathy in human serum (HS)). Sample S is as follows: S0 is the blank sample (i.e., HS). S1 is wild-type monomeric α-syn in HS. S2 is wild-type aggregated α-syn in HS. S3 is phosphorylated (pSer129) α-syn in HS. S4 is aggregated phosphorylated (pSer129) α-syn in HS. S4 is fibril of α-syn in HS. Abeta is oligomeric beta-amyloid in HS. A series of screening experiments were performed. All measurements show the measured signals of the samples in response to different first and second molecular recognition elements (x axis: time (s), y axis: coherent mass density (pg / mm³) 2The diagram shows the following. As indicated by the notations 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 a first molecular recognition element configured to bind to analytes associated with proteinosis. Ab1-Ab6 are similar to those in Figures 13 and 14. The Roman alphabet notations I-X help to identify different second molecular recognition elements. The sensing devices are provided according to step a. described herein. The first unit cells contained a first binding site which was an oligonucleotide. They were exposed to the indicated antibody samples Ab1-Ab6 which contained complementary oligonucleotide chains (i.e., binding units) bound to the antibodies. This resulted in Ab1-Ab6 binding to the first unit cells. The second unit cells contained a second binding site which was similarly an oligonucleotide chain (but different from the oligonucleotide chain of the first binding site). In columns I, II, and III, no additional components are immobilized; therefore, the oligonucleotide chain of the second unit cell forms the second molecular recognition element. In columns IV–X, human serum albumin, linked to an oligonucleotide complementary to the oligonucleotide of the second binding site, is immobilized on the second unit cell. For measurement, different provided sensing devices were exposed to the biological fluid samples S0–S6 as shown (10 μM for S1–S4, and 2 μM for S5 and S6).

[0146] Figure 15 demonstrates that human serum (HS) exhibits a very strong matrix-induced response (i.e., it shows a strong signal triggered by nonspecific binding of components in HS). In columns I–III, where no additional components are immobilized and therefore the oligonucleotide chain of the second unit cell forms the second molecular recognition element, this nonspecific binding signal is very complex and overlaps with any specific binding signal of antibodies against analytes associated with proteinosis. This nonspecific binding is reduced in columns IV–X, where human serum albumin (HSA), identified as one of the nonspecific binding sources, is immobilized in the second unit cell and therefore part of the second molecular recognition element. Furthermore, Ab3 and Ab6 show a distinct response in the presence of aggregated α-syn (S2, column VI).

[0147] Figure 16 shows an immobilization process that can be used in several embodiments of the present invention. The sensing device may comprise a first unit cell 8 and a second unit cell 9. Before immobilization, a first binding site 20 (e.g., an oligonucleotide chain) is bound to the first unit cell 8, and an orthogonal second binding site 21 (e.g., a different oligonucleotide chain) is bound to the second unit cell 9. A first binder unit 18 (e.g., an antibody) is then provided, bound to a linker-forming element 22 (e.g., an oligonucleotide complementary to the binding site 20), and the linker-forming element 22 and the binding site 20 together form a first linker element 16. The first linker element 16 then forms a first molecular recognition element 10 together with the binder unit 18. Similarly, a second molecular recognition element 11 may be formed from a second linker element 17 and a second binder unit 19 by a selective reaction between the second binding site 21 and the second linker-forming element 23. [Explanation of symbols]

[0148] 1. Sensing device 2 carriers 3 Evanescent lighting 4 lattice 5 detection spots 5'a, 5''a detection spots 5'b, 5''b detection spot 5'c, 5''c sensing spot 5'd, 5''d detection spot 6 light source 7 Detectors 8. First unit cell 9. Second unit cell 10. First molecular recognition element 11. Second molecular recognition element 12 Wall structure 13. Analytes of protein disorders 14 Mass-labeled binder 15 Monomeric proteins 16. First linker element 17. Second linker element 18. First Binder Unit 19. Second Binder Unit 20 First binding site 21 Second binding site 22 First linker-forming element 23 Second linker-forming element

Claims

1. A method for screening biological fluid samples for analytes related to protein disorders, a. A step of providing a sensing device (1), wherein the sensing device (1) comprises an evanescent illuminator (3), the evanescent illuminator (3) is configured to generate an evanescent field on a first surface of the evanescent illuminator (3) from a beam of coherent light having a predetermined wavelength, the first surface of the evanescent illuminator (3) comprises one or more sensing spots (5), each sensing spot (5) comprises a first recognition grid having a plurality of first unit cells (8) and a second recognition grid having a plurality of second unit cells (9). The recognition grid comprises a first and a second recognition grid in which the plurality of first unit cells (8) interlock with each other such that the plurality of first unit cells (8) are arranged alternately with the second unit cells (9), a plurality of first molecular recognition elements (10) configured to bind proteinosis-related analytes (13) are bonded to the first unit cells (8), a plurality of second molecular recognition elements (11) different from the first molecular recognition elements (10) are bonded to the second unit cells (9), and the one or more sensing spots (5) each i. At least a portion of the coherent light of the evanescent field is scattered in the plurality of first unit cells (8) to generate a first constructive interference beam having a first phase in the detector (7) of the sensing device (1), ii. At least a portion of the coherent light of the evanescent field is scattered in the plurality of second unit cells (9) to generate a second constructive interference beam having a second phase opposite to the first phase in the detector (7), iii. A step of providing a sensing device (1) wherein the generated first constructive interference beam interferes with the second constructive interference beam in the detector to generate a mass difference-dependent and optionally time-dependent signal in the detector (7), and the signal is configured to be proportional to the square of the mass difference generated by the molecular interaction between the plurality of first molecular recognition elements (10) and the plurality of second molecular recognition elements (11), b. A step of providing a biological fluid sample to one or more sensing spots (5) of the evanescent illuminator (3), c. A step of generating a beam of coherent light having the predetermined wavelength at a predefined beam generation position. d. A method comprising the step of measuring the signal for each of the sensing spots in the detector (7), wherein each signal is proportional to the square of the mass difference generated by the molecular interaction between 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) includes a plurality of sensing spots (5), and one of the plurality of sensing spots (5), in particular the first molecular recognition element (10) of each sensing spot (5), is different from at least some or all of the first molecular recognition elements (10) of the other sensing spots (5).

3. The method according to claim 2, wherein the first molecular recognition elements (10) of the sensing spots (5) differ from at least some or all of the first molecular recognition elements (10) of the other sensing spots (5) in that they are chemically distinct from each other and / or configured to bind to different analytes (13) associated with proteinosis and / or configured to bind to different epitopes of the analytes (13) associated with proteinosis and / or configured to bind to the same analytes (13) associated with proteinosis or to epitopes of the analytes (13) associated with proteinosis with different binding affinities.

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

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

6. The method according to any one of claims 1 to 5, further comprising determining parameters of the analyte (13) related to proteinopathy from the mass difference-dependent and optionally time-dependent signals, and / or determining parameters of aggregates of the analyte (13) related to proteinopathy or co-aggregates with the analyte (13) related to proteinopathy from the mass difference-dependent and optionally time-dependent signals.

7. The method according to claim 6, further comprising the step of determining the presence of the aggregates or coagulates from the determined parameters, preferably the presence of the aggregates or coagulates is determined when the determined parameters exceed a predetermined threshold within a predetermined incubation time.

8. The method according to claim 7, wherein determining the presence of the aggregates or coagulates includes comparing the determined parameters with reference parameters, in particular, reference parameters obtained from different patient populations or healthy subjects.

9. The method according to any one of claims 1 to 8, wherein the analyte (13) related to proteinopathy comprises β-amyloid, tau, α-synuclein, prion protein, fusion sarcoma, wild-type or mutant polyQ huntingtin, ubiquitin, ataxin-3, optinurin, TAR DNA-binding protein 43, neurofibrillary light chain (NfL), soluble or detachable trigger receptor 2 (sTREM2) expressed on myeloid cells, chitinase-3-like protein 1, glial fibrillary acidic protein, and one or more truncated or other post-translational modified forms thereof.

10. The method according to any one of claims 1 to 9, wherein step d is performed over a predetermined measurement time, each signal is measured as a function of the measurement time, preferably during the predetermined measurement time, changes in each signal are measured, the changes being brought about by aggregation, in particular by aggregation of additional materials in the biological fluid sample, such as any residual biological material, e.g., cellular components, mitochondria, cell membranes, nucleic acids, proteins and their fragments.

11. The method according to any one of claims 1 to 10, wherein step d includes a single-point measurement at a specific point in time, particularly an endpoint measurement.

12. The method according to any one of claims 1 to 11, wherein a rinsing step is performed after step b, particularly before or during step d, and / or a treatment step is performed after step b, the treatment step preferably includes the application of a treatment agent such as a detergent, an ionic agent, a solvent, an acid, a base, a flocculation stabilizer and / or a flocculation destabilizer.

13. The method according to any one of claims 1 to 12, wherein, following step b, incubation is carried out with one or more sensing spots and a labeled, in particular mass-labeled or fluorescently labeled binder, preferably the labeled binder is configured to bind to the analyte (13) associated with the proteinosis, in particular to bind to a lysosomal marker, mitochondrial marker, nucleotide, sugar, post-translational modification or mitochondrial DNA of the analyte associated with the proteinosis.

14. The method according to any one of claims 1 to 13, wherein, after step b, a seed amplification assay is performed by adding a binding reagent sample, particularly a protein sample, to one or more sensing spots and measuring each signal over a predetermined measurement time.

15. The method according to any one of claims 1 to 14, further comprising a secondary characterization step for characterizing the state of the analyte (13) associated with proteinosis, or aggregates or coagulates formed by the analyte (13) associated with proteinosis, wherein the secondary characterization step preferably comprises 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 provided to one or more sensing spots (5) of the evanescent illuminator (3).

17. The method according to any one of claims 1 to 16, wherein the signal measured in step d. forms an immunosignature of the biological fluid sample.

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

19. The method according to any one of claims 1 to 18, wherein the plurality of second molecular recognition elements (11) are configured not to bind to the analyte (13) associated with proteinosis.

20. Both the first molecular recognition element (10) and the second molecular recognition element (11) are configured to interact with the same background binding partner, preferably the first molecular recognition element (10) and the second molecular recognition element (11) have essentially the same affinity K for the same background binding partner. Ｄ The method according to claim 19, comprising:

21. The method according to any one of claims 1 to 20, wherein the biological fluid sample includes a background binding partner, and the second molecular recognition element of the sensing device (1) provided in step a. includes or consists 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 of identifying the background binding partners in the biological fluid prior to step a.

24. A method for screening biological fluid samples for analytes related to protein disorders, a. A step of providing a sensing device (1), wherein the sensing device (1) comprises an evanescent illuminator (3), the evanescent illuminator (3) is configured to generate an evanescent field on a first surface of the evanescent illuminator (3) from a beam of coherent light having a predetermined wavelength, the first surface of the evanescent illuminator (3) comprises one or more sensing spots (5), each sensing spot (5) comprises a first recognition grid having a plurality of first unit cells (8) and a second recognition grid having a plurality of second unit cells (9), the first and second recognition grids are interlocked with each other such that the plurality of first unit cells (8) are alternately arranged with the second unit cells (9), a plurality of first coupling portions are coupled to the first unit cells (8), a plurality of second coupling portions different from the first coupling portions are coupled to the second unit cells, and each of the one or more sensing spots (5) is i. At least a portion of the coherent light of the evanescent field is scattered in the plurality of first unit cells (8) to generate a first constructive interference beam having a first phase in the detector (7) of the sensing device (1), ii. At least a portion of the coherent light of the evanescent field is scattered in the plurality of second unit cells (9) to generate a second constructive interference beam having a second phase opposite to the first phase in the detector (7), iii. A step of providing a sensing device (1) wherein the generated first constructive interference beam interferes with the second constructive interference beam in the detector to generate a mass difference dependent and optionally time-dependent signal in the detector (7), and the signal is configured to be proportional to the square of the mass difference generated by the molecular interaction between the plurality of first binding sites and the plurality of second binding sites. b. A process for carrying out a screening cycle, i. Immobilizing a first molecular recognition element or a first binding unit to the first binding site rather than the second binding site, wherein the first molecular recognition element or the first binding unit is configured to bind to the analyte associated with proteinosis. ii. Optionally, immobilizing a second molecular recognition element, a second binding unit, or a background binding partner at the second binding site instead of the first binding site, wherein the immobilized second molecular recognition element, second binding unit, or background binding partner is different from the first molecular recognition element or first binding unit. iii. Providing a biological fluid sample to one or more sensing spots (5) of the evanescent illuminator (3), iv. To generate a beam of coherent light having the predetermined wavelength at a predefined beam generation position. v. Measuring the signal for each of the sensing spots in the detector (7), wherein each signal is proportional to the square of the mass difference generated by the molecular interaction between 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. A step of performing a screening cycle, which includes reactivating the sensing device by optionally cleaning it, by removing the immobilized first molecular recognition element or first binding unit from the first binding site, and optionally removing the immobilized second molecular recognition element or second binding unit or background binding partner from the second binding site. c. A method comprising repeating step b. once or several times using different first molecular recognition elements or first binding units, and optionally using different or the same second molecular recognition element or second binding unit or background binding partner.

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

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

27. A method for screening biological fluid samples for analytes related to protein disorders, a. A step of providing a sensing device (1), wherein the sensing device (1) comprises an evanescent illuminator (3), the evanescent illuminator (3) is configured to generate an evanescent field on a first surface of the evanescent illuminator (3) from a beam of coherent light having a predetermined wavelength, the first surface of the evanescent illuminator (3) comprises a plurality of sensing spots (5), each sensing spot (5) comprises a first recognition grid having a plurality of first unit cells (8) and a second recognition grid having a plurality of second unit cells (9), the first and second recognition grids are configured such that the plurality of first unit cells (8) Multiple first molecular recognition elements (10) are bonded to the first unit cell (8) and are interlocked with each other so as to be alternately arranged with the second unit cell (9) and configured to bind analytes (13) associated with proteinosis; multiple second molecular recognition elements (11) different from the first molecular recognition elements (10) are bonded to the second unit cell (9); each sensing spot is unique in that all sensing spots (5) contain different first molecular recognition elements and optionally different second molecular recognition elements; each sensing spot (5) is i. At least a portion of the coherent light of the evanescent field is scattered in the plurality of first unit cells (8) to generate a first constructive interference beam having a first phase in the detector (7) of the sensing device (1), ii. At least a portion of the coherent light of the evanescent field is scattered in the plurality of second unit cells (9) to generate a second constructive interference beam having a second phase opposite to the first phase in the detector (7), iii. A step of providing a sensing device (1) wherein the generated first constructive interference beam interferes with the second constructive interference beam in the detector to generate a mass difference-dependent and optionally time-dependent signal in the detector (7), and the signal is configured to be proportional to the square of the mass difference generated by the molecular interaction between the plurality of first molecular recognition elements (10) and the plurality of second molecular recognition elements (11), b. A step of providing a biological fluid sample to the plurality of sensing spots (5) of the evanescent illuminator (3), c. A step of generating a beam of coherent light having the predetermined wavelength at a predefined beam generation position. d. A method comprising the step of measuring the signal for each of the sensing spots in the detector (7), wherein each signal is proportional to the square of the mass difference generated by the molecular interaction between the plurality of first molecular recognition elements (10) and the plurality of second molecular recognition elements (11).

28. The method according to claim 27, wherein the first molecular recognition element (10) bound to the first unit cell (8) of at least one sensing spot is configured to selectively bind to a first epitope of the analyte associated with the proteinosis, and the first molecular recognition element (10) bound to the first unit cell (8) of at least another sensing spot is configured to selectively bind to a different epitope of the analyte associated with the proteinosis.

29. The method according to claim 27 or 28, wherein the first molecular recognition element (10) bound to the first unit cell (8) of at least one sensing spot is configured to selectively bind to the aggregated form of the analyte associated with proteinosis, and the first molecular recognition element (10) bound to the first unit cell (8) of at least another sensing spot is configured to selectively bind to the monomeric form of the analyte associated with proteinosis.

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