Molecular probe-functionalized element

Abstract

The present invention provides a molecular probe-functionalized element comprising a solid substrate element, a first layer comprising linker A, a second layer comprising a peptide compound, a third layer comprising linker B, and a fourth layer comprising a molecular probe, wherein the peptide compound is covalently bonded to the solid substrate element via linker A and the molecular probe is covalently bonded to the peptide compound via linker B. The present invention also provides a process for preparing a molecular probe-functionalized element and an apparatus comprising the molecular probe-functionalized element such as an optical biosensor. Further, the present invention provides a method for detecting a biomarker, comprising: a) contacting the molecular probe-functionalized element with a sample suspected of containing a target analyte; and b) detecting the biomarker based on an interaction between the molecular probe and the target analyte. The present invention also provides the use of the molecular probe-functionalized element or apparatus for one or more of a companion diagnostic test; diagnosis of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors in a patient; monitoring the therapy of a patient having Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors; and screening for drugs for the treatment of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors.

Description

Technical Field

[0001] The present invention relates to a molecular probe-functionalized element and a process for preparing a molecular probe-functionalized element. The present invention further relates to an apparatus comprising a molecular probe-functionalized element. For example, the apparatus includes, but is not limited to, a biosensor for detecting a biomarker. The present invention also relates to a method for detecting a biomarker.

Background Art

[0002] Molecular probes are widely used for biochemical, molecular biological, medical, and even non-medical applications. Molecular probes, such as antibodies, nucleic acids, biomolecule pairs and complexes, enzymes, drugs, ligands, small molecules, microorganisms, etc., can interact with a desired target analyte. The target analyte can be widely various, and examples of the target analyte include antibodies, proteins, protein complexes, drugs, toxins, disease biomarkers, cells, such as tumor cells, etc. The biochemical interaction between the molecular probe and the target analyte is converted into a signal that can be detected and / or quantified according to a selected analysis technique. For example, in a biosensor, the biochemical interaction between the molecular probe and the target analyte results in a physicochemical change, which is then converted into a measurable signal via a conversion element. The conversion element may be, for example, an optical conversion element such as those used in surface plasmon resonance (SPR) spectroscopy, attenuated total reflection infrared (ATR-IR) spectroscopy, Raman spectroscopy, UV / Vis spectroscopy, fluorescence spectroscopy, etc.

[0003] In many applications, molecular probes are immobilized on the surface of a solid support of a device such as a biosensor to create a functional surface. As a fluid mixture containing the target analyte passes over the functional surface, an interaction between the molecular probe and the target analyte is enabled, which is then converted into a measurable signal. For example, in an optical biosensor based on ATR-IR immunosensing, the molecular probe is immobilized on a reflective element such as a silicon, germanium, zinc selenide crystal, gold-coated element or fiber to create a functional surface on the reflective element. Then, based on the difference in the measured IR spectra, the interaction with the target analyte can be measured.

[0004] Problems associated with functional surfaces containing immobilized molecular probes are often that non-target components of the analyte mixture bind non-specifically to the surface. Non-specific binding can negatively affect the signal-to-noise ratio and, in the worst case, can make reliable detection of the target analyte impossible. Non-specific binding to the functional surface can be particularly pronounced when the molecular probe is intended to be used to detect larger proteins, lipids or nucleic acids or other compounds present in complex fluids such as body fluids, e.g., CSF, intestinal fluid, ocular fluid, pulmonary fluid, blood, etc.

[0005] Furthermore, it is often difficult to immobilize molecular probes, such as antibodies, proteins or protein complexes, on the surface of a solid support without losing their native functionality. A decrease in the native functionality of the molecular probe can potentially reduce its ability to bind the analyte and, consequently, this is detrimental to the sensitivity and efficiency of the selected analytical technique. Another challenge is to immobilize the molecular probe on the surface in such a way that probe desorption cannot occur, for example, by washing the probe during measurement in a flow chamber.

[0006] Thus, for example, in the detection of biomarkers and / or for use as part of a detection device such as a biosensor, molecular probes as part of a functional surface are still needed in the art. In particular, it is desirable for a molecular probe-functionalized element as a functional surface to exhibit no non-specific binding or a reduction in non-specific binding, especially for particularly complex analyte mixtures. It is further desirable that the immobilized molecular probe is functional and stable and does not tend to detach from the surface.

[0007] One object of the present invention is to provide an improved molecular probe-functionalized element. SUMMARY OF THE INVENTION

[0008] In one aspect, the present invention provides a molecular probe-functionalized element according to independent claim 1. The molecular probe-functionalized element comprises a solid substrate element, a first layer comprising a linker A, a second layer comprising a peptide compound, a third layer comprising a linker B, and a fourth layer comprising a molecular probe and the peptide compound is covalently bonded to the substrate element via the linker A, and the molecular probe is covalently bonded to the peptide compound via the linker B.

[0009] The inventors have surprisingly found that the molecular probe-functionalized element according to the present invention provides a very stable functional surface while maintaining the activity of the molecular probe. The functional surface exhibits a reduction in non-specific binding and an increased binding ability for the target analyte. In the prior art, there is often a trade-off between good blocking of the surface and the activity of the molecular probe, but the molecular probe-functionalized element provided by the present invention achieves good blocking and good activity of the molecular probe. Using the molecular probe-functionalized element of the present invention, the signal-to-noise ratio can be improved.

[0010] In particular, the present inventors have found that a more native-like functional molecular probe can be immobilized on a surface by (i) covalently blocking the surface of a substrate element with a peptide compound and then (ii) covalently attaching a molecular probe (e.g., an antibody) to the peptide compound via a linker. Furthermore, it has been found that non-specific binding from an analyte mixture (e.g., a body fluid) occurs less on a surface blocked with a peptide.

[0011] Thus, the peptide compound of the second layer can, on the one hand, form a peptide blocking layer on the surface of the substrate element to reduce non-specific binding and, on the other hand, serve as a platform for stably linking a molecular probe to the substrate element by a linker compound. Accordingly, the molecular probe-functionalized element according to the present invention is particularly useful for detecting biomarkers and / or for devices for detecting biomarkers, such as optical biosensors, e.g., biosensors based on ATR-IR immunoassay.

[0012] Furthermore, the molecular probe-functionalized element can be prepared by a relatively simple process under ambient conditions.

[0013] In one aspect, the present invention provides an apparatus, preferably a biosensor, comprising a molecular probe-functionalized element according to the present invention.

[0014] In one aspect, the present invention provides a process for preparing a molecular probe-functionalized element according to the present invention. The process comprises a) providing a solid substrate element; b) preparing a linker A precursor covalently bonded to the surface of the solid substrate element; c) reacting the linker A precursor with a peptide compound to covalently bond the peptide compound to the solid substrate element via the linker A; d) preparing a linker B precursor covalently bonded to the peptide compound; e) reacting the linker B precursor with a molecular probe to covalently attach the molecular probe to the peptide compound via the linker B; comprising.

[0015] In one aspect, the present invention provides a method for detecting a biomarker. The method comprises a) contacting a molecular probe-functionalized element according to the present invention with a sample suspected of containing a target analyte, optionally wherein the sample is a blood or CSF sample; b) detecting a biomarker based on the interaction between the molecular probe and the target analyte, optionally wherein the interaction between the molecular probe and the target analyte is detected by infrared spectroscopy, and optionally wherein the biomarker is the maximum value of the amide band of the target analyte; comprising.

[0016] In one aspect, the present invention companion diagnostic tests, diagnosis of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors in a patient, monitoring of therapy in a patient having Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors, and screening of drugs for the treatment of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors provides the use of a molecular probe-functionalized element according to the present invention or a device according to the present invention for one or more of the above.

[0017] "Peptide compound" in the context of the present invention is a compound comprising at least two amino acids, optionally at least four amino acids (which are linked by one peptide bond, optionally multiple peptide bonds). In this context, "amino acid" should be understood broadly as a compound containing a carboxylic acid group and an amino group.

[0018] Where the specification and claims define a subject matter as "comprising" certain features, this is to be construed as meaning that the subject matter includes those features but does not exclude other unspecified features. For the purposes of the present invention, the terms "essentially consisting of" and "consisting of" are considered to be embodiments as in the case of the term "comprising". Hereinafter, when a subject matter is defined as comprising at least a certain number of features, it should also be understood that this discloses, optionally, a subject matter consisting (essentially) of only those features.

[0019] Whenever the terms "including" or "having" are used, these terms are meant to be equivalent to "comprising" as defined above.

[0020] When an indefinite or definite article, e.g., "a", "an" or "the", is used in reference to a singular noun, this includes the plural form of that noun unless otherwise specified. Terms such as "can be obtained" or "is obtained" are used interchangeably. This means, for example, that the term "is obtained" does not indicate that, for example, an embodiment has to be obtained by a series of steps following the term "is obtained", unless otherwise specified in the context, and this is so even if such a restrictive understanding were always included as a preferred embodiment in the term "is obtained".

[0021] Preferred embodiments of the present invention are defined in the dependent claims.

Mode for Carrying Out the Invention

[0022] The present invention will be described in more detail below.

[0023] Molecular probe-functionalized element according to the present invention In one aspect, the present invention relates to a solid substrate element, a first layer comprising a linker A, a second layer comprising a peptide compound, a third layer comprising a linker B, and a fourth layer comprising a molecular probe to provide a molecular probe-functionalized element comprising, wherein the peptide compound is covalently bonded to the substrate element via the linker A, and the molecular probe is covalently bonded to the peptide compound via the linker B.

[0024] According to one embodiment, the molecular probe-functionalized element is obtainable or available by a process according to the present invention.

[0025] According to one embodiment, there is provided a molecular probe-functionalized element consisting of a solid substrate element, a first layer, a second layer, a third layer and a fourth layer as defined herein.

[0026] The molecular probe-functionalized element according to the present invention can be understood in that a plurality of linker A, and optionally a matrix compound form a first layer on the surface of the substrate element, a plurality of peptide compounds form a second layer on the first layer, a plurality of linker B form a third layer on the second layer, and a plurality of molecular probes form a fourth layer on the third layer. The first and third layers each containing linker A and B can be understood as a connecting layer connecting the solid substrate element to the peptide compound of the second layer and the peptide compound to the molecular probe. The second layer containing the peptide compound can have the function of a blocking layer.

[0027] The term "layer" in the context of the present invention should not be construed as limiting the first layer, the second layer, the third layer and the fourth layer to a specific dimension or a specific state of aggregation.

[0028] Solid substrate element The molecular probe-functionalized element according to the present invention includes a solid substrate element. The substrate element is a solid support covalently bonded to linker A.

[0029] The solid substrate element may be a solid support suitable for use in an apparatus for detecting a biomarker. The solid substrate element may be a solid support suitable for use in a biosensor such as an optical biosensor, an acoustic biosensor, or a field effect biosensor. The solid substrate element may be a quartz crystal microbalance (QCM), a semiconductor, or an optical element suitable for use in an optical biosensor.

[0030] According to one embodiment, the solid substrate element is a waveguide (e.g., an SPR waveguide or an ATR waveguide) suitable for use in an optical biosensor. According to an exemplary embodiment, the solid substrate element is an internal reflection element. According to a preferred embodiment of the present invention, the solid substrate element is an internal reflection element suitable for use in ATR-IR spectroscopy.

[0031] According to one embodiment, the solid substrate element is an optical element. According to one embodiment, the solid substrate element is an optical element suitable for use in ATR-IR spectroscopy, transmission IR spectroscopy, SPR spectroscopy, or surface-enhanced infrared absorption (SEIRA) spectroscopy, preferably ATR-IR spectroscopy and transmission IR spectroscopy, more preferably ATR-IR spectroscopy.

[0032] According to a preferred embodiment, the solid substrate element is preferably an IR-transmissive optical element suitable for use in ATR-IR spectroscopy, transmission IR spectroscopy, SPR spectroscopy, or surface-enhanced infrared absorption (SEIRA) spectroscopy, more preferably ATR-IR spectroscopy and transmission IR spectroscopy, most preferably ATR-IR spectroscopy.

[0033] Preferably, the IR-transmissive optical element is in the range of 40 to 13000 cm -1 , more preferably in the range of 500 to 3000 cm -1 , for example, 1400 to 1800 cm -1It is at least transmissive to infrared rays having a wavenumber within a range. In a particularly preferred embodiment, the optical element, preferably an IR transmissive optical element, is an ATR-IR waveguide.

[0034] According to one embodiment, the solid substrate element is a substance and / or crystal selected from the group consisting of plastic, glass, diamond, gold, silver, palladium, germanium, silicon, silicon dioxide, silver halide, GaAs, ZnSe, ZnS, thallium(I) mixed halide (e.g., KRS-5), and AMTIR. "AMTIR" is an amorphous substance that is known in the art to transmit infrared rays. A suitable AMTIR can be Ge 33 As 12 Se 55 (AMTIR-1), and preferably is a silicon crystal or a germanium crystal, more preferably a silicon crystal.

[0035] According to a preferred embodiment, the solid substrate element is a silicon crystal or a germanium crystal, preferably a silicon crystal.

[0036] The solid substrate element can be at least partially surface-modified, for example, by an oxide layer (e.g., a silicon dioxide layer). The surface modification can assist in the attachment of linker A.

[0037] According to one embodiment of the present invention, at least a part of the solid substrate element has an oxide surface layer, and the peptide is covalently bonded to the oxide surface layer via linker A. The oxide surface layer is preferably a metal oxide surface layer or a metalloid oxide surface layer. "Metalloid oxide" is an oxide of an element selected from the group consisting of boron, silicon, germanium, arsenic, antimony, and tellurium.

[0038] According to one embodiment of the present invention, at least a part of the solid substrate element has a silicon dioxide surface layer, and the peptide is covalently bonded to the silicon dioxide surface layer via linker A.

[0039] According to a preferred embodiment of the present invention, the solid substrate element is a silicon crystal, at least a part of the silicon crystal has a silicon dioxide surface layer, and the peptide compound is covalently bonded to the silicon dioxide surface layer via a linker A. In this case, it is more preferable that the linker A contains a silyl group covalently bonded to the silicon dioxide surface.

[0040] The solid substrate element may be covalently bonded to the surface attachment group of the linker A. For example, the surface attachment group of the linker A may be a silicon-containing group or a sulfur-containing group. The surface attachment group of the linker A can be selected in consideration of the material of the solid substrate element. For example, when the solid substrate element is a silicon crystal at least partially surface-modified with silicon dioxide or a germanium crystal at least partially surface-oxidized, the surface attachment group of the linker A may be a silicon-containing group. When the solid substrate element is a germanium crystal, the surface attachment group of the linker A may be a sulfur-containing group.

[0041] The first layer containing linker A The molecular probe-functionalized element according to the present invention further includes a first layer containing linker A.

[0042] Linker A covalently bonds the peptide compound to the solid substrate element. This can be understood in that the linker A has a surface attachment group covalently bonded to the solid substrate element and a peptide attachment group covalently bonded to the peptide compound, and the surface attachment group is covalently bonded to the peptide attachment group via a linking portion.

[0043] Linker A is not particularly limited with respect to its chemical structure as long as the linker A is suitable for covalently bonding the peptide compound defined herein to the solid substrate element.

[0044] The surface attachment group of the linker A can be selected in consideration of the material of the solid substrate element defined above herein.

[0045] Linker A can include a surface-attached group that is a silicon- or sulfur-containing group. According to one embodiment of the present invention, Linker A includes a surface-attached group covalently bonded to a solid substrate element, and the surface-attached group is a silicon-containing group or a sulfur-containing group.

[0046] The surface-attached group may have the formula: BE-Y 1 - / wherein BE represents the attachment of Linker A to the solid substrate element; In the formula, BE represents the attachment of Linker A to the solid substrate element; Y 1 is S or -O-Si(R 2 )-, and in the formula, R 2 is independently selected from OH, alkyl, alkoxy, OBE, and -O-Si x respectively, and Si x is the silicon atom of another Linker A or the silicon atom of any additional silane compound attached to the surface of the solid substrate element, and " / " indicates the attachment point to the remainder of Linker A.

[0047] According to one embodiment, the solid substrate element is a silicon crystal that is optionally at least partially surface-modified by silicon dioxide, and the surface-attached group has the formula: BE-Y 1 - / wherein BE represents the attachment of Linker A to the solid substrate element; In the formula, BE represents the attachment of Linker A to the solid substrate element; Y 1 is -O-Si(R 2 )-, and in the formula, R 2 is independently selected from OH, alkyl, alkoxy, OBE, and -O-Si x respectively, and Si x is the silicon atom of another Linker A or the silicon atom of any optional additional silane compound attached to the surface of the solid substrate element, and " / " indicates the attachment point to the remainder of Linker A.

[0048] According to one embodiment, linker A includes an alkylene oxide unit or a poly(alkylene oxide) unit. Preferably, linker A includes an ethylene glycol unit or a polyethylene glycol unit. For example, linker A can include a -(OCH2CH2) k - group, where k is an integer from 1 to 100, preferably from 1 to 50, more preferably from 1 to 16.

[0049] According to one embodiment, linker A includes a surface attachment group covalently bonded to a solid substrate element and a peptide attachment group covalently bonded to a peptide compound, the surface attachment group being covalently bonded to the peptide attachment group via a linking moiety, the linking moiety including a -(OCH2CH2) k - group, where k is an integer from 1 to 100, preferably from 1 to 50, more preferably from 1 to 16.

[0050] Furthermore, or alternatively, linker A can include a group that can be obtained by a bioorthogonal ligation reaction. Bioorthogonal ligation reactions are known to those skilled in the art. Those skilled in the art are also familiar with the chemical structures of the functional groups that can be obtained by said reactions. Examples of bioorthogonal ligation reactions include, but are not limited to, the reaction between an azide and a phosphine or phosphite (“Staudinger ligation”), the [2+3]-addition cyclization between an azide and an alkyne (“click reaction”), between an azide and a cycloalkyne (“copper-free click reaction”), between a nitrone and a cycloalkyne, the [2+4]-addition cyclization between a tetrazine and a trans-alkene (“tetrazine ligation”), oxime / hydrazone formation from aldehydes and ketones, etc. Thus, the bioorthogonal ligation reaction can be one of the following reactions: (i) the reaction between an azide and a phosphine or phosphite; (ii) the reaction between an azide and an alkyne (including cycloalkynes); (iii) the reaction between an azide and a cycloalkyne; (iv) the reaction between a tetrazine and a trans-alkene; (v) an oxime and / or hydrazone formation reaction.

[0051] According to one embodiment, linker A contains an optionally substituted N - heterocyclic group, and the N - heterocyclic group is selected from triazole (e.g., 1,2,3 - triazole), dihydropyridazine, and pyridazine.

[0052] Preferably, linker A contains an optionally substituted triazole group that can be obtained by [2 + 3] - addition cyclization (“click ligation”) between an azide compound and an alkyne compound, or linker A contains an optionally substituted dihydropyridazine that can be obtained by [2 + 4] - addition cyclization (“click ligation”) between a tetrazine compound and a trans - alkene compound.

[0053] According to one embodiment, linker A contains a surface - attaching group covalently bonded to a solid substrate element and a peptide - attaching group covalently bonded to a peptide compound. The surface - attaching group is covalently bonded to the peptide - attaching group via a connecting portion. The connecting portion contains a group that can be obtained by a bio - orthogonal ligation reaction. Preferably, the connecting portion contains an optionally substituted N - heterocyclic group, and the N - heterocyclic group is selected from triazole (e.g., 1,2,3 - triazole), dihydropyridazine, and pyridazine.

[0054] Linker A can contain a peptide - attaching group, which can be any group suitable for attaching the linker to a peptide compound, optionally to an amino, thiol, or alcohol group of the peptide compound, and optionally to an amino group of the peptide compound (e.g., the amino group of a lysine residue).

[0055] The peptide - attaching group can be an ester group, an amide group, or a succinimide group or an open - ring product of a succinimide group, and is optionally an amide group.

[0056] The peptide - attaching group can be one of the following:

[0057]

Chemical formula

[0058] According to one embodiment of the present invention, linker A is a surface attachment group covalently bonded to the solid substrate element, and a peptide attachment group covalently bonded to the peptide compound and includes the surface attachment group is covalently bonded to the peptide attachment group via a linking moiety; the surface attachment group is a silicon or sulfur-containing group, the peptide attachment group is an ester group, an amide group, a succinimide group or an open-ring product thereof, preferably, the peptide attachment group is one of the following:

[0059]

Chemical formula

[0060] According to one embodiment of the present invention, linker A is a surface attachment group covalently bonded to the solid substrate element, and a peptide attachment group covalently bonded to the peptide compound and includes the surface attachment group is covalently bonded to the peptide attachment group via a linking moiety; the surface attachment group is a silicon or sulfur-containing group, the linking moiety includes an optionally substituted N-heterocyclic group selected from triazole (e.g., 1,2,3-triazole), dihydropyridazine and pyridazine, which can optionally be obtained by an additional cyclization reaction (e.g., [2+3]- or [2+4]-addition cyclization); The peptide attachment group is an ester group, an amide group, a succinimide group, or a ring-opening product thereof, and preferably, the peptide attachment group is one of the following:

[0061]

Chemical formula

[0062] According to one embodiment of the present invention, linker A has a surface attachment group covalently bonded to the solid substrate element, and a peptide attachment group covalently bonded to the peptide compound and includes The surface attachment group is covalently bonded to the peptide attachment group via a linking moiety; The surface attachment group is a silicon- or sulfur-containing group, The linking moiety includes a -(OCH2CH2) k - group, where k is an integer from 1 to 100, preferably from 1 to 50, more preferably from 1 to 16; The peptide attachment group is an ester group, an amide group, a succinimide group, or a ring-opening product thereof, and preferably, the peptide attachment group is one of the following:

[0063]

Chemical formula

[0064] According to a preferred embodiment of the present invention, linker A has a surface attachment group covalently bonded to the solid substrate element, and A peptide attachment group covalently bonded to a peptide compound comprising the surface attachment group is covalently bonded to the peptide attachment group via a linking moiety; the surface attachment group is a silicon or sulfur-containing group, the linking moiety comprises (i) an optionally substituted N-heterocyclic group selected from triazole (e.g., 1,2,3-triazole), dihydropyridazine and pyridazine, which can optionally be obtained by an addition cyclization reaction (e.g., [2+3]- or [2+4]-addition cyclization), and / or (ii) a -(OCH2CH2) k - group (wherein k is an integer from 1 to 100, preferably from 1 to 50, more preferably from 1 to 16), preferably, the linking moiety comprises features (i) and (ii); the peptide attachment group is an ester group, an amide group or a succinimide group or an open-ring product thereof, preferably, the peptide attachment group is one of the following:

[0065]

Chemical formula

[0066] The inventors have found that it is possible to provide better and denser functionalization of the surface of a substrate element by binding the surface of the solid substrate element to a peptide compound via a linker A, which is prepared sequentially by click ligation and thus contains an optionally substituted N-heterocyclic group selected from triazole (e.g., 1,2,3-triazole), dihydropyridazine and pyridazine in its linking moiety.

[0067] According to a preferred embodiment of the present invention, the linker A has a structure according to any one of formulas (LA-I) to (LA-III-b) or a salt thereof, preferably has a structure according to formula (LA-II) or (LA-II-b) or a salt thereof:

[0068]

Chemical formula

[0069] According to a preferred embodiment, the linking moiety L 1 in any one of the above formulas (LA-I) to (LA-III-b) contains an ethylene glycol group or a PEG group, more preferably an (OCH2CH2) k - group (where k is an integer from 1 to 100, preferably from 1 to 50, more preferably from 1 to 16).

[0070] Any additional silane compound adhering to the surface of the solid substrate element may be a matrix compound as defined below in this specification.

[0071] According to one embodiment, linker A has a structure according to formula (LA-IV) or a salt thereof:

[0072]

Chemical formula

[0073] When the substituent can be "bond", this means "covalent bond". As will be understood by those skilled in the art, when two or more adjacent substituents are selected to be bonds, this means that the two adjacent substituents form one bond. For example, X 5a and X 6a are both selected to be bonds, this means that X 5a and X 6a form one bond that binds a methylene group ("CH2-") to Y 2 . This understanding applies to each embodiment disclosed herein where two or more adjacent substituents can be selected to be bonds.

[0074] According to a preferred embodiment, the linker A has a structure according to formula (LA-V) or a salt thereof:

[0075]

Chemical formula

[0076] According to one embodiment, the linker A has a structure according to formula (LA-VI) or a salt thereof:

[0077]

Chemical formula

[0078] According to a preferred embodiment, linker A has a structure according to formula (LA-VII) or a salt thereof or a [2+3]-addition cyclization positional isomer thereof:

[0079]

Chemical formula

[0080]

Chem.

[0081] According to one embodiment, linker A has a structure according to formula (LA-VII) or a salt thereof or a [2+3]-addition cyclization positional isomer thereof:

[0082]

Chem.

[0083]

Chemical formula

[0084] According to one embodiment, linker A has a structure according to formula (LA-VII) or a salt thereof or a [2+3]-addition cycloisomer thereof:

[0085]

Chemical formula

[0086]

Chemical formula

[0087] According to a more specific embodiment, linker A has a structure according to formula (LA-VII) or a salt thereof or a [2+3]-addition cyclization positional isomer thereof:

[0088]

Chemical formula

[0089]

Chemical formula

[0090] According to one embodiment, linker A has a structure according to formula (LA-VII) or its salts or its [2+3]-addition cycloisomers:

[0091]

Chemical formula

[0092]

Chem.

[0093] According to one embodiment, linker A has a structure according to formula (LA-IX) or a salt thereof or a [2+3]-addition cycloisomer thereof:

[0094]

Chem.

[0095] [Chemical formula] is one of them, where Q 1 is NH, O or S of the amino acid of the peptide compound, and PEP is a peptide compound.

[0096] According to one embodiment, linker A has a structure represented by formula (LA-X) or a salt thereof or a [2+3]-addition cyclized positional isomer thereof:

[0097] [Chemical formula] where BE represents the attachment of linker A to the solid substrate element; n 1f is an integer from 1 to 16, optionally 11, n 2f is an integer from 1 to 16, Y 2 is

[0098] [Chemical formula] is one of them, where Q 1 is NH, O or S of the amino acid of the peptide compound, and PEP is a peptide compound.

[0099] When linker A has a structure represented by any one of formulas (LA-I) to (LA-VII) or a salt thereof or a [2+3]-addition cyclized positional isomer thereof, and the Y 1 group is -Si(R 2 )-, the solid substrate element is preferably a silicon crystal or a germanium crystal, preferably a silicon crystal, more preferably a silicon crystal having at least a part of its surface as silicon dioxide, and the Y 1 group is a silicon crystal covalently bonded to silicon dioxide.

[0100] Alternatively, linker A has a structure according to any one of formulas (LA-I) to (LA-VII), or a salt thereof, or a [2+3]-addition cyclization positional isomer thereof, and when the Y 1 group is -S-, the solid substrate element is preferably a germanium crystal.

[0101] According to one embodiment, linker A can be obtained or is obtained by steps a) to c) of the process according to the present invention. According to one embodiment, linker A can be obtained or is obtained by steps a), b1) to b4), and c) of the process according to the present invention.

[0102] Please refer to the section "Process according to the present invention" below.

[0103] Matrix compound in some cases In addition to linker A, the first layer can further contain a matrix compound covalently bonded to the solid substrate element.

[0104] The matrix compound may be a compound containing a silicon-containing group covalently bonded to the solid substrate element. The matrix compound is preferably a compound containing a PEG group or an mPEG group and a silicon-containing group, and the silicon-containing group is covalently bonded to the solid substrate element.

[0105] According to one embodiment, the matrix compound is a PEG-containing or mPEG-containing silane, preferably an mPEG-containing silane. Further or alternatively, the matrix compound may be an optionally substituted alkyl sulfide, preferably an optionally substituted C1-C30 alkyl sulfide.

[0106] Therefore, according to one embodiment, the matrix compound is (i) a PEG-containing or mPEG-containing silane, preferably an mPEG-containing silane, and / or (ii) Optionally substituted alkyl sulfide, preferably optionally substituted C1-C30 alkyl sulfide It is.

[0107] According to one embodiment, the first layer contains a matrix compound covalently bonded to the solid substrate element. The matrix compound is a compound according to formula (MA-I) or a salt thereof: (MA-I): BE-Y-X, In the formula, BE indicates the attachment of the compound to the solid substrate element; Y is S or -O-Si(R 2g ), where R 2g is independently selected from OH, alkyl, alkoxy, OBE and -O-Si y respectively, and Si y is the silicon atom of linker A or the silicon atom of another silane attached to the surface of the solid substrate element. X is a group containing a PEG group, an mPEG group or an alkyl group (for example, a C1-C30 alkyl group).

[0108] According to one embodiment, the matrix compound is a compound according to formula (MA-II) or a salt thereof:

[0109]

Chemical formula

[0110] The inventors have found that further compounds can function as a matrix on the surface of the substrate element, which further reduces the non-specific binding of the medium or sample components to the surface. Furthermore, by using the matrix compound in combination with linker A, it becomes possible to finely adjust the amount of linker A binding to the surface of the substrate element and to space linker A on the surface of the substrate element. Therefore, by attaching a "matrix compound" in addition to linker A to the surface of a substrate element such as an ATR waveguide (e.g., a silicon crystal), the performance for detecting biomarkers can be improved.

[0111] According to one embodiment, the matrix compound is a compound according to formula (MA-II) or a salt thereof:

[0112]

Chemical formula

[0113] According to one embodiment, the matrix compound is a compound according to formula (MA-II) or a salt thereof:

[0114]

Chemical formula

[0115] According to one embodiment, the matrix compound is a compound represented by formula (MA-II) or a salt thereof:

[0116]

Chemical formula

[0117] When the compound represented by formula (MA-II) has a Y 2g group which is -Si(R 1g )-, the solid substrate element is preferably a silicon crystal or a germanium crystal, preferably a silicon crystal, more preferably a silicon crystal in which at least a part of the silicon crystal has a silicon dioxide surface and the Y 1g group is covalently bonded to the silicon dioxide.

[0118] Alternatively, when the compound represented by formula (MA-II) has a Y 1g group which is -S-, the solid substrate element is preferably a germanium crystal.

[0119] A second layer containing a peptide compound The molecular probe functionalized element according to the present invention includes a second layer containing a peptide compound. The peptide compound is covalently bonded to the solid substrate element via linker A and to the molecular probe via linker B. Preferably, the second layer has the function of a blocking layer.

[0120] The second layer can further include a peptide compound that is covalently bonded to the solid substrate element via linker A but is not attached to the molecular probe. In other words, the second layer can include a peptide compound that is not functionalized by the molecular probe via linker B but functions solely as part of a blocking layer.

[0121] Thus, according to a preferred embodiment, the second layer includes a peptide compound that is covalently bonded to the solid substrate element via linker A but is not attached to the molecular probe.

[0122] The second layer can include a single type of peptide compound or a mixture of peptide compounds. According to one embodiment, the second layer includes or consists of a single type of peptide compound.

[0123] According to a preferred embodiment, the second layer includes or consists of a mixture of peptide compounds. When the second layer includes or consists of a mixture of peptide compounds, different peptide compounds may be attached to the solid substrate element via linker A.

[0124] According to a preferred embodiment, the peptide compound of the second layer is selected from the group consisting of peptides, substituted peptides, proteins, protein fragments, and mixtures thereof. The substituted peptide may be, for example, a pegylated peptide.

[0125] The peptide compound is preferably a peptide compound that has no binding affinity or essentially no binding affinity for the target analyte of the molecular probe or the fluid containing the target analyte, such as a body fluid (e.g., intestinal fluid, ocular fluid, pulmonary fluid, blood or CSF). The peptide compound is preferably a peptide compound that is inert to the target analyte of the molecular probe or the fluid containing the target analyte, such as a body fluid (e.g., intestinal fluid, ocular fluid, pulmonary fluid, blood or CSF).

[0126] The peptide compound may be an optionally substituted natural, semi-synthetic or synthetic peptide, optionally having an amino acid chain length between 2 and 600 amino acids, optionally between 2 and 400 amino acids, optionally between 2 and 200 amino acids, optionally between 2 and 100 amino acids, and optionally between 4 and 100 amino acids. The peptide compound may be a natural, semi-synthetic or synthetic peptide having an amino acid chain length between 2 and 100 amino acids, optionally between 4 and 100 amino acids. Preferably, the peptide compound comprises one or more amino acids containing an H2N, HO or HS group in its / their side chain.

[0127] The peptide compound may be a protein fragment. The protein fragment can be obtained or potentially obtained by protein hydrolysis. Thus, the peptide compound can be obtained or potentially obtained from protein hydrolysis products. For example, the peptide compound can be obtained or potentially obtained from hydrolysis products of albumin, casein, BSA, serum proteins (e.g., sera of animals, such as sera of pigs, horses or goats), milk proteins or mixtures thereof. Preferably, the peptide compound can be obtained or is obtained from casein hydrolysis products.

[0128] The peptide compound may be an optionally substituted peptide, optionally having an amino acid chain length between 2 and 600 amino acids, optionally between 2 and 400 amino acids, optionally between 2 and 200 amino acids (e.g., 2 - 100 or 4 - 100), and the peptide can be or is obtained from a protein hydrolysis product. The peptide compound may be an optionally substituted peptide having an amino acid chain length between 2 and 600 amino acids, optionally between 2 and 400 amino acids, optionally between 2 and 200 amino acids (e.g., 2 - 100 or 4 - 100), and the peptide can be or is obtained from a protein hydrolysis product, and the protein is selected from the group consisting of albumin, casein, BSA, serum protein, milk protein, and mixtures thereof. Preferably, the peptide compound is an optionally substituted peptide having an amino acid chain length between 2 and 100 amino acids, and the peptide can be or is obtained from a casein hydrolysis product. The casein hydrolysis product may be a hydrolysis product of high - purity casein. A suitable casein hydrolysis product is "The blocking solution" commercially available from Candor Bioscience GmbH.

[0129] According to one embodiment, the second layer contains a mixture of peptide compounds, and the mixture of peptide compounds is a mixture of peptides that can be or are obtained from a protein hydrolysis product, and the peptides are optionally substituted, for example, pegylated.

[0130] According to one embodiment, the second layer contains a mixture of peptide compounds, and the mixture of peptide compounds is a mixture of peptides that can be or are obtained from a casein hydrolysis product, and the peptides are optionally substituted, for example, pegylated peptides.

[0131] The third layer containing linker B The molecular probe-functionalized element according to the present invention includes a third layer containing a linker B. The linker B is covalently bonded to a peptide compound and a molecular probe. The linker B is not particularly limited with respect to its chemical structure as long as the linker B is suitable for covalently bonding the peptide compound defined herein to the molecular probe.

[0132] According to one embodiment, the linker B includes a peptide attachment group covalently bonded to the peptide compound and a molecular probe attachment group covalently bonded to the molecular probe, and the peptide attachment group is covalently bonded to the molecular probe attachment group by a linking moiety, and the linking moiety includes a group that can be obtained by a bioorthogonal ligation reaction.

[0133] Preferably, the linking moiety includes an optionally substituted N-heterocyclic group, and the N-heterocyclic group is optionally selected from triazole (e.g., 1,2,3-triazole), dihydropyridazine, and pyridazine.

[0134] Preferably, the linking moiety can be obtained by [2+3]-addition cyclization ("click ligation") between an azide compound and an alkyne compound, or includes an optionally substituted triazole group that can be obtained or is obtained, or the linking moiety can be obtained by [2+4]-addition cyclization ("click ligation") between a tetrazine compound and a trans-alkene compound, or includes an optionally substituted dihydropyridazine that can be obtained or is obtained.

[0135] According to one embodiment, the linker B is a peptide attachment group covalently bonded to the peptide compound, a molecular probe attachment group covalently bonded to the molecular probe and includes the peptide attachment group is covalently bonded to the molecular probe attachment group via a linking moiety; the linking moiety includes an optionally substituted N-heterocyclic group (optionally, can be obtained or is obtained by an addition cyclization reaction) selected from triazole, dihydropyridazine, and pyridazine, The peptide attachment group is an ester group, an amide group, a succinimide group, or a ring-opened product thereof.

[0136] According to one embodiment, linker B has a structure represented by any one of formulas (LB-I) to (LB-VII) or a salt thereof:

[0137] [Chemical formula] In the formula, Y 3 is an ester group, an amide group, a succinimide group, or a ring-opened product thereof, and each of these is covalently bonded to the peptide compound. Preferably, Y 3 is

[0138] [Chemical formula] or one of their salts. In the formula, Q 1 is NH, O, or S of an amino acid of the peptide compound, PEP is a peptide compound, L 3 and L 4 are each a linking moiety when present, Y 4 is an ester group, an amide group, a succinimide group, or a ring-opened product thereof, and each of these is covalently bonded to the molecular probe, MP is a molecular probe, Ring B is an optionally substituted 8-membered carbocyclic ring or an optionally substituted 8-membered heterocyclic ring when present; R 11 is H, an optionally substituted alkyl, or an optionally substituted aryl when present.

[0139] According to one embodiment, linker B has a structure represented by any one of formulas (LB-VIII) to (LB-XIV) or a salt thereof:

[0140] [Chemical formula] In the formula, Y 3 is an ester group, an amide group, a succinimide group or a ring-opening product thereof, and each of these is covalently bonded to the peptide compound. Preferably, Y 3 is

[0141] [Chemical formula] or one of their salts. In the formula, Q 1 is NH, O or S of the amino acid of the peptide compound, PEP is a peptide compound, L 3 and L 4 , if present, are each a linking moiety, Y 4 is an ester group, an amide group, a succinimide group or a ring-opening product thereof, and each of these is covalently bonded to the molecular probe, MP is a molecular probe, Ring B, if present, is an optionally substituted 8-membered carbocyclic ring or an optionally substituted 8-membered heterocyclic ring; R 11 , if present, is H, optionally substituted alkyl or optionally substituted aryl; X 1h is a bond or (CH2)n 1h wherein n 1h is an integer between 1 and 10, X 2h is a bond or (OCH2CH2)n 2h wherein n 2h is an integer between 1 and 15, X 3h is a bond, C(O) or (CH2)n 3h wherein n 3h is an integer between 1 and 10.

[0142] According to one embodiment, linker B has a structure represented by formula (LB-X) or (LB-XIII) or a salt thereof:

[0143]

Chemical formula

[0144]

Chemical formula

[0145] According to one embodiment, linker B has a structure represented by formula (LB-X) or a salt thereof:

[0146]

Chemical formula

[0147]

Chemical formula

[0148] According to one embodiment, linker B has a structure according to formula (LB-XIII) or their salts:

[0149]

Chemical formula

[0150]

Chemical formula

[0151] According to one embodiment, linker B has a structure of formula (LB-XV) or their salts or their [2+3] addition cyclization positional isomers:

[0152]

Chemical formula

[0153]

Chemical formula

[0154] According to one embodiment, linker B has a structure of formula (LB-XVI) or a salt thereof or a [2+3] addition cyclization positional isomer thereof:

[0155]

Chemical formula

[0156]

Chemical formula

[0157] According to one embodiment, linker B has a structure of formula (LB-XV) or a salt thereof or a [2+3] addition cyclization positional isomer thereof:

[0158]

Chemical formula

[0159]

Chemical formula

[0160] A fourth layer comprising a molecular probe The molecular probe-functionalized element comprises a fourth layer comprising a molecular probe. The molecular probe is covalently bonded to the peptide compound by a linker B.

[0161] The molecular probe is not particularly limited and can be selected by those skilled in the art according to the target analyte to be detected and / or analyzed, and / or the target chemical interaction to be detected or analyzed. The molecular probe-functionalized element can also include two or more different molecular probes, such as antibodies and the like.

[0162] According to one embodiment, the molecular probe is an antigen, an antibody, an antibody fragment, a fusion protein with an antibody or an antibody fragment, a conjugate with an antibody conjugate or an antibody fragment, a protein complex optionally containing an antibody fragment or their fusion proteins, an anticalin, a nanobody, an organic small molecule, a drug, a nucleic acid, an aptamer, a lipid, a carbohydrate, a peptide, or a mixture thereof.

[0163] According to one embodiment, the molecular probe is an antigen-binding protein optionally selected from an antibody, an antibody fragment, a fusion protein with an antibody or an antibody fragment, a conjugate with an antibody conjugate or an antibody fragment, or a protein complex optionally containing an antibody fragment or their fusion proteins, an anticalin, a nanobody, and mixtures thereof.

[0164] According to one embodiment, the molecular probe is an anti-amyloid β antibody, an anti-alpha-synuclein antibody, an anti-tau antibody, an anti-TDP-43 antibody, or a fragment, fusion protein and / or conjugate of the above antibodies. According to one embodiment, the molecular probe is an anti-amyloid β antibody, an anti-alpha-synuclein antibody, an anti-tau antibody, an anti-TDP-43 antibody, or a fragment, fusion protein and / or conjugate of the above antibodies and / or a mixture of the above antibodies. The anti-amyloid β antibody can be selected from the following list: 2E9, H31L21, 11A50-B10, 12B2, 4G8, 11H3, MOAB-2, anti-Aβ25-35, A8978, 5C3, 8G7, 6G12, 1E8 and 32A1. The anti-alpha-synuclein antibody may be 4B12, AKS5946, S5566. The anti-tau antibody may be tau-5 or tau396. The anti-TDP-43 antibody may be 1HCLC.

[0165] According to one embodiment, the molecular probe is an anti-amyloid β antibody, or a fragment, fusion protein, and / or conjugate thereof.

[0166] According to one embodiment, the molecular probe is an anti-alpha-synuclein antibody, or a fragment, fusion protein, and / or conjugate thereof.

[0167] According to one embodiment, the molecular probe can form a complex with a protein associated with or capable of causing a proteopathy. A proteopathy is a class of diseases in which one or more proteins are misfolded and / or structurally abnormal, which can disrupt the function of cells, tissues, and / or organs. Proteopathies and proteins associated with proteopathies are known to those skilled in the art and include, but are not limited to, amyloid beta peptide, tau protein, alpha-synuclein, TDP-43, and huntingtin.

[0168] According to one embodiment, the molecular probe can form a complex with a target analyte selected from the group consisting of amyloid beta (Aβ) peptide and its isoforms, alpha-synuclein, tau protein, TDP-43, human islet amyloid polypeptide (hIAPP), prion protein, and p53, and optionally with a target analyte selected from the group consisting of amyloid beta (Aβ) peptide and its isoforms, alpha-synuclein, tau protein, and TDP-43. The amyloid beta (Aβ) peptide may be a monomeric, oligomeric, or protofibrillar amyloid beta (Aβ) peptide.

[0169] The device according to the present invention In one aspect, the present invention provides a device comprising a molecular probe-functionalized element according to the present invention.

[0170] Preferably, the device is a biosensor. Biosensors are well known in the art. A biosensor is an analytical device for detecting a target analyte. A biosensor includes a bioreceptor element (molecular probe) and a transducer element. The bioreceptor element (molecular probe) can interact with the target analyte to create a physicochemical change, which is converted into a signal measurable by the transducer element.

[0171] According to a preferred embodiment, the device is an optical biosensor. The optical biosensor may be based on surface plasmon resonance (SPR) microscopy, infrared ATR spectroscopy, Raman spectroscopy, colorimetric microscopy, fluorescence microscopy, luminescence microscopy, or a combination of these techniques. The optical biosensor can include a flow chamber and a pump for passing a sample stream over the molecular probe functionalized element according to the invention.

[0172] According to a preferred embodiment, the device is an SPR or ATR-IR based biosensor, more preferably, the device is an ATR-IR based biosensor. Such devices are known in the art and are described, for example, in Nabers et al, Anal. Chem. 2016, 88, 27552762.

[0173] The process according to the invention In one aspect, the invention is a process for preparing a molecular probe functionalized element according to the invention. The process comprises a) providing a solid substrate element; and b) preparing a linker A precursor covalently bound to the surface of the solid substrate element; and c) reacting the linker A precursor with a peptide compound to covalently bind the peptide compound to the solid substrate element via the linker A; and d) preparing a linker B precursor covalently bound to the peptide compound; and e) reacting the linker B precursor with a molecular probe to covalently bond the molecular probe to the peptide via the linker B; comprising.

[0174] Step a) In step a), a solid substrate element is provided. For the definition of the solid substrate element, reference is made to the definition, embodiments and preferred embodiments of the solid substrate element as defined herein for the molecular probe-functionalized element according to the present invention.

[0175] According to one embodiment, the solid substrate element is obtained by oxidizing at least a part of the surface of an IR-transmissive optical element, preferably an ATR-IR waveguide (for example, a silicon crystal or a germanium crystal). During the oxidation treatment (for example, chemical oxidant or plasma treatment), at least a part of the surface is converted into an oxide layer, which can improve the attachment of the linker compound in subsequent step b).

[0176] According to a preferred embodiment, the solid substrate element is obtained by oxidizing at least a part of the surface of a silicon crystal, for example, by chemical oxidant or plasma treatment. During the oxidation treatment, at least a part of the surface of the silicon crystal is converted into silicon dioxide. The surface of the silicon crystal can be polished before the plasma treatment.

[0177] Step b) In step b), a linker A precursor covalently bonded to the surface of the solid substrate element is prepared.

[0178] Preferably, the linker A precursor contains a peptide reaction group, preferably an amine reaction group (for example, an active ester), which enables covalent bonding of a peptide compound. "Peptide reaction group" is known in the art. Such groups are used to attach a compound to a functional residue (for example, an amino acid residue) of a peptide, such as an amino group (for example, of a lysine residue), a thiol (for example, of cysteine), an alcohol (for example, of serine), etc.

[0179] The linker A precursor can be prepared stepwise.

[0180] Preferably, the linker A precursor is first reacted with the surface of the solid substrate element with linker A1 to obtain a surface that is at least partially functionalized by click reaction groups, and then the surface that is at least partially functionalized is reacted with linker A2 by click ligation to obtain a linker A precursor covalently bonded to the surface of the solid substrate element.

[0181] "Click reaction groups" are well known in the art. Click reaction groups include, but are not limited to, alkynes, strained cycloalkynes (e.g., cyclooctynes where one or more carbon atoms of the ring are substituted by heteroatoms such as N), strained cycloalkenes (e.g., trans-cyclooctene), azides, tetrazines, nitrones, etc. Click reaction groups can react with each other in specific combinations ("click reaction" or "click ligation"), which are all well known to those skilled in the art (e.g., azide and alkyne or azide and strained alkyne in the presence of a copper catalyst in some cases).

[0182] According to a preferred embodiment, step b) is to obtain a linker A precursor covalently attached to the surface of the substrate element, b1) (i) preparing a linker A1 containing a silyl group or a thiol group, and (ii) an azide group or an optionally substituted tetrazine group, preferably an azide group; b2) optionally, activating the linker A1 prepared in step b1); b3) reacting the linker A1 prepared in step b1) or the compound obtained in step b2) with the solid substrate element to obtain a surface that is at least partially functionalized; b4) Reacting the surface of the at least partially functionalized solid substrate element obtained in step b3) with a linker A2 containing (i) an alkyne group or a trans-alkene group, and (ii) a peptide reactive group selected from carboxylic acid, ester, active ester and maleimide comprises

[0183] The inventors have surprisingly discovered that by functionalizing the surface of a solid substrate element (e.g., a waveguide for an optical biosensor such as a silicon crystal) with a click reaction group such as an azide or tetrazine group, it is possible to obtain a stably and highly densely functionalized surface. The functionalized surface can be prepared and stored before using it to prepare the molecular probe functionalized element according to the present invention. This is particularly advantageous for preparing it shortly before using the highly reactive molecular probe functionalized element, and thus, this improves the compatibility of the molecular probe functionalized element according to an embodiment of the present invention for implementation in a point-of-care (POC) testing device.

[0184] In step b1), linker A1 is preferably prepared, and linker A1 contains (i) a silyl group or a thiol group, and (ii) an azide group or an optionally substituted tetrazine group, preferably an azide group.

[0185] According to one embodiment, linker A1 has a structure according to formula (LA1-I) or a salt thereof:

[0186]

Chemical formula

[0187]

Chem.

[0188] According to one embodiment, linker A1 has a structure according to formula (LA1-I) or a salt thereof:

[0189]

Chem.

[0190]

Chem.

[0191] According to one embodiment, linker A1 has a structure according to formula (LA1-II) or a salt thereof:

[0192]

Chem.

[0193]

Chemical formula

[0194] According to one embodiment, the linker A1 has a structure according to formula (LA1-II) or a salt thereof:

[0195]

Chemical formula

[0196]

Chem.

[0197] In a specific embodiment, linker A1 has the following structure:

[0198]

Chem.

[0199] In optional step b2), the linker A1 prepared in b1) is activated. The activation step b2) can convert the linker A1 into an intermediate that is more reactive towards the surface of the solid substrate element. The specific conditions of the activation step b2) depend on the chemical structure of the linker A1 and can be selected by those skilled in the art.

[0200] In step b3), the linker A1 prepared in step b1) or the compound obtained in step b2) is reacted with the substrate element to obtain a surface that is at least partially functionalized. Preferably, the linker A1 is selected to obtain an azide-functionalized surface in step b3).

[0201] In step b4), the surface of the at least partially functionalized solid substrate element obtained in step b3) is reacted with a linker A2 containing (i) an alkyne group or a trans-alkene group, and (ii) a peptide reaction group selected from carboxylic acid, carboxylic acid ester, active ester, and maleimide to obtain a linker A precursor that is covalently attached to the surface of the solid substrate element.

[0202] The alkyne group (i) of Linker A2 can be selected from optionally substituted 8-membered cycloalkynes, optionally containing heteroatoms in the 8-membered ring. Such compounds are known to react with azides in strain-promoted click reactions. The strain-promoted click reaction is a reaction between a strained internal cyclic alkyne and an azide and is known to those skilled in the art. The alkyne group (i) of Linker A2 can be, but is not limited to, DBCO, DIBO, DIFO or BCN. These abbreviations are well-known in the art. For illustration, DBCO is

[0203]

Chem.

[0204]

Chem.

[0205]

Chem.

[0206] "Active ester" is well-known in the art and can react with an amino group to form an amide bond.

[0207] According to one embodiment, Linker A2 has a structure according to formula (LA2-I) or a salt thereof:

[0208]

Chem.

[0209]

Chem.

[0210] “N-linked maleimide” means that the linker L is covalently bonded to the nitrogen of maleimide.

[0211] According to one embodiment, the linker A2 has a structure according to formula (LA2-II) or a salt thereof:

[0212]

Chemical formula

[0213]

Chemical formula

[0214] According to one embodiment, the linker A2 has a structure according to formula (LA2-II) or a salt thereof:

[0215]

Chemical formula

[0216]

Chemical formula

[0217]

Chemical formula

[0218] According to a specific example, linker A2 has the following structure:

[0219]

Chemical formula

[0220] In step b4), a linker A precursor that covalently binds to the surface of the substrate element and contains a peptide reactive group (e.g., an amine reactive group such as an active ester) is preferably obtained. The reactivity of the peptide reactive group can be utilized in subsequent step c) of the process according to the present invention to covalently bind a peptide to the substrate element via linker A, as defined above herein for the molecular probe functionalized element of the present invention.

[0221] It is also possible to swap the click reaction groups of linker A1 and linker A2. In such a case, linker A1 can contain an alkyne or trans-alkene group, and linker A2 can contain an azide group or an optionally substituted tetrazine group. Thus, according to another embodiment, step b) is to obtain a linker A precursor that covalently attaches to the surface of the substrate element. Step b1) of preparing a linker A1 comprising (i) a silyl group or a thiol group, and (ii) an alkyne group or a trans-alkene group b2) Optionally, step of activating the linker A1 prepared in b1) b3) Step of reacting the linker A1 prepared in step b1) or the compound obtained in step b2) with a solid substrate element to obtain a surface that is at least partially functionalized b4) Step of reacting the surface of the at least partially functionalized solid substrate element obtained in step b3) with a linker A2 comprising (i) an azide group or an optionally substituted tetrazine group, preferably an azide group, and (ii) a peptide reactive group selected from carboxylic acids, esters, active esters and maleimides which comprises.

[0222] The process of the present invention further comprises the step of reacting a matrix precursor compound with a solid substrate element to covalently attach the matrix compound to the surface of the substrate element, if any. For the definition of the matrix compound, reference is made to the definition, embodiments and preferred embodiments of the matrix compound as defined herein for the molecular probe functionalized element according to the present invention.

[0223] This step can be carried out before, during and / or after step b), preferably during step b).

[0224] This step can be carried out simultaneously with step b1), optionally step b2) and step b3). When this step is carried out simultaneously with step b1), optionally step b2) and step b3), the matrix precursor compound can be prepared in the form of a mixture with linker A1 in step b1), and then this mixture can be used in optionally step b2) and step b3 as defined above. The mixture can have a molar ratio of linker A1:matrix precursor compound in the range of 1:99 to 99.9:0.1, optionally 25:75 to 99.9:0.1, optionally 50:50 to 99.9:0.1, optionally 75:25 to 99.9:0.1, optionally 90:10 to 99:1.

[0225] The matrix precursor compound may be a compound containing (i) a silyl group and (ii) a PEG group or an mPEG group, or a compound containing (i) a thiol group and (ii) an optionally substituted alkyl group (e.g., an optionally substituted C1-C30 alkyl group).

[0226] The matrix precursor compound may have a structure according to formula (MA2-I) or a salt thereof:

[0227]

Chemical formula

[0228] The matrix precursor compound may have a structure according to formula (MA2-I) or a salt thereof:

[0229]

Chemical formula

[0230] Specific embodiments of the matrix precursor compound have the following structures:

[0231]

Chemical formula

[0232] Step c) In step c), the linker A precursor is reacted with the peptide compound to covalently bond the peptide compound to the solid substrate element via the linker A. For the definitions of the peptide compound and the linker A, reference is made to the definitions, embodiments and preferred embodiments of the peptide compound and the linker A as defined herein for the molecular probe functionalized element of the present invention.

[0233] The reaction of the peptide compound with the activated surface obtained in step b) or step b4) can block the surface of the solid substrate element. Thus, according to one embodiment, step c) is a blocking step.

[0234] The peptide compound is prepared in pure form or as part of a mixture such as a protein hydrolysate.

[0235] According to one embodiment, the peptide compound is prepared as part of a protein hydrolysate and optionally as part of a hydrolysate of albumin, casein, BSA, serum proteins (e.g., animal sera such as porcine, equine or caprine sera), milk proteins or mixtures thereof. Preferably, the peptide is prepared in the form of a casein hydrolysate. The protein hydrolysate containing the peptide compound can be contacted with the linker A precursor obtained in step b) or b4) to react the peptide compound with the linker A precursor. Preferably, the peptide compound is prepared as part of a casein hydrolysate.

[0236] Step d) In step d), a linker B precursor covalently bonded to the peptide compound is prepared.

[0237] Preferably, the linker B precursor contains a click reaction group selected from, for example, alkyne, strained cycloalkyne (e.g., cyclooctyne, where one or more carbon atoms in the ring may be substituted by a heteroatom such as N), strained cycloalkene (e.g., trans-cyclooctene), azide, tetrazine, nitrone, etc., thereby enabling covalent attachment of the molecular probe by click ligation.

[0238] The linker B precursor can be prepared by reacting a peptide compound with linker B1 as defined hereinafter in this specification.

[0239] According to one embodiment, linker B1 contains (i) a peptide reaction group (e.g., an amine reaction group) selected from carboxylic acid, ester, active ester, and maleimide, and (ii) a click reaction group selected from alkyne, strained cycloalkyne, strained cycloalkene, azide, and tetrazine.

[0240] According to one embodiment, linker B1 has a structure according to formula (LB1-I) or a salt thereof:

[0241]

Chemical formula

[0242]

Chemical formula

[0243] According to one embodiment, linker B1 has a structure according to formula (LB1-II) or a salt thereof:

[0244]

Chemical formula

[0245]

Chemical formula

[0246] According to one embodiment, linker B1 has a structure according to formula (LB1-II) or a salt thereof:

[0247]

Chemical formula

[0248]

Chemical formula

[0249] According to a specific embodiment, linker B1 has one of the following structures:

[0250]

Chemical formula

[0251] After step c) and / or step d), there may be a reactive linker A precursor remaining on the surface of the solid substrate element. In such a case, after step c) and / or step d), it is possible, and preferably also possible, to react the surface with a small molecule such as an amine compound (e.g., ethanolamine) to quench the remaining reactive linker A precursor.

[0252] According to one embodiment, the process includes a further step of adding a quenching reagent between step c) and step d) and / or between step d) and step e). The quenching reagent can be any reagent that reacts with the peptide reactive group (e.g., the active ester). For example, the quenching reagent may be an amine compound, such as ethanolamine.

[0253] It is possible, and preferably also possible, to add a quenching reagent before reacting the molecular probe with the linker B precursor in step e). According to one embodiment, the process includes a further step of adding a quenching reagent between step d) and step e). According to one embodiment, the process includes adding a quenching reagent (e.g., an amine compound such as ethanolamine) before step e).

[0254] Step e) In step e), the linker B precursor is reacted with the molecular probe to covalently attach the molecular probe to the peptide compound via the linker B. For the definitions of the molecular probe and the linker B, reference is made to the definitions, embodiments and preferred embodiments of the molecular probe and the linker B as defined herein for the molecular probe-functionalized element of the present invention.

[0255] The molecular probe can be activated before reacting it with the linker B precursor. The molecular probe can be activated by attaching a click reaction group suitable for reaction with the click reaction group of the linker B precursor onto the molecular probe. Those skilled in the art know the combinations of click reaction groups that react with each other in click reactions.

[0256] According to one embodiment, the molecular probe is activated prior to step e) by reacting the molecular probe with a linker B2 comprising a click reaction group selected from alkynes, strained cycloalkynes (e.g., cyclooctyne, where optionally one or more carbon atoms of the ring are substituted by heteroatoms such as N), strained cycloalkenes (e.g., trans-cyclooctene), azides, and tetrazines.

[0257] According to one embodiment, the molecular probe is activated prior to step e) by reacting the molecular probe with a linker B comprising a click reaction group selected from strained cycloalkynes (e.g., cyclooctyne, where optionally one or more carbon atoms of the ring are substituted by heteroatoms such as N) and azides. The molecular probe can be activated prior to step e) by reacting the molecular probe with NHS-azide or NHS-DBCO. NHS-azide and NHS-DBCO can react with the amines of the molecular probe, e.g., the lysine residues of the molecular probe.

[0258] The method according to the present invention In one aspect, the present invention provides a method for detecting a biomarker. The method comprises a) contacting a molecular probe-functionalized element according to the present invention with a sample suspected of containing a target analyte; b) detecting a biomarker based on an interaction between the molecular probe and the target analyte comprising.

[0259] The sample may be a body fluid, such as, but not limited to, intestinal fluid, ocular fluid, pulmonary fluid, blood or cerebrospinal fluid (CSF) sample. According to one embodiment, the sample is a blood or CSF sample. The sample can be contacted with the molecular probe-functionalized element in a flow chamber.

[0260] According to one embodiment, the target analyte can be selected from the group consisting of amyloid beta (Aβ) peptide, alpha-synuclein, tau protein, TDP-43, human islet amyloid polypeptide (hIAPP), prion protein and p53, and optionally, amyloid beta (Aβ) peptide and its isoforms, alpha-synuclein, tau protein and TDP-43. The amyloid beta (Aβ) peptide may be a monomeric, oligomeric or protofibrillar amyloid beta (Aβ) peptide.

[0261] The interaction between the molecular probe and the target analyte can be detected by any suitable means for detecting biochemical interactions. This interaction can be detected by spectroscopic methods, such as, but not limited to, infrared spectroscopy, UV / Vis spectroscopy, fluorescence spectroscopy, etc.

[0262] According to one embodiment, the interaction between the molecular probe and the target analyte is detected by infrared spectroscopy, preferably by ATR-IR spectroscopy.

[0263] According to one embodiment, the interaction between the molecular probe and the target analyte is detected by infrared spectroscopy, preferably by ATR-IR spectroscopy, and the biomarker is the maximum value of the amide band of the target analyte. The maximum value of the amide band may be the maximum value of the amide band of amyloid beta peptide, and optionally, it is in the range of 1600-1700 cm -1 , optionally 1620-1670 cm -1 , optionally 1640-1647 cm -1 .

[0264] Such methods are known in the art and are described, for example, in Nabers et al, Anal. Chem. 2016, 88, 2755-2762.

[0265] Use according to the present invention In one aspect, the present invention provides companion diagnostic tests, diagnosis of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors in a patient, monitoring the therapy of a patient having Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors, and screening of drugs for the treatment of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors for one or more of the above, the use of the molecular probe functionalized element according to the present invention or the device according to the present invention.

[0266] The tumor is not limited to a specific tumor and can be various types of tumors.

[0267] In one embodiment, the molecular probe functionalized element or device is used for companion diagnostic tests.

[0268] In one embodiment, the molecular probe functionalized element or device is used for the diagnosis of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors in a patient. In one embodiment, the molecular probe functionalized element or device is used for monitoring the therapy of a patient having Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors in a patient. In one embodiment, the molecular probe functionalized element or device is used for screening drugs for the treatment of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors in a patient.

[0269] According to one embodiment, the molecular probe functionalized element or device is used for the diagnosis of a patient's protein misfolding disease and / or for monitoring the therapy of a patient having a protein misfolding disease.

[0270] According to one embodiment, the molecular probe functionalized element or device is used for the diagnosis of Alzheimer's disease and / or for monitoring the therapy of a patient having Alzheimer's disease.

[0271] According to one embodiment, companion diagnostic test, diagnosis of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors in a patient, monitoring the therapy of a patient having Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors, and screening drugs for the treatment of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors There is provided a molecular probe functionalized element according to the present invention or a device according to the present invention for one or more of the uses of.

Brief Description of the Drawings

[0272]

Figure 1

Figure 2

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Figure 4

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Figure 6

Figure 7

Examples

[0273] The present invention will be further described below by specific examples. The examples are not intended to be construed as limiting the present invention in any way.

[0274] A. Molecular probe-functionalized element according to an embodiment of the invention and process for preparing the same Solid substrate element: silicon ATR crystal (internal reflection element), trapezoidal, incident angle 45°, 52 mm × 20 mm × 2 mm (tolerance: + / -0.1 mm), optically polished Linker A1: 1-Azido-N-(3-(triethoxysilyl)propyl)-3,6,9,12,15-pentaoxaoctadecane-18-amide Matrix precursor compound: 3-(2-(2-Methoxyethoxy)ethoxy)-N-(3-(triethoxysilyl)propyl)propanamide Linker A2: 2,5-Dioxopyrrolidin-1-yl 6-{2-azatricyclo[10.4.0.0 4,9 hexadeca-4,6,8,12,14,16-hexaen-10-in-2-yl}-6-oxohexanoate Peptide compound: Casein fragment blocking solution prepared from bovine casein (Sigma Aldrich / Art.-Nr.: C3400) by alkaline hydrolysis Linker B1: 2,5-Dioxopyrrolidin-1-yl 1-{2-azatricyclo[10.4.0.0 4,9 hexadeca-4,6,8,12,14,16-hexaen-10-in-2-yl}-1,4-dioxo-7,10,13,16,19,22,25,28,31,34,37,40,43-tridecaoxa-3-azatetracontane-46-oate Linker B2: 2,5-Dioxopyrrolidin-1-yl 1-azido-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-oate Molecular probe: Anti-β-amyloid (13-28) antibody, mouse monoclonal, clone BAM90.1, purified from hybridoma cell culture (Sigma Aldrich / Art.-Nr.: A8978) Device: ATR-IR-biosensor Target analyte: Amyloid beta (13-28)-BSA conjugate

[0275] Pretreatment of silicon crystal (process step a)) The surface was activated by oxidation treatment to generate a stable silicon dioxide layer.

[0276] Silanization of silicon crystal (process step b)) For the silane treatment of silicon crystals, a mixture of linker A1 and matrix precursor compound in 2-propanol was prepared and brought into contact with a selected surface of the silicon crystals. Subsequently, the crystals were rinsed with H2O and dried under nitrogen. After completion of the silane treatment, the ATR-FTIR difference spectrum (before and after silane treatment) showed an azide band at 2110 cm -1 indicating the azide band.

[0277] NHS activation of the azide moiety on the silanized surface (process step b)) The previously silanized crystal surface was reacted with linker A2 in a SPAAC click reaction. As understood by those skilled in the art, the coupling occurs by the reaction of the DBCO group and the azide group on the silanized surface. After the SPAAC click reaction, reactive NHS groups were obtained on the silanized silicon crystals, which can react with the peptide compounds in the casein fragment blocking solution.

[0278] After completion of the SPAAC click reaction, the ATR-IR difference spectrum (before silane treatment / after click reaction) showed three NHS bands at 1738 cm -1 , 1782 cm -1 and 1815 cm -1 . The azide band at 2110 cm -1 was no longer visible in the difference spectrum. The ATR-IR spectrum is shown in Figure 1.

[0279] Generation of azide-labeled antibody (preparation for process step e)) An anti-amyloid beta antibody was reacted with linker B2 to prepare an azide-labeled anti-amyloid beta antibody. The IR spectrum of the azide-labeled antibody is shown in Figure 2.

[0280] Generation of Aβ-13-28-BSA conjugate A 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution and hydroxy-2,5-dioxopyrrolidine-3-sulfonic acid were mixed with 1 ml of a BSA solution (10 mg / ml) (Sigma Aldrich, Art.No.: A9413) to a 14-fold molar excess for both substances and subsequently incubated at room temperature for 15 minutes. After changing the buffer, 0.5 ml of Aβ13-28 (2 mg / ml in PBS buffer) was added to the activated BSA. This was followed by incubation at room temperature for 2 hours and subsequently size exclusion chromatography was performed.

[0281] Surface blocking and immobilization of antibodies on the blocking layer (process steps c) - e)) After silanization and NHS activation by SPAAC click reaction (i.e., process step b)), protein-reactive silicon crystals were obtained. In subsequent surface functionalization, the NHS-functionalized crystal surface was first blocked with a casein fragment solution. A covalent bond was achieved by an amide bond between the lysine of the casein peptide or the primary amine group at the N-terminus of the casein peptide and the NHS functional group of the silicon crystal.

[0282] The NHS-functionalized crystal was placed on the crystal holder of an ATR flow-through cuvette with the top (long side) facing up. The lid of the cuvette with a silicone seal inserted was placed on the crystal. The cuvette was placed in the cuvette holder of the IR spectrometer. The sample compartment of the spectrometer was sealed and purged with dry air. The flow system containing the cuvette was flushed with water and then with PBS buffer. Subsequently, the casein fragment blocking solution was circulated over the crystal surface, followed by a washing step using PBS in flow-through mode.

[0283] During the blocking process, a complete IR spectrum was continuously recorded with reference to a PBS background. The blocking process was characterized by an increase in the absorbance of amide 2 (1550 cm-1) and simultaneously a decrease in the absorbance of NHS (1740 cm -1) was monitored by following its decrease over time (in time). Figure 3 shows the corresponding IR spectra and kinetics.

[0284] Next, the excess NHS groups were quenched with an ethanolamine solution, followed by additional washing steps.

[0285] Subsequently, the peptide blocking layer was DBCO-functionalized by reacting it with NHS- and DBCO-containing linker B1 in flow-through mode.

[0286] In the next step, the previously prepared azide-labeled anti-amyloid beta antibody was immobilized on the DBCO-functionalized peptide blocking layer by means of a SPAAC click reaction between the DBCO moiety and the azide group in flow-through mode. During antibody attachment, the complete IR spectrum was continuously recorded and compared to the background before antibody attachment. An indicator of successful immobilization of the antibody on the surface is the amide 2 signal (1550 cm -1 )

[0287] The functionalized surface prepared by this process corresponds to a molecular probe-functionalized element according to one embodiment of the present invention.

[0288] B. Stability tests Stability tests are exemplarily shown for two different peptide-blocking ATR crystal surfaces.

[0289] The first stability test was performed using a peptide-blocking ATR silicon crystal surface prepared by (1) preparing an NHS-activated surface by the process according to the present invention (steps a) and b) of the above invention example); and (2) blocking the NHS-activated surface with a casein fragment blocking solution (step c) of the above invention example). For the stability test, the peptide-blocking ATR crystal surface was not linked to a molecular probe.

[0290] The second stability test was performed using the method for NHS-silanization of germanium crystals described in the prior art (U.S. Patent Application Publication No. 20200240908), followed by blocking with the same casein fragment solution, using a peptide-blocked ATR crystal surface.

[0291] Subsequently, both surfaces were washed with PBS for 2 hours in a flow-through mode (washing). During both steps, continuous IR spectra were recorded. The results are shown in FIGS. 4 and 5, where the graph "NHS-activated azide-silane surface of the present invention" corresponds to the surface prepared according to the present invention, and the graph "Conventional NHS-silanization" corresponds to the surface of a comparative example prepared by the method of the prior art (U.S. Patent Application Publication No. 20200240908).

[0292] FIG. 4 shows a comparison of the kinetic profiles of the amide 2 band in both stability tests. Looking at the kinetic profile of the amide 2 band in the washing step, a clear difference can be observed between the examples and the comparative example prepared according to steps a) to c) of the present invention. The blocking layer on the NHS-activated surface according to steps a) to c) of the present invention remains completely stable during the entire 2-hour washing step. For the NHS-functionalized surface of the comparative example, during the washing step, a very strong disappearance of the blocking material can first be observed, and then the blocking material continues to disappear at a constant rate of about 4 μAU / min.

[0293] FIG. 5 further illustrates this fact. It shows the relative amount of blocking material on the surface over a 1-hour PBS wash. Within 1 hour, on the surface prepared by the prior art method, about 7% of the blocking layer disappears. In comparison, on the surface prepared according to the present invention, the blocking layer does not disappear at all or hardly at all during the entire time. Due to the fact that experiments performed on the blocked surface (e.g., further functionalization or ultimately detection of analytes with molecular probes) take at least 2 hours, the disappearance of 7% of the blocking material per hour had a very bad impact on the quality of the experiment.

[0294] The stability test shows that the molecular probe-functionalized element of the present invention provides a much more stable functionalized surface, which leads to, for example, an improvement in the quality of the detection of target analytes.

[0295] C. Inertness test As in a further exemplary embodiment, the above-described peptide-blocked ATR crystal surface for the stability test was subjected to an inertness test.

[0296] This inertness test was performed on the surface prepared as precisely as above for the stability test. Subsequently, 300 μl of human CSF (cerebrospinal fluid) was added to the blocked surface to observe possible non-specific protein adsorption (NSP adsorption) on the surface. For this purpose, CSF was circulated on the surface for 1 hour (circulation (circ)). Subsequently, the surface was washed with PBS for 2 hours in flow-through mode (washing). During both steps, the IR spectrum was continuously recorded.

[0297] The IR spectra are shown in FIG. 6. The spectrum "blocking layer immobilized on the NHS-activated azide-silane of the present invention" corresponds to the surface prepared by steps a) to c) of the present invention, and the spectrum "blocking layer immobilized on conventional NHS-silane functionalization" corresponds to the surface of a comparative example prepared by the method of the prior art (U.S. Patent Application Publication No. 20200240908).

[0298] FIG. 6 shows that the surface prepared by steps a) to c) of the present invention has significantly improved characteristics regarding non-specific protein adsorption from complex body fluids such as CSF. The degree of non-specific protein adsorption is reduced by half. Furthermore, the shape of the band and the position of the maximum of the amide I band indicate that the surface of the comparative example binds non-specifically to a significantly larger amount of beta-sheet structure proteins. In contrast, the surface prepared according to the present invention shows only slight non-specific binding of non-beta-sheet structure proteins.

[0299] The inactivity test shows that the molecular probe functionalized element of the present invention is much more inactive than the functionalized surface of the comparative example with respect to, for example, non-specific binding of proteins from body fluids. Consequently, this improves the detection of target analytes from complex samples when using the molecular probe functionalized element of the present invention.

[0300] D. Test of Activity of Molecular Probe - Activity of Antibody (i) Two antibody activity tests were exemplarily performed using the molecular probe functionalized element (Example of the present invention) according to an embodiment of the present invention prepared as described in Section A above, and (ii) using the molecular probe functionalized element of the comparative example (comparative example).

[0301] The molecular probe functionalized element of the comparative example was prepared using a conventional blocking technique in which an antibody was first attached to the NHS-activated surface, followed by surface blocking. The probe element of the comparative example was prepared as follows: (1) The same process steps a) and b) were performed to obtain an NHS-activated surface by SPAAC click reaction; (2) an anti-amyloid beta antibody was covalently bound onto the surface by NHS coupling of the antibody and the NHS-activated surface; (3) the remaining NHS groups on the NHS-activated surface were reacted with a casein fragment blocking solution.

[0302] The steps of antibody binding and surface blocking for the example of the present invention and the comparative example were analyzed by IR spectroscopy. In the comparative example, the anti-β-amyloid (13-28) antibody (A8978) was first covalently bound. In the next blocking step, a blocking peptide was bound to saturate free surface patches.

[0303] In the example of the present invention, the surface was first inactivated by stable blocking with a peptide compound. Subsequently, azide linker immobilization was performed. Subsequently, the linker-modified anti-β-amyloid (13-28) antibody was immobilized onto the surface in two steps.

[0304] The Aβ-13-28-BSA conjugate was circulated on the test surface, bound to the immobilized antibody, and subsequently a washing step was performed. The activity test shows that the molecular probe-functionalized element (the surface of the present invention) according to an embodiment of the present invention is significantly more active than the molecular probe-functionalized element of the comparative example (the surface of the comparative example). Figure 7 shows the IR spectrum recorded approximately 5 minutes after the start of the washing step. In the spectrum, an increase in activity for the example of the present invention can be clearly recognized. Furthermore, in the example of the present invention, it can be recognized that a significantly improved signal-to-noise ratio is achieved by the increase in signal intensity without a change in noise. The spectrum shows a maximum value of the amide 1 band at 1655 wavenumbers, as expected for the antigen being used.

Claims

1. A solid substrate element, a first layer containing linker A, a second layer containing a peptide compound, a third layer containing linker B, and a fourth layer containing a molecular probe A molecular probe-functionalized element comprising: The peptide compound is covalently bonded to the solid substrate element via linker A, and the molecular probe is covalently bonded to the peptide compound via linker B. A molecular probe-functionalized element.

2. The molecular probe-functionalized element according to claim 1, wherein the solid substrate element is an IR-transmissive optical element, preferably an attenuated total reflection infrared (ATR-IR) waveguide.

3. The molecular probe-functionalized element according to claim 1 or 2, wherein at least a part of the solid substrate element has an oxide surface layer, and the peptide is covalently bonded to the oxide surface layer via linker A.

4. The molecular probe-functionalized element according to any one of claims 1 to 3, wherein linker A contains a surface attachment group covalently bonded to the solid substrate element, and the surface attachment group is a silicon-containing group or a sulfur-containing group.

5. Linker A is a surface attachment group covalently bonded to the solid substrate element, a peptide attachment group covalently bonded to the peptide compound including, The surface attachment group is covalently bonded to the peptide attachment group via a linking moiety, and the linking moiety contains a -(OCH ２ CH ２ ) ｋ - group, where k is an integer from 1 to 100, preferably from 1 to 50, more preferably from 1 to 16. The molecular probe-functionalized element according to any one of claims 1 to 4.

6. Linker A is a surface attachment group covalently bonded to the solid substrate element, and a peptide attachment group covalently bonded to the peptide compound including, The surface attachment group is covalently bonded to the peptide attachment group via a linking moiety, The molecular probe-functionalized element according to any one of claims 1 to 5, wherein the linking moiety is a group obtainable by a bioorthogonal ligation reaction, preferably an N-heterocyclic group optionally substituted when selected from triazole, dihydropyridazine and pyridazine.

7. The first layer further contains a matrix compound covalently bonded to the solid substrate element, and the matrix compound is (i) a PEG-containing or mPEG-containing silane, and / or (ii) an optionally substituted alkyl sulfide The molecular probe-functionalized element according to any one of claims 1 to 6.

8. The peptide compound in the second layer is selected from the group consisting of peptides, substituted peptides, proteins, protein fragments and mixtures thereof, or The second layer contains a mixture of peptide compounds, and the mixture of peptide compounds can be obtained from a protein hydrolysis product or is a mixture of peptides obtained, and the peptides are optionally substituted, according to any one of claims 1 to 7. The molecular probe functionalized element described.

9. The linker B is a peptide attachment group covalently bonded to the peptide compound, a molecular probe attachment group covalently bonded to the molecular probe including the peptide attachment group is covalently bonded to the molecular probe attachment group by a linking moiety, the linking moiety is a group that can be obtained by a bioorthogonal ligation reaction, preferably including an optionally substituted N - heterocyclic group, and the N - heterocyclic group is selected from triazole (for example, 1,2,3 - triazole), dihydropyridazine and pyridazine, the molecular probe functionalized element according to any one of claims 1 to 8.

10. The molecular probe is an antigen, an antibody, an antibody fragment, an antibody fusion protein or a fusion protein with an antibody fragment, an antibody conjugate or a conjugate with an antibody fragment, a protein complex optionally containing an antibody fragment or a fusion protein thereof, an anticalin, a nanobody, an organic small molecule, a drug, a nucleic acid, an aptamer, a lipid, a carbohydrate, a peptide or a mixture thereof, according to any one of claims 1 to 9. The molecular probe functionalized element described.

11. The molecular probe can form a complex with a target analyte selected from the group consisting of amyloid beta (Aβ) peptide and its isoforms, alpha - synuclein, tau protein, TDP - 43, human islet amyloid polypeptide (hIAPP), prion protein and p53, according to any one of claims 1 to 10. The molecular probe functionalized element described.

12. An apparatus, preferably a biosensor, comprising the molecular probe functionalized element according to any one of claims 1 to 11.

13. A method for preparing the molecular probe functionalized element according to any one of claims 1 to 11, comprising a) preparing a solid substrate element; and b) preparing a linker A precursor covalently bonded to the surface of the solid substrate element; c) reacting the linker A precursor with a peptide compound to covalently bond the peptide compound to the solid substrate element via the linker A; d) preparing a linker B precursor covalently bonded to the peptide compound; e) reacting the linker B precursor with a molecular probe to covalently bond the molecular probe to the peptide compound via the linker B A method comprising the steps.

14. A method for detecting a biomarker, comprising: a) contacting the molecular probe-functionalized element according to any one of claims 1 to 11 with a sample suspected of containing a target analyte, optionally, the sample being a sample of intestinal fluid, ocular fluid, pulmonary fluid, blood or CSF; b) detecting the biomarker based on the interaction between the molecular probe and the target analyte, optionally, the interaction between the molecular probe and the target analyte being detected by infrared spectroscopy, and optionally, the biomarker being the maximum value of the amide band of the target analyte A method comprising the steps.

15. Companion diagnostic test; Diagnosis of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors in a patient; Monitoring the therapy of a patient having Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors; and Screening of drugs for the treatment of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, diabetes, Huntington's, prion disease or tumors Use of the molecular probe-functionalized element according to any one of claims 1 to 11 or the device according to claim 12 for one or more of the above.

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