Detection sensor

Abstract

The nanostructures can interact non-specifically with biomolecules, reducing the sensitivity of the assay and leading to false positive responses, or worse, the biomolecules can completely block the sensor surface, rendering the sensor ineffective. Thus, there remains a need for more sensitive sensors.SOLUTION: A sensor for determining the presence of at least one target molecule in a sample is disclosed. The sensor comprises a layer formed of high aspect ratio molecular structures (HARM-structures) and a blocking layer on the layer formed of HARM-structures, wherein the blocking layer is formed of a plurality of casein micelles, wherein the casein micelles comprise primary amines, wherein at least a part of the primary amines are functionalized with an amine-reactive cross-linker, and wherein cavities are formed between the plurality of casein micelles.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a sensor for determining the presence of at least one target molecule in a sample. The present disclosure also relates to a method for determining the presence of at least one target molecule in a sample. The present disclosure also relates to a process for preparing the sensor. The present disclosure also relates to a kit for determining the presence of at least one target molecule in a sample. [Background technology]

[0002] Biological sensors detect target biomolecules by using biological recognition elements, such as antibodies or aptamers, that interact with the target biomolecule. The interaction is detected and measured by various means, such as electrical, optical, or thermal signals. Modern sensors use nanostructures that offer large surface areas and enhanced electronic properties, facilitating rapid and accurate signal transduction.

[0003] Non-Patent Document 1 discloses vertically aligned arrays of single-walled carbon nanotubes on pyrolytic graphite surfaces with surfactant / casein blocking to minimize nonspecific binding for use as enzyme-linked assays (biosensors).

[0004] Non-Patent Document 2 discloses a label-free electrochemical aptasensor based on functionalized carbon nanotubes, in which an amino-terminated aptamer reacts with the carboxyl groups of carbon nanotubes.

[0005] Non-Patent Document 3 discloses inkjet-printed carbon nanotube forest arrays printed on indium tin oxide electrodes.

[0006] However, nanostructures can interact nonspecifically with biomolecules, reducing the sensitivity of the assay and resulting in false positives, or worse, the biomolecules can completely block the sensor surface, rendering the sensor ineffective. Thus, there remains a need for more sensitive sensors. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Yu et al.(2005)(DOI:10.1039 / b502124c) [Non-patent document 2] Venegas et al.(2023)(DOI:10.3390 / chemosensors11020117) [Non-patent document 3] Venkatanarayanan et al.(2011)(DOI:10.1016 / j.bios.2011.10.022) Summary of the Invention

[0008] A sensor for determining the presence of at least one target molecule in a sample is disclosed. The sensor comprises: i. a layer formed of a high aspect ratio molecular structure (HARM structure), wherein the layer formed of the HARM structure is configured to generate an electrical signal when in contact with a signal molecule indicative of the presence of at least one target molecule upon application of an electrical potential to the sensor, the magnitude of the electrical signal correlating with the concentration of the at least one target molecule in the sample; ii. A blocking layer formed from a plurality of casein micelles on the layer formed from the HARM structure; Equipped with the casein micelles comprise primary amines, at least a portion of which are functionalized with an amine-reactive crosslinker to provide at least one linking group and allow for binding thereto; Cavities are formed between multiple casein micelles, allowing signal molecules to diffuse through the blocking layer into the layer formed by the HARM structure and generate an electrical signal.

[0009] Further disclosed is a method for determining the presence of at least one target molecule in a sample, the method comprising: contacting a sensor disclosed herein having a biorecognition element attached to a linking group with a sample, thereby allowing binding of at least one target molecule to the sample; contacting the sensor with at least one detection probe capable of selectively binding to at least one target molecule, thereby allowing binding of the at least one detection probe to the at least one target molecule; subjecting the sensor to an enzyme-linked assay to obtain a signal molecule indicative of the presence of at least one target molecule, the signal molecule diffusing through the blocking layer to the layer formed by the HARM structure, thereby generating an electrical signal which is quantified by a means for measuring the electrical signal; determining a concentration of at least one target molecule in the sample based on the quantified electrical signal; Includes.

[0010] Further disclosed is a process for preparing a sensor for determining the presence of at least one target molecule in a sample, the process comprising: i. Providing a layer formed of a HARM structure; ii. providing a blocking layer formed from a plurality of casein micelles on the layer formed from the HARM structure, the casein micelles comprising primary amines; iii. functionalizing at least a portion of the primary amines with an amine-reactive crosslinker, thereby providing at least one linking group to which attachment is possible; Includes.

[0011] Further disclosed is a kit for determining the presence of at least one target molecule in a sample. a sensor as disclosed herein and a sensor as disclosed herein having at least one biorecognition element capable of selectively binding to at least one target molecule, or a biorecognition element attached to a linking group; at least one detection probe capable of selectively binding to at least one target molecule; Instructions for use and Equipped with. [Brief explanation of the drawings]

[0012] The accompanying drawings, which are included to provide a further understanding of the invention and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0013] [Figure 1] 1A-C show schematic diagrams of an exemplary embodiment of a sensor for determining the presence of at least one target molecule in a sample, according to an exemplary embodiment. [Figure 2] 2A-B show a schematic flow chart of a method for determining the presence of at least one target molecule in a sample, according to an exemplary embodiment. [Figure 3] 3A-B show a schematic flow chart of a process for preparing a sensor for determining the presence of at least one target molecule in a sample, according to an exemplary embodiment. [Figure 4] FIG. 4 shows the experimental results related to Example 1. [Figure 5] FIG. 5 shows the experimental results related to Example 1. [Figure 6] FIG. 6 shows the experimental results related to Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present disclosure relates to a sensor for determining the presence of at least one target molecule in a sample. i. a layer formed of a high aspect ratio molecular structure (HARM structure), wherein the layer formed of the HARM structure is configured to generate an electrical signal when in contact with a signal molecule indicative of the presence of at least one target molecule upon application of an electrical potential to the sensor, the magnitude of the electrical signal correlating with the concentration of the at least one target molecule in the sample; ii. A blocking layer formed from a plurality of casein micelles on the layer formed from the HARM structure; Equipped with the casein micelles comprise primary amines, at least a portion of which are functionalized with an amine-reactive crosslinker to provide at least one linking group and allow for binding thereto; Cavities are formed between multiple casein micelles, allowing signal molecules to diffuse through the blocking layer into the layer formed by the HARM structure and generate an electrical signal.

[0015] In such sensors, the layer formed by the HARM structure is blocked with multiple casein micelles to prevent nonspecific binding of biomolecules, such as DNA, RNA, proteins, enzymes, or other biomolecules, enabling accurate detection of target molecules. The blocking layer may contain multiple casein micelles. The casein micelles may be of various sizes and may form various interactions with other casein micelles or the layer formed by the HARM structure. The casein micelles should maintain their micellar morphology to prevent nonspecific binding and enable accurate detection of target molecules. Complete blocking of the layer formed by the HARM structure prevents the layer formed by the HARM structure from interacting with signal molecules. The blocking layer may also include cavities formed between the multiple casein micelles to allow signal molecules to diffuse through the blocking layer into the layer formed by the HARM structure. The casein micelles may be further functionalized with various click chemistry molecules that can bind to primary amines on the surface of the casein micelles. This allows for the covalent or non-covalent attachment of biorecognition elements to the casein micelles without compromising the ability of the blocking layer to prevent non-specific binding of target molecules. The sensor may be multiplexed, i.e., used to determine one or more target molecules in a sample.

[0016] The present disclosure further relates to a method for determining the presence of at least one target molecule in a sample, the method comprising: contacting a sensor disclosed herein having a biorecognition element attached to a linking group with a sample, thereby allowing binding of at least one target molecule to the sample; contacting the sensor with at least one detection probe capable of selectively binding to at least one target molecule, thereby allowing binding of the at least one detection probe to the at least one target molecule; subjecting the sensor to an enzyme-linked assay to obtain a signal molecule indicative of the presence of at least one target molecule, the signal molecule diffusing through the blocking layer to the layer formed by the HARM structure, thereby generating an electrical signal which is quantified by a means for measuring the electrical signal; determining a concentration of at least one target molecule in the sample based on the quantified electrical signal; Includes.

[0017] It is understood that washing with an appropriate buffer may be performed between the above steps to improve the sensitivity of the determination.

[0018] In such methods, the amount of at least one target molecule may be quantified by quantifying the amount of a signal molecule. This method may use a sensor with a biorecognition element already bound to it. Optionally, the sensor and the at least one biorecognition element are provided separately, and the sensor should be contacted with the biorecognition element before proceeding with the method. The described methods may be specific to at least one target molecule and provide a quantitative measure of the concentration of the at least one target molecule. When the signal molecule contacts the layer formed of the HARM structure, an electrical signal is generated, which can be quantified by measuring the electrical signal. The amount of the at least one target molecule may be related to the quantitative measurement of the electrical signal (e.g., the amount of the signal molecule). Because the sensor comprises a layer formed of a HARM structure and a blocking layer of multiple casein micelles, quantification is sensitive, and the blocking layer of multiple casein micelles may prevent nonspecific binding of molecules in the sample, the at least one target molecule, the at least one biorecognition element, the at least one detection probe, or any component of an enzyme-linked assay to the HARM structure.

[0019] The present disclosure further relates to a process for preparing a sensor for determining the presence of at least one target molecule in a sample, the process comprising: i. Providing a layer formed of a HARM structure; ii. providing a blocking layer formed from a plurality of casein micelles on the layer formed from the HARM structure, the casein micelles comprising primary amines; iii. functionalizing at least a portion of the primary amines with an amine-reactive crosslinker, thereby providing at least one linking group to which attachment is possible; Includes.

[0020] Such a process may be used to provide or prepare highly sensitive sensors comprising a layer formed of a HARM structure and a functionalized blocking layer of multiple casein micelles. The resulting sensors may be multiplexed.

[0021] The present disclosure further relates to a kit for determining the presence of at least one target molecule in a sample. The kit comprises: a sensor as disclosed herein and a sensor as disclosed herein having at least one biorecognition element capable of selectively binding to at least one target molecule, or a biorecognition element attached to a linking group; at least one detection probe capable of selectively binding to at least one target molecule; Instructions for use and Equipped with.

[0022] Such a kit may be used to quantify at least one target molecule from a sample, or the kit may be used to determine the presence of at least one target molecule in a sample.

[0023] The sensor includes a layer formed of a HARM structure. The layer formed of a HARM structure may be a conductive layer of a HARM structure. In the context of this specification, the expression "HARM structure", unless otherwise specified, should be understood herein as referring to a "nanostructure", i.e., a structure having one or more characteristic dimensions on the nanometer scale, i.e., a structure of about 100 nanometers or less. "High aspect ratio" refers to the dimensions of the conductive structure in two perpendicular directions being significantly different in order of magnitude. For example, a nanostructure may have a length tens, hundreds, or even thousands of times greater than its thickness and / or width. In a layer formed of a HARM structure, a large number of said nanostructures are interconnected with each other to form a network of interconnected molecules. Considered on a macroscopic scale, a HARMS network forms a solid monolithic material in which the individual molecular structures are unoriented or non-oriented, i.e., substantially randomly oriented or oriented.

[0024] In one embodiment, the HARM structure is a conductive HARM structure. In one embodiment, the HARM structure is a carbon nanostructure. In one embodiment, the HARM structure comprises carbon nanotubes (CNTs), carbon nanobuds (CNBs), carbon nanoribbons, or any combination thereof. In one embodiment, the HARM structure comprises carbon nanotubes and / or carbon nanobuds. In one embodiment, the HARM structure comprises carbon nanotubes. Carbon nanobuds, sometimes referred to as carbon nanobud molecules, have fullerene or fullerene-like molecules covalently bonded to the side of a tubular carbon molecule.

[0025] In one embodiment, the HARM structure comprises or is a nanostructure made from carbon, gold, silver, graphene, or a conducting polymer, hi one embodiment, the HARM structure comprises or is a metal nanowire, such as a silver nanowire and / or a gold nanowire.

[0026] In one embodiment, the layers formed of the HARM structure are attached to a support. The support may be any type of support suitable for attaching the layers of the HARM structure. The support may be formed from a polymer, a metal, silicon, glass, a ceramic material, or any combination thereof. In one embodiment, the layers formed of the HARM structure are deposited on a substrate. The substrate may act as the support.

[0027] The form of the support may vary. The support may have the form of a frame. In one embodiment, the support has the form of a frame, and the layer formed of the HARM structure is a free-standing layer formed of the HARM structure attached to the frame. The frame may support the free-standing layer formed of the HARM structure at its outer edges, so as to form unsupported, free-standing regions of the free-standing layer formed of the HARM structure. The support locations may be located anywhere within the structure, as long as they provide sufficient support for the free-standing layer formed of the HARM structure. For example, they may be on the sides of the free-standing layer formed of the HARM structure, in areas near corners, or adjacent to each other along the sides. Any larger area including multiple support points is also intended to be encompassed by this aspect, for example, if the frame has a continuous circular shape and the free-standing regions are within the circle. The frame may also have any other elongated, continuous shape. In one embodiment, the frame is shaped as a circle, square, triangle, rectangle, oval, or polygon. The support may be a solid support on which the layer formed of the HARM structure is provided. The support may be a mesh on which a layer formed of the HARM structure is provided. The HARM structure may be transferred to a pre-fabricated mesh, or the mesh may be formed after the layer formed of the HARM structure is formed. A potential or current may be applied to the sensor. The potential may be negative. The potential enables the layer formed of the HARM structure to generate an electrical signal in the presence of a signal molecule. The electrical signal may be measured by various means. The electrical signal may be measured by a means selected from the group consisting of amperometry, potentiometry, conductometry, voltammetry, coulometry, impedance measurement, or chronoamperometry. Since the HARM structure is electrically conductive or can be electrically conductive to drive an electrochemical reaction, when an electrochemical substance is in contact with the layer formed of the HARM structure, a transfer of electrons occurs, which may cause a change in the current, electrical signal, electrical potential, and / or impedance of the sensor. The sensor coupled with a means for measuring the electrical signal may be used to quantify the concentration of the signal molecule.Several means may be used to measure the electrical signal, such as amperometry or voltammetry. When the sensor is used to determine the presence of at least one target molecule in a sample, the concentration of the signal molecule measured by the means for measuring current may be correlated with the concentration of the at least one target molecule in the sample.

[0028] In the context of this specification, the term "sensor" may refer to a device or component that detects, measures, or records biological substances, such as molecules. The sensor may be an electrode or a biosensor. The sensor may include a layer formed of a HARM structure and a blocking layer. The sensor may be multiplexed.

[0029] In one embodiment, the sensor is configured to determine the presence of at least one target molecule in the sample. In one embodiment, the sensor is configured to determine the presence of one target molecule in the sample. In one embodiment, the sensor is configured to determine the presence of multiple target molecules in the sample. In one embodiment, the sensor is a biosensor. In one embodiment, the sensor is an electrode.

[0030] In one embodiment, at least one target molecule comprises or is a single-stranded DNA molecule, a single-stranded RNA molecule, a double-stranded DNA molecule, a double-stranded RNA molecule, a protein, a peptide, a lipid, a hormone, a pathogen, or a pharmaceutical compound. In one embodiment, at least one target molecule is selected from the group consisting of a single-stranded DNA molecule, a single-stranded RNA molecule, a double-stranded DNA molecule, a double-stranded RNA molecule, a protein, a peptide, a lipid, a hormone, a pathogen, and a pharmaceutical compound. In one embodiment, multiple target molecules are independently selected from the group consisting of a single-stranded DNA molecule, a single-stranded RNA molecule, a double-stranded DNA molecule, a double-stranded RNA molecule, a protein, a peptide, a lipid, a hormone, a pathogen, and a pharmaceutical compound.

[0031] In the context of this specification, a "target molecule" may refer to, for example, a biomolecule that is indicative of health, a biomolecule that is indicative of a disease or condition, a biomolecule that is indicative of infection (i.e., a pathogen or toxin), or a biomolecule that is in any other way useful for quantification. A target molecule may be, for example, a single-stranded DNA molecule, a single-stranded RNA molecule, a double-stranded DNA molecule, a double-stranded RNA molecule, a protein, a peptide, an enzyme, a metabolite, a lipid, a carbohydrate, a hormone, a pathogen, a toxin, or a pharmaceutical compound.

[0032] In the context of this specification, a "signal molecule" may refer to a molecule that undergoes an oxidation or reduction reaction, thereby being in an oxidized or reduced state and facilitating the transfer of electrons when in contact with a layer formed by a HARM structure. A signal molecule may be generated when an enzyme catalyzes its substrate. A signal molecule may be generated by an enzyme. A signal molecule may be catalyzed by an enzyme. For example, a signal molecule may be a benzidine oxide such as 3,3',5,5'-tetramethylbenzidine or 3,3'-diaminobenzidine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), o-phenylenediamine dihydrochloride, p-nitrophenyl phosphate, 5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium, ferrocyanide, ferrocyanide, hydrogen peroxide, luminol, or Amplifu Red. There are several options for the signal molecule.

[0033] In the context of this specification, the phrase "multiple casein micelles" or "casein micelles" may refer to the colloidal spherical aggregates of casein proteins, calcium phosphate, and water typically found in bovine milk. Casein proteins are a family of phosphoproteins, including caseins αS1, αS2, β, and κ. Casein proteins can form globular protein and calcium phosphate complexes (micelles) composed of various ratios of casein proteins. Casein proteins may form linear and branched chains through various protein-protein and protein-calcium phosphate interactions. The outermost layer of a casein micelle may be rich in κ-casein, which is relatively more hydrophilic, while the inner complex may contain more α-casein and β-casein, which are relatively more hydrophobic. Overall, the casein micelle may contain a variety of casein protein compositions. Casein micelles can be extracted from bovine milk in various sizes, and milk or casein extracts may be filtered to obtain specific casein micelle sizes. To be able to form casein micelles, the casein proteins must be in their native state (e.g., functional state in which the proteins maintain their native three-dimensional structure). Denaturation of casein proteins can alter the structure of the casein micelles or the soil-repellent properties of the casein micelles. By "casein micelle" or "casein micelles," it is to be understood that the casein micelles have a quaternary structure comprising casein proteins, calcium phosphate, and water. If the casein proteins are denatured or cross-linked, or if the casein micelles are in contact with an emulsifier or surfactant, the quaternary structure may not be fully functional. The casein micelles may also be non-conductive.

[0034] The expression that a blocking layer is "on" a layer formed of a HARM structure should be understood in the context of this specification to mean that a blocking layer formed of a plurality of casein micelles is attached to the surface of the layer formed of a HARM structure, unless otherwise specified.

[0035] In the context of this specification, the expression "layer" may refer to a structure whose lateral dimensions are substantially greater than its thickness. In this sense, a layer may be considered to be a "thin" structure. A layer as a structure may be porous, channeled, permeable, uneven, non-uniform, or may be formed in a disordered manner from multiple subunits, such as casein micelles. A layer as a structure may also be flat, impermeable, solid, or sealed.

[0036] In the context of this specification, the expression "blocking layer" may refer to a layer that serves to prevent non-specific binding of molecules. A blocking layer may reduce current leakage. A blocking layer may improve the sensitivity of a sensor. A blocking layer may reduce the available reaction surface area or reduce the electrochemical rate, leading to a decrease in the amplitude of the unblocked current on the sensor compared to an unblocked sensor. A blocking layer may also improve the sensitivity of a sensor. A blocking layer may be formed of a plurality of casein micelles. A blocking layer may be non-conductive.

[0037] In the context of this specification, the expression "electrically conductive" may refer to the ability of a material or component to allow an electric current or electrical signal to flow with minimal resistance.

[0038] In the context of this specification, the expression "non-conductive" may refer to the property of a material or component to resist the flow of electric current or electrical signals, effectively acting as an insulator.

[0039] In the context of this specification, the term "anti-fouling" may refer to the property of a material to resist binding to its surface. For example, casein micelles or their blocking layer may be anti-fouling not only to other proteins, enzymes, nucleic acids, but also to biomolecules, including but not limited to biotin.

[0040] When used as a blocking layer on a layer formed by a HARM structure, casein micelles interact primarily with either the HARM structure, or casein micelles, calcium phosphate, or water, but not with other molecules. That is, casein micelles are antifouling supramolecules for proteins, enzymes, and nucleic acids, but also for other biomolecules, including but not limited to biotin. Casein micelles may be tightly bound to the HARM structure. Casein micelles may also be bound to the HARM structure by other covalent or non-covalent means. Casein micelles may sterically block other molecules from reaching the layer formed by the HARM structure.

[0041] Furthermore, the surface proteins of casein micelles may contain multiple functional groups, including primary amines. Some of these functional groups may be required for interaction with other casein proteins, for micelle aggregation, for interaction with the layer formed by the HARM structure, or for the stability of the blocking layer. The primary amines can be functionalized with click chemistry compounds, such as amine-reactive crosslinkers, without impairing the ability of the casein micelles to bind to other casein micelles or the layer formed by the HARM structure or affecting the antifouling properties of the blocking layer. In one embodiment, the casein micelles are nonconductive. In one embodiment, the blocking layer is nonconductive. In one embodiment, the blocking layer does not contribute to the electrochemical reaction occurring on the sensor surface. In one embodiment, the blocking layer is nonconductive and does not contribute to the electrochemical reaction occurring on the sensor surface.

[0042] When multiple casein micelles are bound onto a layer formed from a HARM structure, they form cavities. The cavities allow signal molecules to diffuse through the blocking layer into the layer formed from the HARM structure to generate an electrical signal or current, while preventing non-specific binding of other molecules to the layer formed from the HARM structure. The binding of the HARM structure to the casein micelles and the formation of the porous blocking layer or its cavities may not require chemical or physical treatment of the micelles. This may allow the surface of the layer formed from the HARM structure to be blocked with casein micelles to prevent non-specific binding of molecules, and further provides the additional ability to have diffusion cavities, as well as the possibility of functionalizing the casein micelles with, for example, click chemistry compounds.

[0043] In the context of this specification, the expression "cavity" may refer to a distinct path through which particles or molecules may pass through the blocking layer formed from casein micelles. Casein micelles may form cavities when they interact with other casein micelles or with the layer formed from the HARM structure. The cavities may be through-holes. The cavities may be channels. The cavities may form a direct path from the HARM structure to the layer formed, or may be uneven or tortuous, creating a more complex passageway that may follow a non-linear path from the HARM structure to the layer formed. It should be understood that to form cavities, at least some of the casein micelles may be bonded in a way that allows their formation. However, not all casein micelles in the blocking layer may be involved in the formation of cavities.

[0044] In the context of this specification, the term "click chemistry" may refer to the use of chemical compounds to link two molecular entities together. For example, in the case of a protein, a specific functional group in an amino acid side chain may be contacted with a compound capable of covalently binding to the specific functional group. The functional group present in the compound then binds to the protein, facilitating the further linking of another molecule. The compound may be, for example, an amine-reactive crosslinker that binds to a primary amine in a lysine residue or the N-terminal amine of a protein and introduces an additional functional group (linking group). Other molecules with appropriate functional groups may be bound to the linking group and thereby covalently bound to the protein.

[0045] In the context of this specification, the expression "amine-reactive crosslinker" may refer to a compound that can covalently bind to a primary amine and introduce a linking group such as an azide, carbohydrate, carboxyl, phosphine, or sulfhydryl group. Amine-reactive crosslinkers may have additional chemical properties, such as being cleavable or non-cleavable by a cleavage enzyme, or being soluble or insoluble in water. The choice of which click chemistry to use may be context-dependent (i.e., some target molecules are best captured by specific click chemistries), as known to those skilled in the art.

[0046] In the context of this specification, the expression "linking group" may refer to a functional group provided by a cross-linking molecule, such as an amine-reactive cross-linker. The linking group may be, for example, a thiol group, an amine group, a maleimide group, an azide group, or an epoxy group.

[0047] In the context of this specification, the term "functionalized" may refer to a chemical reaction in which a molecule, such as DNA, RNA, or protein, is treated with a click chemistry compound for crosslinking, such as to provide the molecule with a specific functional group. The functional group may be introduced, for example, by a maleimide linker, an azide linker, an alkyne linker, or a thiol linker, or by a click chemistry-specific crosslinker or reagent. In the context of this specification, functionalization is compatible with users of amine-reactive crosslinkers, and thus can provide for attachment to linking groups as part of the same click chemistry.

[0048] In the context of this specification, the term "biorecognition element" may refer to a biorecognition element that is compatible with an amine-reactive crosslinker. The biorecognition element may be chemically or biologically modified (i.e., functionalized) to be compatible with an amine-reactive crosslinker. The biorecognition element may contain a functional group that is compatible with an amine-reactive crosslinker without chemical or biological modification. For example, if the amine-reactive crosslinker is an amine-thiol crosslinker, the biorecognition element may be thiolated DNA. The biorecognition element may also be a functionalized biorecognition element. The biorecognition element may be, for example, a functionalized oligonucleotide probe, a functionalized nucleotide probe, a functionalized DNA probe, a functionalized RNA probe, an antibody, a functionalized antibody, a bispecific antibody, a functionalized bispecific antibody, an antibody fusion protein, a functionalized antibody fusion protein, an antibody fragment thereof, a functionalized antibody fragment thereof, or a functionalized molecularly imprinted polymer.

[0049] In the context of this specification, the expression "probe" may refer to a single- or double-stranded nucleotide or oligonucleotide sequence that is specific for or has high affinity to a target nucleotide sequence. A probe may be an aptamer.

[0050] In the context of this specification, the expression "antibody" may refer to a protein that specifically binds to a target protein or a protein that has high affinity for a target protein. An antibody may be a monoclonal antibody, a polyclonal antibody, or a humanized antibody. In the context of antibodies, the present disclosure may refer to a single-chain variable fragment or antibody fragment thereof, which refers to the specific binding region of the antibody. The specific binding region of the antibody may, for example, be conjugated to another protein.

[0051] In one embodiment, the amine-reactive crosslinker has a molecular length of 1-20 Å, or 1-50 Å, or 1-100 Å, or 1-10,000 Å, or 2-20,000 Å. In one embodiment, the amine-reactive crosslinker is selected from the group consisting of an amine-amine crosslinker, an amine-sulfhydryl crosslinker, an amine-phosphine crosslinker, an amine-alkyne crosslinker, an amine-azide crosslinker, an amine-streptavidin crosslinker, an amine-biotin crosslinker, or an amine-carboxyl crosslinker. In one embodiment, the amine-reactive crosslinker is an amine-sulfhydryl crosslinker. In one embodiment, the amine-reactive crosslinker is N-γ-maleimidobutyryl-oxysuccinimide ester.

[0052] Casein micelles can be effective in preventing the binding of nucleotides (DNA / RNA) to layers formed by HARM structures. For example, DNA has a slight negative charge and spontaneously binds nonspecifically to HARM structures, preventing the use of sensors due to high background currents, regardless of the presence or absence of target molecules. While casein micelles can prevent nonspecific binding of nucleotides, they still allow molecules smaller than the cavity width (such as signal molecules) to diffuse to the surface of the layer formed by HARM structures. Casein micelles can be particularly useful in preventing the nonspecific binding of DNA and RNA to layers formed by HARM structures. Furthermore, a blocking layer formed from multiple casein micelles can also prevent the nonspecific binding of proteins, enzymes, peptides, and other biomolecules to layers formed by HARM structures.

[0053] Casein micelles of a particular size may be suitable for blocking nanoporous HARM structures with a particular pore size. It should be understood that the size of casein micelles that provides the best performance depends on the pore diameter of the HARM structure. Typically, nanomaterials have pore diameters between 1 and 200 nm. For example, a nanoporous HARM structure with a pore diameter of 30 nm can be usefully blocked with casein micelles with diameters ranging from 0.5 to 30 nm. Blocking a layer formed with a HARM structure with a pore diameter of approximately 30 nm with casein micelles with diameters ranging from 0.5 to 30 nm, or 2 to 10 nm, may result in a sensor with higher sensitivity than using casein micelles with diameters ranging up to 450 nm. A more sensitive sensor may enable detection of target molecules even at very low concentrations, which may enable, for example, detection of DNA from patient samples without the need for DNA concentration or amplification procedures. In one embodiment, the diameter of the casein micelles is selected based on the pore diameter of the layer formed with the HARM structure. In one embodiment, the pore size of the layer formed from the HARM structure is in the range of 1 to 200 nm, and the casein micelles have diameters in the range of 0.5 to 200 nm, or 0.5 to 100 nm, or 0.5 to 30 nm, or 0.7 to 20 nm, or 1 to 15 nm, or 2 to 10 nm. In one embodiment, the pore size of the layer formed from the HARM structure is 30 nm, and the casein micelles have diameters in the range of 0.5 to 30 nm, or 0.7 to 20 nm, or 1 to 15 nm, or 2 to 10 nm.

[0054] It should be understood that while casein micelles can be filtered to a particular size, some casein micelles may not be at that size because filtration is usually not perfect and has an inherent error rate with any filtration method. Thus, when a size range is discussed, it should be understood that not all casein micelles will fall within the size range, but the majority will. While a blocking layer may not rely on every casein micelle being of a particle size, the blocking layer will be more effective if most of the casein micelles are within a preferred size range. It should be understood that a proportion of casein micelles larger or smaller than the preferred size range may not interfere with sensor function, but may reduce the sensitivity of the sensor.

[0055] In one embodiment, the casein micelles have a diameter in the range of 0.5 to 450 nm, or 0.5 to 200 nm, or 0.5 nm to 100 nm, or 0.5 to 30 nm, or 0.7 to 20 nm, or 1 to 15 nm, or 2 to 10 nm. In one embodiment, the casein micelles have a diameter in the range of 0.5 to 30 nm, or 0.7 to 20 nm, or 1 to 15 nm, or 2 to 10 nm. In one embodiment, the casein micelles have a diameter in the range of 0.5 to 30 nm, or 0.7 to 20 nm, or 1 to 25 nm, or 1 to 15 nm, or 1 to 10 nm, or 2 to 30 nm, or 2 to 20 nm, or 2 to 10 nm. In one embodiment, at least 1%, or at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or essentially all, of the casein micelles have a diameter in the range of 0.5-30 nm, or 0.7-20 nm, or 1-15 nm, or 2-10 nm. In one embodiment, at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or essentially all, of the casein micelles have a diameter in the range of 0.5-30 nm, or 0.7-20 nm, or 1-15 nm, or 2-10 nm. In one embodiment, at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or essentially all, of the casein micelles have a diameter in the range of 0.5 to 30 nm, or 0.7 to 20 nm, or 1 to 25 nm, or 1 to 15 nm, or 1 to 10 nm, or 2 to 30 nm, or 2 to 20 nm, or 2 to 10 nm.In one embodiment, at least 1%, or at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or essentially all, of the casein micelles have a diameter in the range of 0.5 to 30 nm, or 0.7 to 20 nm, or 1 to 25 nm, or 1 to 15 nm, or 1 to 10 nm, or 2 to 30 nm, or 2 to 20 nm, or 2 to 10 nm.

[0056] In the context of this specification, the terms "uniform" or "essentially uniform" may refer to objects of the same size, or to objects in which a majority of the objects are the same size. Objects of the same size may be provided by filtration. The filtration may be incomplete. Conversely, the term "heterogeneous" may refer to each object having an individual size that may differ from other objects.

[0057] The use of uniformly or non-uniformly sized casein micelles can affect the structure of the blocking layer. Using uniformly sized casein micelles with a diameter of less than 2 nm on a HARM structure layer can prevent the formation of cavities for signal molecules. On the other hand, the use of non-uniformly sized casein micelles may allow the use of very small casein micelles, even those with diameters less than 2 nm, such as in the 0.5-30 nm range, while still leaving diffusion cavities for signal molecules. In one embodiment, the casein micelles are essentially uniformly sized or non-uniformly sized. In one embodiment, the casein micelles are uniformly sized, having diameters in the range of 2-30 nm, 2-20 nm, or 2-10 nm. In one embodiment, the casein micelles are non-uniformly sized, having diameters in the range of 0.5-30 nm, 0.7-20 nm, 1-25 nm, 1-15 nm, 1-10 nm, or 2-10 nm.

[0058] The sensor may include a blocking layer formed from a plurality of casein micelles, which may contain multiple functional groups, including primary amines. When functionalized with an amine-reactive crosslinker, a linking group is provided at the primary amine site, allowing binding thereto. The sensor may further include at least one biorecognition element functionalized with a linker molecule or by chemical reaction, for example, using a specific enzyme, whereby the functionalization is compatible with the linking group. This should be understood, for example, if an amine-sulfhydryl crosslinker is used to provide the linking group, the functionalization should provide a free thiol group, for example, for alternative click chemistry. When the sensor containing the linking group contacts the at least one biorecognition element, a bond is formed between the linking group and the at least one biorecognition element, thereby binding the at least one biorecognition element to the casein micelles and bound by the sensor. The biorecognition element may be designed to be capable of selectively or specifically binding to at least one target molecule. The biorecognition element may include a functional group compatible with the linker molecule without chemical modification. When the sensor is contacted with at least one such biorecognition element, the sensor may bind to the linking group.

[0059] In the context of this specification, the phrases "selective binding" or "selectively bind" may refer to the ability of a probe or biorecognition element to bind to or with high affinity to a group of similar or otherwise related targets, such as proteins of the same protein family or different variants of the same gene or several RNA transcripts of the same gene.

[0060] In the context of this specification, the phrases "specific binding" or "specifically binds" may refer to the ability of a probe or biorecognition element to specifically bind to a single target, such as a single protein in the presence of members of the same protein family, or a single target, such as a single gene variant in the presence of other variants.

[0061] The biorecognition element may be any biorecognition element suitable for the target molecule. When the target molecule is single-stranded DNA, the biorecognition element may be a functionalized DNA oligonucleotide or aptamer, such as a thiolated DNA oligonucleotide or aptamer. When the target molecule is a protein, the biorecognition element may be a functionalized antibody, such as a biotinylated antibody or aptamer. In one embodiment, the biorecognition element is selected from the group consisting of a functionalized oligonucleotide probe, a functionalized nucleotide probe, a functionalized DNA probe, a functionalized RNA probe, an antibody, a functionalized antibody, a bispecific antibody, a functionalized bispecific antibody, an antibody fusion protein, a functionalized antibody fusion protein, an antibody fragment thereof, a functionalized antibody fragment thereof, or a functionalized molecularly imprinted polymer. In one embodiment, the biorecognition element is selected from the group consisting of an oligonucleotide probe, a nucleotide probe, a DNA probe, an RNA probe, a functionalized oligonucleotide probe, a functionalized nucleotide probe, a functionalized DNA probe, a functionalized RNA probe, an antibody, a functionalized antibody, a bispecific antibody, a functionalized bispecific antibody, an antibody fusion protein, a functionalized antibody fusion protein, an antibody fragment thereof, a functionalized antibody fragment thereof, a molecularly imprinted polymer, and a functionalized molecularly imprinted polymer.

[0062] In one embodiment, the sensor is a multiplexed sensor comprising a plurality of biorecognition elements independently selected from the group consisting of oligonucleotide probes, nucleotide probes, DNA probes, RNA probes, functionalized oligonucleotide probes, functionalized nucleotide probes, functionalized DNA probes, functionalized RNA probes, antibodies, functionalized antibodies, bispecific antibodies, functionalized bispecific antibodies, antibody fusion proteins, functionalized antibody fusion proteins, antibody fragments thereof, functionalized antibody fragments thereof, molecularly imprinted polymers, and functionalized molecularly imprinted polymers.

[0063] In one embodiment, the sensor further comprises at least one biorecognition element attached to at least one linking group capable of selectively binding at least one target molecule, hi one embodiment, the sensor further comprises a plurality of functionalized biorecognition elements attached to a plurality of linking groups capable of selectively binding a plurality of target molecules.

[0064] The disclosed method for determining the presence of at least one target molecule in a sample may include contacting a sensor with the sample, thereby allowing binding of the at least one target molecule to the sample. The sample may be in liquid form. The sample may or may not be processed prior to contacting the sensor. Processing may include, for example, adjusting pH, adjusting temperature, fractionation, concentrating the at least one target molecule, amplifying the at least one target molecule, homogenizing, filtering, concentrating, storing, or other common sample processing steps. Processing steps depend on the type of sample and the at least one target molecule and are known to those skilled in the art.

[0065] The sample may be a bodily fluid such as whole blood, serum, plasma, urine, saliva, cerebrospinal fluid, lymph, interstitial fluid, mucus, synovial fluid, sweat, semen, breast milk, or a combination thereof. The sample may be, for example, reconstituted dried blood. The sample may be obtained from a subject as part of normal clinical practice. The sample may be provided by a subject as part of normal clinical practice. The sample may be obtained from a subject prior to determining the presence of at least one target molecule in the sample. The sample may be obtained from a subject which may be a mammal, such as a dog, cat, rat, primate, or human. The sample may be obtained from a human subject.

[0066] In one embodiment, the sample is a bodily fluid. In one embodiment, the sample is whole blood, serum, plasma, urine, saliva, cerebrospinal fluid, lymph, interstitial fluid, mucus, synovial fluid, sweat, semen, breast milk, or a combination thereof. In one embodiment, the sample is obtained from a mammal. In one embodiment, the sample is obtained from a human. In one embodiment, the sample is a bodily fluid obtained from a mammal. In one embodiment, the sample is a bodily fluid obtained from a human. In one embodiment, the sample is whole blood, serum, plasma, urine, saliva, cerebrospinal fluid, lymph, interstitial fluid, mucus, synovial fluid, sweat, semen, breast milk, or a combination thereof obtained from a human.

[0067] A method for determining the presence of at least one target molecule in a sample may include contacting a sensor with at least one detection probe. The at least one detection probe can selectively or specifically bind to the at least one target molecule. Once the at least one target molecule is bound, it may bind to the sensor. The at least one detection probe may be functionalized, for example, with an additional functional group or other modification. The at least one detection probe may be any detection probe suitable for the target molecule. In one embodiment, the at least one detection probe is selected from the group consisting of an oligonucleotide probe, a nucleotide probe, a DNA probe, an RNA probe, a functionalized oligonucleotide probe, a functionalized nucleotide probe, a functionalized DNA probe, a functionalized RNA probe, an antibody, a bispecific antibody, an antibody fusion protein, an antibody fragment thereof, and a molecularly imprinted polymer. In one embodiment, the at least one biorecognition element and the at least one detection probe are independently selected from the group consisting of oligonucleotide probes, nucleotide probes, DNA probes, RNA probes, functionalized oligonucleotide probes, functionalized nucleotide probes, functionalized DNA probes, functionalized RNA probes, antibodies, functionalized antibodies, bispecific antibodies, functionalized bispecific antibodies, antibody fusion proteins, functionalized antibody fusion proteins, antibody fragments thereof, functionalized antibody fragments thereof, molecularly imprinted polymers, and functionalized molecularly imprinted polymers. In one embodiment, the plurality of detection probes are independently selected from the group consisting of oligonucleotide probes, nucleotide probes, DNA probes, RNA probes, functionalized oligonucleotide probes, functionalized nucleotide probes, functionalized DNA probes, functionalized RNA probes, antibodies, functionalized antibodies, bispecific antibodies, functionalized bispecific antibodies, antibody fusion proteins, functionalized antibody fusion proteins, antibody fragments thereof, functionalized antibody fragments thereof, molecularly imprinted polymers, and functionalized molecularly imprinted polymers.In one embodiment, the plurality of biorecognition elements and the plurality of detection probes are independently selected from the group selected from the group consisting of oligonucleotide probes, nucleotide probes, DNA probes, RNA probes, functionalized oligonucleotide probes, functionalized nucleotide probes, functionalized DNA probes, functionalized RNA probes, antibodies, functionalized antibodies, bispecific antibodies, functionalized bispecific antibodies, antibody fusion proteins, functionalized antibody fusion proteins, antibody fragments thereof, functionalized antibody fragments thereof, molecularly imprinted polymers, and functionalized molecularly imprinted polymers.

[0068] In the context of this specification, the term "enzyme-linked assay" may refer to a process in which a sensor is contacted with an enzyme and, optionally, an enzyme substrate. An enzyme-linked assay may involve the use of an enzyme and a corresponding enzyme substrate, where the enzyme catalyzes a signal molecule. An enzyme-linked assay may also involve the use of an enzyme without a substrate, where the enzyme produces a signal molecule.

[0069] A method for determining the presence of at least one target molecule in a sample may include subjecting a sensor to an enzyme-linked assay to react with or obtain a signal molecule. The enzyme-linked assay may involve the use of an enzyme and a corresponding enzyme substrate. The enzyme may catalyze a reaction that converts the enzyme substrate into the signal molecule. The conversion may be, for example, oxidation. The enzyme may be part of a detection probe (fusion protein), or the enzyme may include a recombinant region specific for a modification on the detection probe. The enzyme-linked assay may include providing the enzyme such that the amount of enzyme is relative to the amount of the target molecule, thereby providing a means for accurate quantification. In one embodiment, the enzyme includes horseradish peroxidase or alkaline phosphatase. In one embodiment, the detection probe is a biotinylated nucleotide probe, the enzyme is streptavidin-conjugated horseradish peroxidase, the enzyme substrate is 3,3',5,5'-tetramethylbenzidine, and the signal molecule is the oxidized or reduced form of 3,3',5,5'-tetramethylbenzidine. In one embodiment, the detection probe is an antibody conjugated to horseradish peroxidase, the enzyme substrate is 3,3',5,5'-tetramethylbenzidine, and the signal molecule is oxidized or reduced 3,3',5,5'-tetramethylbenzidine.

[0070] The above-described sensors and methods, which include determining the concentration of at least one target molecule in a sample, may be used to provide a diagnosis, monitor a disease or condition, and, for example, track the prognosis, remission, recurrence, and / or effectiveness of a treatment in the subject. The disease or contraindication may be any disease or condition. The sensors and methods may enable quantification of a diagnostic or prognostic biomarker (target molecule). The sensors and methods may also be used as part of a treatment to determine further clinical trials or a treatment plan for the subject. The further diagnostic test may be selected from the group consisting of ultrasound, diagnostic x-ray, magnetic resonance imaging, immunohistochemistry, electrocardiogram, blood test, or biopsy, and the treatment may be selected from the group consisting of drug therapy, surgery, chemotherapy, hormone therapy, radiation therapy, biological therapy such as immunotherapy, small molecule therapy, or antibody therapy, or a combination thereof. When the sensors or methods are used to provide a diagnosis, monitor a disease or condition, and, for example, track the prognosis, remission, recurrence, and / or effectiveness of a treatment in the subject, or as part of a treatment, the use may include quantifying at least one target molecule and comparing the quantified value to a reference value. The reference value may be derived from a representative group of healthy subjects or subjects with a condition or disease. For example, the quantitative value of at least one target molecule in a sample may be increased or decreased compared to a value obtained from healthy subjects, or the quantitative value of at least one target molecule in a sample may be increased or decreased compared to a value obtained from a subject with a condition or disease, or the quantitative value of at least one target molecule in a sample may be increased or decreased compared to a value from a control sample. An increase or decrease in the quantitative value of at least one target molecule or multiple target molecules may be indicative of a disease or condition, or a risk of developing a disease or condition, or a change in state in a disease or condition, e.g., remission or recurrence, or health, or the effectiveness of a treatment. A difference in fold change, e.g., a 1.5-fold, 2-fold, or 3-fold increase or decrease in the quantitative value of at least one target molecule, may be considered a biomarker indicative of diagnosis, risk of a disease or condition, remission, recurrence, and / or the effectiveness of a treatment in a subject.The at least one target molecule may or may not be associated with a known disease or condition. Determining the presence of the at least one target molecule in the sample may be performed in vitro. Determining the presence of the at least one target molecule in the sample may be more sensitive than alternative methods when using the disclosed sensors. Determining the presence of the at least one target molecule in the sample may be improved compared to alternative methods when using undisclosed sensors. In one embodiment, the method is for determining the diagnosis, prognosis, remission, or recurrence of a disease or condition, comprising determining a quantitative value of at least one target molecule in a biological sample obtained from a subject and comparing the quantitative value of the at least one target molecule with a control sample or control value, wherein an increase or decrease in the quantitative value of the at least one target molecule compared to the control sample or value indicates the diagnosis, prognosis, remission, or recurrence of the disease or condition. In one embodiment, the method is a method for determining the risk of developing a disease or condition, comprising the steps of determining a quantitative value of at least one target molecule in a biological sample obtained from the subject, and comparing the quantitative value of the at least one target molecule to a control sample or control value, wherein an increase or decrease in the quantitative value of the at least one target molecule compared to the control sample or value indicates the risk of developing the disease or condition.

[0071] The process for preparing a sensor for determining the presence of at least one target molecule in a sample may be used to prepare or produce the sensor. The sensor may be a multiplexed sensor. In one embodiment, the HARM structure is a carbon nanostructure. In one embodiment, the HARM structure is a carbon nanotube or a carbon nanobud. In one embodiment, the casein micelle or its blocking layer is not treated with any additive. In one embodiment, the casein micelle or its blocking layer is not treated with an added substance. In one embodiment, the casein micelle or its blocking layer is not mixed with any added substance. In one embodiment, the casein micelle or its blocking layer is not subjected to a physical treatment. In one embodiment, the plurality of casein micelles have essentially uniform or heterogeneous sizes. In one embodiment, the functionalization is performed using an amine-sulfhydryl cross-linking agent. In one embodiment, the process further includes providing the sensor with at least one biorecognition element capable of selectively or specifically binding to at least one target molecule, wherein the at least one biorecognition element is contacted with the sensor and thereby bound to at least one linking group.

[0072] Such a process provides a sensor having attached thereto at least one specific or selective biorecognition element for at least one target molecule, which may be used to determine the presence of at least one target molecule in a sample or as part of a method such as those disclosed herein.

[0073] The present disclosure further relates to a kit for determining the presence of at least one target molecule in a sample. The kit comprises: a sensor as disclosed herein and a sensor as disclosed herein having at least one biorecognition element capable of selectively binding to at least one target molecule, or a biorecognition element attached to a linking group; at least one detection probe capable of selectively binding to at least one target molecule; Instructions for use and Equipped with.

[0074] In one embodiment, the kit comprises a sensor disclosed in the present disclosure and at least one biorecognition element capable of selectively binding to at least one target molecule, or a sensor having a biorecognition element bound to the sensor, and instructions for use.

[0075] Some of the materials needed for quantification or determination may be common laboratory materials or reagents and may not be included in the kit, such as pipette tips, tubes, purified water, or buffers. Some reagents for quantification or determination may have to be obtained separately, such as enzymes or enzyme substrates for enzyme-linked assays, or means for measuring electrical signals.

[0076] The methods and processes disclosed herein have the additional utility of providing highly sensitive sensors for determining the presence of at least one target molecule in a sample. Sensitivity is achieved by using multiple casein micelles as a blocking layer. Casein micelles are characterized by being antifouling molecules that prevent nonspecific binding of molecules in the sample, thereby allowing the sensor to have a low background signal. Casein micelles are characterized by their ability to bind only to layers formed from HARM structures and other casein micelles, forming a strong blocking layer while still allowing the formation of cavities that allow signal molecules to pass through to the layer formed from the HARM structures. Furthermore, casein micelles contain surface functional groups, such as primary amines, that can be modified by click chemistry without interfering with their ability to bind to other casein micelles or layers formed from HARM structures, allowing the surface functional groups to be functionalized, for example, to attach biorecognition elements. In summary, the blocking layer comprising multiple casein micelles blocks nonspecific binding, allows specific binding to functionalized groups, and forms cavities that allow the passage of signal molecules into the layer formed by the HARM structure, thereby providing the additional utility of providing a highly sensitive sensor for determining the presence of at least one target molecule in a sample. [Example]

[0077] Reference will now be made in detail to various embodiments, examples of which are illustrated in the accompanying drawings.

[0078] The following description discloses some embodiments in such detail that one skilled in the art can utilize the embodiments based on the present disclosure. Not every step or feature of the embodiments is discussed in detail, and many steps or features will be apparent to one skilled in the art based on this specification.

[0079] For the sake of brevity, in the following exemplary embodiment, item numbers are maintained when repeating components.

[0080] 1A-C show schematic diagrams of an exemplary embodiment of a sensor for determining the presence of at least one target molecule in a sample, according to an exemplary embodiment.

[0081] FIG. 1A shows a sensor 100 for determining the presence of at least one target molecule in a sample, comprising a layer formed of a HARM structure 101 and a blocking layer 102 on the layer formed of the HARM structure 101. The blocking layer 102 is composed of casein micelles 103. The layer of the HARM structure 101 is configured to generate an electrical signal upon contact with a signal molecule indicating the presence of the at least one target molecule when an electric potential is applied to the layer formed of the HARM structure 101, the magnitude of the electrical signal being correlated with the concentration of the at least one target molecule in the sample. Cavities 104 are formed between the casein micelles 103, allowing the signal molecule to diffuse through the blocking layer 102 into the layer formed of the HARM structure 101 and generate an electrical signal. The casein micelles 103 contain primary amines 105.

[0082] 1B shows a sensor 100 for determining the presence of at least one target molecule in a sample, comprising a layer formed of a HARM structure 101 and a blocking layer 102 formed of casein micelles 103, with cavities 104 formed between the casein micelles 103. The casein micelles 103 comprise primary amines 105, at least a portion of which are functionalized with an amine-reactive crosslinker, thereby providing at least one linking group 106.

[0083] 1C shows a sensor 100 for determining the presence of at least one target molecule in a sample, comprising a layer formed of a HARM structure 101 and a blocking layer 102 formed of casein micelles 103, with cavities 104 formed between the casein micelles 103. The casein micelles 103 comprise primary amines 105, at least a portion of which are functionalized with an amine-reactive crosslinker to provide at least one linking group 106. The sensor 100 is contacted with at least one biorecognition element 107. The at least one biorecognition element 107 is functionalized to bind the linking group 106, thereby binding the sensor. The biorecognition element 107 is capable of selectively binding at least one target molecule.

[0084] 1A-C, the casein micelles 103 that form the blocking layer 102 form cavities 104. The casein micelles can be in a configuration that does not form cavities, for example, because the casein micelles are too large or too densely packed. The casein micelles 103 are antifouling molecules that prevent, for example, biorecognition elements 107 from binding to the casein micelles 103. However, when the casein micelles 103 are functionalized with an amine-reactive crosslinker, linking groups 106 are provided that can form bonds with the biorecognition elements 107.

[0085] FIG. 2 shows a schematic flow chart of a method for determining the presence of at least one target molecule in a sample, according to an exemplary embodiment.

[0086] (FIG. 2A) In one embodiment, a sensor such as that described in FIG. 1C is contacted with a sample in operation 201a. The sensor includes a layer of a HARM structure, a blocking layer, and at least one biorecognition element capable of binding at least one target molecule. When the sensor is contacted with a sample containing the target molecule and incubated, the target molecule binds to the at least one biorecognition element, thereby binding to the sensor. The sensor may then be washed. In operation 202a, the sensor is contacted with at least one detection probe and incubated. The at least one detection probe is capable of specifically binding to at least one target molecule, thereby binding to the sensor. The sensor may then be washed to remove unbound detection probe. In operation 203a, the sensor is subjected to an enzyme-linked assay. In an enzyme-linked assay, the sensor is contacted with an enzyme capable of binding at least one detection probe, thereby binding it to the sensor. The sensor may then be washed to remove unbound enzyme. An enzyme substrate may then be added. The enzyme catalyzes a chemical reaction, such as oxidation, that converts the enzyme substrate into a signal molecule. For example, the signal molecule can be an oxidase substrate. When the signal molecule contacts a layer of the HARM structure (FIG. 1, 101) of the sensor (FIG. 1, 100) to which an electric potential is applied, an electric current or electrical signal is generated. In operation 204a, the concentration of at least one target molecule can be determined. When the signal molecule contacts a layer of the HARM structure (FIG. 1, 101) of the sensor (FIG. 1, 100) to which an electric potential is applied, an electric signal is generated. The electric signal or current may be measured and quantified with a means for measuring the electric signal. The magnitude of the electric signal correlates with the concentration of at least one target molecule in the sample.

[0087] FIG. 2B) In one embodiment, the sensor is contacted with the sample and the detection probes in operation 201b. The sample and at least one detection probe may be premixed, and then the mixture may be contacted with the sensor and incubated. The at least one target molecule and the detection probe bind to the sensor. The sensor may then be washed. In operation 202b, the sensor is subjected to an enzyme-linked assay. In an enzyme-linked assay, the sensor is contacted with an enzyme capable of binding at least one detection probe, thereby binding it to the sensor. In operation 203b, the concentration of the at least one target molecule can be determined using a means for detecting an electrical signal.

[0088] 3A shows a schematic flowchart of a process for preparing a sensor for determining the presence of at least one target molecule in a sample, according to an exemplary embodiment. A layer formed of a HARM structure is provided in operation 301a, and the HARM structure may be freestanding or attached to a base or frame. The HARM structure may be, for example, carbon nanotubes. In operation 302a, a solution containing a plurality of casein micelles is provided. The solution is contacted with the layer formed of the HARM structure to provide a blocking layer. The casein micelles contain primary amines, which can be functionalized with an amine-reactive crosslinker in operation 303a, thereby providing at least one linking group and enabling binding thereto.

[0089] FIG. 3B shows a schematic flowchart of a process for preparing a sensor for determining the presence of at least one target molecule in a sample, according to an exemplary embodiment. A layer formed of a HARM structure is provided in operation 301b. The HARM structure may be freestanding or attached to a base or frame. The HARM structure may be, for example, carbon nanotubes. In operation 302b, a solution containing a plurality of casein micelles is provided. The solution is contacted with the layer formed of the HARM structure to provide a blocking layer. The casein micelles contain primary amines, which may be functionalized with an amine-reactive crosslinker in operation 303b to provide at least one linking group and allow binding thereto. In operation 304b, at least one biorecognition element is contacted with the sensor and incubated. The at least one biorecognition element is compatible with, e.g., functionalized with, the linking group. The at least one biorecognition element binds to the at least one linking group and is immobilized on the sensor. The sensor may then be washed with an appropriate buffer. Here, the sensor comprises a biorecognition element capable of binding to at least one target molecule.

[0090] Example 1 - Preparation and Testing of a Sensor to Determine the Presence of at Least One Target Molecule In this example, a series of experiments were performed to examine how different layers formed in the HARM structure can be blocked with different types of blocking layers and how the effect of the blocking layers affects non-specific binding of biomolecules.

[0091] Nonspecific binding of biomolecules affects the sensitivity of the sensor by generating a larger background signal. Nonspecific binding of biomolecules was evaluated by incubating the sensor with a detection probe and streptavidin-conjugated polyHRP. In this example, the detection probe was biotinylated DNA, allowing the streptavidin-conjugated polyHRP to bind. Nonspecific binding of either the detection probe and / or the streptavidin-conjugated polyHRP causes a high background current.

[0092] First, the test sensors were prepared. Carbon nanotubes (CNT, Canatu) and high aspect ratio graphene (3D-Graphene, Integrated Graphene) were used as the HARM structure, and a layer formed with the HARM structure was formed. Then, different blocking layers were formed on the layer formed with either carbon nanotubes (CNT) or graphene nanostructures. The following samples were prepared: [Table 1]

[0093] The reference sensor did not have a blocking layer on top of the HARM structure. The blocking layer labeled "small casein" was formed using a blocking solution of casein micelles with diameters ranging from 2 to 10 nm.

[0094] The blocking layer labeled "Casein Large" was formed with a blocking solution of casein micelles of undetermined diameter, except for the solution filtered through a 0.45 μm filter. This blocking solution contained casein micelles naturally present in milk with diameters up to 450 nm, which were then filtered to a size cutoff. A comparative sensor was formed by using bovine serum albumin (BSA) as the blocking layer, and another comparative sensor was formed by using a blocking solution of lignin in dimethyl sulfoxide solution. Each different sensor type was formed in triplicate (n = 21).

[0095] The sensor was incubated with the blocking solution for 2 hours. After incubation, the prepared sensor was washed to remove excess blocking molecules and then dried in air.

[0096] The sensors were then incubated with a biotinylated detection probe DNA solution (prepared in 1x PBS at a concentration of 2.5 μM) followed by washing, and then the sensors were incubated with streptavidin-conjugated poly-HRP and washed.

[0097] Finally, the sensor was contacted with a liquid substrate system "electrolyte" containing 3,3',5,5'-tetramethylbenzidine (TMB), into which an Ag / AgCl pseudo-reference electrode and a carbon rod counter electrode were placed. The electrochemical reduction reaction of oxidized TMB catalyzed by polyHRP was analyzed using chronoamperometry with a Reference 1010E potentiostat (Gamry) at a holding potential of -0.2 V (vs. Ag / AgCl). Current was measured for 60 s, and reduction current data were analyzed from the point when the system had already reached steady state (t = 55 s). Measurements were performed on three sensors, and average values ​​for each sensor type were obtained. In background measurements, the recorded current resulted from nonspecific binding of the detection probe and / or streptavidin-conjugated polyHRP, because they were prepared in clean buffer (1x PBS) in the absence of target or interfering molecules. Therefore, this experimental procedure emphasizes the sensor signal component arising from nonspecific binding.

[0098] To obtain signal-to-background values, sensors (n = 21) were blocked with small caseins and then functionalized to immobilize biorecognition elements on them in a similar manner to that described above. These sensors were then incubated with target DNA, followed by administration of a detection probe and streptavidin-conjugated poly-HRP. In Figure 4, the signals generated from these sensors are labeled "target."

[0099] After blocking the sensor, N-γ-maleimidobutyryl-oxysulfosuccinimide ester (sulfo-GMBS) in PBS solution was drop-cast onto the sensor, and the formed system was incubated to allow a click chemistry reaction to occur between the N-hydroxysuccinimide ester (NHS) provided by sulfo-GMBS and the primary amine.

[0100] Next, the sensor was washed, and then a biorecognition element (here, thiolated DNA) solution (2.5 μM concentration prepared in PBS) was incubated on the sensor to allow a click chemistry reaction between the sulfo-GMBS' maleimide group and the biorecognition element thiol group. After incubation, the sensor was washed, and the sensor was incubated with a sample to hybridize the biorecognition element with the target molecule (here, DNA) (5 nM concentration prepared in 1× PBS), and then the sensor was washed.

[0101] Finally, biotinylated detection probes and streptavidin-conjugated poly-HRP were incubated on the sensors as described above. Finally, the sensors were loaded into the TMB liquid substrate system "electrolyte" and analyzed as described above.

[0102] The results are shown in Figures 4 to 6.

[0103] Figure 4 shows chronoamperometry traces of the studied sensors. The target current signal is shown in gray, and the background signal is shown in black. The x-axis displays the measured current in nA, and the y-axis displays time in seconds. The displayed measured current reflects the average of measurements from triplicate sensors for each study type. As can be seen in Figure 4, the "CNT, small casein" sensor exhibited the lowest background signal compared to the other sensors, more specifically, approximately 50% less than the "large casein" and "BSA" blocking sensors. Lignin was found to function poorly as a blocking layer, resulting in a high background signal and a low target signal. The current measured with the "graphene, small casein" sensor exhibited a 40-fold increase in background current compared to the "CNT, small casein" sensor, indicating that carbon nanotubes as the material for the layer formed in the HARM structure are more sensitive and that the detection probe and / or streptavidin-conjugated polyHRP remain anchored inside the 3D graphene despite effective washing.

[0104] Figure 5 shows the current signal from nonspecific binding of DNA and protein. The current signal was larger in the unblocked sensor, regardless of the layer material or HARM structural material. The carbon nanotube blocking layer labeled "small casein" provided 52% more efficient blocking compared to "large casein" and 50% more efficient blocking compared to BSA. Lignin was the least efficient at blocking nonspecific binding of DNA and protein, producing a background current 346% larger than the "small casein" sensor.

[0105] In Figure 6, the signal-to-background (SNB) ratio between the target signal and the background signal is visualized. The SNB was significantly improved by blocking nonspecific binding, and the SNB ratio was highest for the sensor blocked with "small casein," primarily due to the significantly improved blocking efficiency against nonspecific binding of biomolecules. The SNB for the "small casein" sample was approximately 2-fold compared to "large casein" (2.12-fold) or BSA (1.98-fold) blocking, but the improvement in SNB was even more significant compared to the lignin-blocked (7.82-fold) and unblocked sensors (27.2-fold).

[0106] It is obvious to those skilled in the art that with the advancement of technology, the basic idea can be implemented in various ways. Therefore, the embodiments are not limited to the above examples, but instead may vary within the scope of the claims.

[0107] The above-described embodiments may be used in any combination with each other. Some of the embodiments may be combined together to form further embodiments. A sensor, method, process, use, or kit may include at least one of the embodiments described herein. It should be understood that the benefits and advantages described above may relate to one embodiment or to several embodiments. The embodiments are not limited to those that solve any or all of the stated problems or have any or all of the stated benefits and advantages. It should be further understood that a reference to "an" or "an" item refers to one or more of those items. The term "comprising" is used herein to mean including the feature or act that follows it without excluding the presence of one or more additional features or acts.

Claims

1. A sensor (100) for determining the presence of at least one target molecule in a sample, comprising: i. a layer (101) formed of a high aspect ratio molecular structure (HARM structure), wherein the layer (101) formed of the HARM structure (101) is configured to generate an electrical signal when a potential is applied to the sensor (100) when in contact with a signal molecule indicative of the presence of the at least one target molecule, the magnitude of the electrical signal correlating with the concentration of the at least one target molecule in the sample; ii. a blocking layer (102) formed from a plurality of casein micelles (103) on the layer formed from the HARM structure (101); The present invention is characterized by comprising: the casein micelles (103) comprise primary amines (105), at least a portion of the primary (105) amines being functionalized with an amine-reactive crosslinker, thereby providing and allowing binding to at least one linking group (106); - A sensor (100) in which cavities (104) are formed between the casein micelles (103), allowing the signal molecule to diffuse through the blocking layer (102) into the layer formed by the HARM structure (101) to generate the electrical signal.

2. The sensor (100) of claim 1, wherein the HARM-structure (101) is a carbon nanostructure.

3. 3. The sensor (100) of claim 1 or 2, wherein the casein micelles (103) are essentially uniformly sized or non-uniformly sized.

4. 4. The sensor (100) of any one of claims 1 to 3, wherein at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or essentially all, of the casein micelles (103) have a diameter in the range of 0.5 to 30 nm, or 0.7 to 20 nm, or 1 to 15 nm, or 2 to 10 nm.

5. The sensor (100) of any one of claims 1 to 4, further comprising at least one biorecognition element (107) coupled to at least one linking group (106) and capable of selectively binding to the at least one target molecule.

6. 1. A method for determining the presence of at least one target molecule in a sample, comprising: - contacting the sensor (100) of claim 5 with the sample, thereby allowing binding of the at least one target molecule to the sample; - contacting the sensor (100) with at least one detection probe capable of selectively binding to the at least one target molecule, thereby allowing binding of the at least one detection probe to the at least one target molecule; - subjecting said sensor (100) to an enzyme-linked assay to obtain signal molecules indicative of the presence of said at least one target molecule, said signal molecules diffusing through said blocking layer (102) into said layer formed by HARM-structures (101), thereby generating an electrical signal which is quantified by means of measuring said electrical signal; - determining the concentration of the at least one target molecule in the sample based on the quantified electrical signal; A method comprising:

7. 7. The method of claim 6, wherein the at least one detection probe and the at least one biorecognition element (107) of claim 5 are independently selected from the group consisting of an oligonucleotide probe, a nucleotide probe, a DNA probe, an RNA probe, a functionalized oligonucleotide probe, a functionalized nucleotide probe, a functionalized DNA probe, a functionalized RNA probe, an antibody, a functionalized antibody, a bispecific antibody, a functionalized bispecific antibody, an antibody fusion protein, a functionalized antibody fusion protein, an antibody fragment thereof, a functionalized antibody fragment thereof, a molecularly imprinted polymer, and a functionalized molecularly imprinted polymer.

8. The sensor (100) according to any one of claims 1 to 5, wherein the at least one target molecule is selected from the group consisting of a single-stranded DNA molecule, a single-stranded RNA molecule, a double-stranded DNA molecule, a double-stranded RNA molecule, a protein, a peptide, a lipid, a hormone, a pathogen, and a pharmaceutical compound. The method according to any one of claims 6 to 7.

9. The method according to any one of claims 6 to 8, wherein the sample is a body fluid.

10. The method of any one of claims 6 to 9, wherein the sample is obtained from a mammal.

11. 1. A process for preparing a sensor (100) for determining the presence of at least one target molecule in a sample, comprising: i. Providing a layer formed of a HARM-structure (101); ii. Providing a blocking layer (102) formed from a plurality of casein micelles (103) on the layer formed from HARM-structures (101), wherein the casein micelles (103) contain primary amines (105); iii. functionalizing at least a portion of said primary amines (105) with an amine-reactive crosslinker, thereby providing and enabling bonding to at least one linking group (106); A process comprising:

12. 12. The process of claim 11, wherein the casein micelles (103) are essentially uniform or heterogeneous in size.

13. 13. The process of claim 11 or 12, wherein at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or essentially all, of the casein micelles (103) have a diameter in the range of 0.5 to 30 nm, or 0.7 to 20 nm, or 1 to 15 nm, or 2 to 10 nm.

14. 14. The process according to any one of claims 11 to 13, further comprising providing the sensor (100) with at least one biorecognition element (107) capable of selectively binding to at least one target molecule, the at least one biorecognition element (107) being brought into contact with the sensor (100) and thereby binding to the at least one linking group (106).

15. 1. A kit for determining the presence of at least one target molecule in a sample, comprising: a sensor (100) according to any one of claims 1 to 4 and at least one biorecognition element (107) capable of selectively binding to said at least one target molecule, or a sensor (100) according to claim 5, at least one detection probe capable of selectively binding to said at least one target molecule; ・Instruction manual and A kit comprising:

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