CN105339791A - Molecular nets on solid phases - Google Patents

Abstract

Disclosed herein is a covalently linked multilayered three-dimensional matrix (referred to as a molecular net) comprising capture molecules, linkers, and spacers for the specific and sensitive capture of analytes from a sample. Also disclosed herein is a molecular net for the specific and sensitive capture of more than one type of analyte from a sample, the molecular net comprising a covalently linked multilayered three-dimensional matrix comprising more than one One type of capture molecule and more than one type of linker, and may include one or more spacers. Molecular Nets can include pseudorandom properties. The use of different capture molecules, linkers, and spacers in Molecular Nets can give Molecular Nets unique binding properties. The porosity, binding affinity, size exclusion, filtration, concentration, and signal amplification capabilities of a Molecular Net can vary and depend on the nature of the components used in the manufacture of the Molecular Net. Uses of Molecular Nets may include analyte capture, analyte enrichment, analyte purification, analyte detection, analyte measurement, and analyte delivery. Molecular Nets can be used in the liquid phase or on solid phases such as nanomaterials, modified metal surfaces, nanospheres, microspheres, microtiter plates, glass slides, pipettes, cassettes, cartridges, discs, Probes, lateral flow devices, microfluidic devices, microfluidic devices, fiber optics, and others.

Description

Molecular Nets on Solid Phase

Cross References to Related Applications

This application claims the benefit of U.S. Provisional Application Serial No. 61/783,189, filed March 14, 2013, and is the result of U.S. Patent Application Serial No. 13/511,364, filed March 22, 2012 and U.S. A continuation-in-part of patent application Serial No. 13/938,055, the entire contents of which are hereby incorporated by reference.

Background technique

Existing current strategies for solid-phase analyte capture, analyte detection, and analyte measurement use a monolayer of capture molecules adsorbed or covalently bonded to a surface for direct real-time sensing, or in an indirect detection modality Used in conjunction with a secondary detection step, these strategies are well known in the art. Both direct and indirect methods show limitations in sensitivity, specificity, signal-to-noise ratio and/or cost.

There is a need for an analyte capture technology for solid-phase surfaces or devices that can selectively capture analytes from complex samples with little or no sample preparation and maximize the concentration of said selected analytes. Oriented in a manner that maximizes capture analyte measurement and/or detection in a manner that is compatible with most technologies.

Contents of the invention

Devices for capturing analytes are described. In one embodiment, a device can include a solid phase and a molecular net coupled to at least a portion of a surface of the solid phase. The molecular net may comprise a plurality of capture molecules of at least one type linked to each other by a plurality of linker molecules of various types so as to form a covalently linked multilayered three-dimensional matrix. The capture molecules can be configured to bind the analyte.

A method of making a device for capturing an analyte is also described. In one embodiment, a method can include providing a solid phase, and disposing a molecular net on at least a portion of a surface of the solid phase. The molecular net may comprise a plurality of capture molecules of at least one type linked to each other by a plurality of linker molecules of various types so as to form a covalently linked multilayered three-dimensional matrix. The capture molecules can be configured to bind the analyte.

Methods of measuring the amount of an analyte in a sample are also described. In one embodiment, a method can include providing one or more devices, each device comprising a solid phase and a molecular net covering at least a portion of a surface of the solid phase. The molecular net may comprise a plurality of capture molecules of at least one type linked to each other by a plurality of linker molecules of various types so as to form a covalently linked multilayered three-dimensional matrix. The capture molecules are configured to bind the analyte. The method also includes exposing the devices to the sample and allowing at least a portion of the analyte to bind to capture molecules of molecular nets of the devices.

Description of drawings

Figure 1 shows a comparison of conventional conjugated microparticles and molecular net microparticles in IgG purification;

Figure 2 shows the conjugation of traditional capture molecules to microparticles and the corresponding analyte measurement capabilities;

Figure 3 shows the effectiveness of Molecular Net microparticles in measuring analytes;

Figure 4 shows the effectiveness of molecular nets with topology in measuring analytes;

Figure 5 shows a comparison of conventional conjugated microparticles and Molecular Net microparticles in TauELISA;

Figure 6 shows a comparison of conventional conjugated microparticles and Molecular Net microparticles in a TSH Luminex sandwich immunoassay;

Figure 7 shows an exemplary molecular net on a particle;

Figure 8 shows exemplary Molecular Net topological features on particles;

Figure 9 shows an exemplary molecular net for analyte delivery.

detailed description

The construction and use of a covalently linked pseudorandom multilayer three-dimensional matrix is shown in U.S. Patent Serial Nos. 61/281,991, 61/337,257, 61/340,287, 61/343,467, 61/410,837, 61/489,646, and 61/489,648 enabling rapid and specific capture of proteins, nucleic acids, carbohydrates, lipids, cells, or other analytes from unprocessed samples, and showing that the use of such molecular nets may be a significant improvement over conventional analyte binding methods, these Each of the patents is hereby incorporated by reference. Molecular Net Design and Fabrication

Molecular Net properties can be conferred by: the capture molecules selected for use (examples of capture molecules may include antibodies, nucleic acid probes, enzymes, recombinant proteins, peptides, and others); the resulting specificity conferred by the capture molecules; The size and number of capture molecules selected; the placement and spacing of capture molecules within the Molecular Net layer or layers; the combination of capture molecules; the order in which capture molecules can be used; and the ratio of capture molecules to linker and spacer molecules used.

Molecular Net properties can also be conferred by: the linker molecule chosen for use (examples of linker molecules include homobifunctional, heterobifunctional, trifunctional and multifunctional types); the chemical specificity of the linker molecule; The Angstrom length of the linker molecules; the combination of linker molecules; the order in which linker molecules can be used; and the ratio of capture molecules to linker and spacer molecules used.

Molecular Net properties can also be imparted by: the spacer molecule chosen for use (examples of spacer molecules include PEG, polymers, nucleic acids, albumin, Fc regions, peptides, and others); the chemical properties of the spacer molecule; The size and number; the order in which spacer molecules can be used; and the ratio of spacer molecules to linker and capture molecules used.

The placement and spacing of capture molecules, linker molecules, and spacer molecules can: give characteristic topologies on the Molecular Net surface; give characteristic densities in each layer of a Molecular Net; give Molecular Nets characteristic porosity; eliminate spatial constraints and thus steric hindrance; improving binding capacity; reducing non-specific binding; enabling binding of multiple forms of analyte (eg, simultaneous capture of degraded analyte, intact analyte, and complexed analyte), and others.

Pores in molecular nets can be random, pseudorandom, or irregularly distributed. The pores of molecular nets can be used to filter samples; they can be used to distinguish binding potential molecules in samples by size exclusion; they can be used to realize the binding of macromolecules or cells due to the reduction of steric hindrance, or others. The pores of the Molecular Net contain multiple capture molecules, multiple linker molecules, and may contain multiple spacer molecules. Traditional methods of creating porosity in solid phases involve mechanical modification of the solid surface and employ methods such as laser etching, lamination, lithography, laser printing, or other methods to create pores, pores, or other structures in the solid surface . This solid surface is then prepared for receiving conjugated capture molecules. Using Molecular Nets eliminates the need for mechanical modification of the surface and is therefore more cost-effective. Furthermore, traditional methods are still hampered by the problem of high non-specific binding and require capture chemicals bound to mechanically modified solid phases, which is not an improvement. Furthermore, flexibility can be imparted into Molecular Nets due to the size exclusion properties imparted by the porosity built up in each layer of Molecular Nets compared to traditional trapping formats. In some layers, pore diameters and depths may be similar or may vary depending on the application. In some layers, the pore size may vary, and the variation in pore size may depend on the application.

Pores that may be imparted on a molecular net may include, but are not limited to, picopores, nanopores, micropores, filters, meshes, pockets, or others. Pores can be imparted into the Molecular Net by selecting and incorporating specific capture, linker, and spacer molecules into each layer of the Molecular Net. Porosity can also be imparted into molecular nets by the selection and incorporation of specific capture molecules, linker molecules and spacer molecules used to make the continuous layer.

Molecular Net pores can range in diameter from about 6 Angstroms to greater than about 1 um, based on the nature of the capture molecules, linkers, and spacers used in the layer. In some cases, the pores of the Molecular Net can include a range of pore diameters. Exemplary diameter ranges may be from about 5 nm to about 50 nm, from about 10 nm to about 100 nm, from about 50 nm to about 200 nm, from about 250 nm to about 500 nm, from about 500 nm to about 1 um, and from about 800 nm to about 1.5 um.

In some cases, capture molecules can be used to create pores in molecular nets. In these examples, the capture molecules can be pre-linked to each other prior to their incorporation into the Molecular Net layer. In some cases, linkers can be selected based on the Angstrom length of the spacer. In some examples, extenders can be used to link a first linker to a second linker to create a long multifunctional linker. In some cases, spacers can be used to create pores in the Molecular Net. It is also possible to pre-attach the spacers to each other before incorporating the spacers into the Molecular Net layer. In other examples, inert physical plugs can be used to construct apertures whereby each physical plug can be placed on a previously constructed layer while a new layer is constructed. After curing, these physical plugs can be removed, thus leaving pores of a specific diameter.

The flexible nature of Molecular Nets enables the use of many types of capture molecules. In some examples, a Molecular Net includes a single type of capture molecule. In other examples, the Molecular Net includes multiple types of capture molecules. In some examples, the use of more than one monoclonal antibody during the manufacture of the Molecular Net enables the Molecular Net to bind more than one epitope of the analyte. The use of more than one type of epitope-specific capture molecule enables improved capture of Molecular Net analytes and correlates to the performance of the Molecular Net. In some examples, the use of more than one nucleic acid sequence can be used in a manufacturing process to generate a molecular network capable of binding more than one epitope of an analyte. Examples of benefit depend on the use of Molecular Nets, and may include, when used in an assay, minimum level of detection, sensitivity, positive predictive value, negative predictive value, ability to function on degraded samples, effect on various populations Functional ability and other aspects of improved performance; may include improved performance in binding capacity, purity, binding kinetics, target analyte depletion, and other conveniences when used as a purification tool; or others.

In some examples, the use of capture molecules for mutually identified analytes can be used in a Molecular Net and correlated with the performance of the Molecular Net. The use of mutually confirmed capture molecules in molecular nets can be used in a confirmed manner whereby capture of more than one analyte can provide statistically more significant positive results; can provide more stable test results; can provide Additional information about the sample; and others. The use of mutually validated capture molecules in Molecular Nets can also be used to qualify a sample, or can be used as a control in a test, or can be used to measure more than one relevant molecular variable related to a disease state, or can be used to measure related disease states More than one relevant molecular variable for treatment.

Examples of mutually confirming analytes that Molecular Nets can be fabricated for simultaneous capture from a sample can include: gene sequences and corresponding protein products (e.g., cancer-associated SNPs in BRCA1 and BRCA1 proteins); mRNA and corresponding protein products products (e.g., human lactase mRNA and lactase protein); gene sequences and corresponding mRNA products (e.g., disease-associated SNPs in LMNA and pre-spliced or spliced lamin A/C mRNA); miRNAs and associated mRNA or protein products (miR9 and REST, or CoREST mRNA, or miR9 and REST protein); small molecule drugs and drug targets (tofacitinib and Janus kinase); epitope-specific biologics and corresponding targets (such as anti - TNF antibodies and circulating TNF cytokines); epitope-specific antibodies, epitope-specific T cells and/or epitope-specific B cells, etc. (e.g., anti-DNA autoantibodies, anti-DNA CD4 ⁺ T cells and/or anti-DNAB cells); or others. Examples of benefit are use dependent and can relate to test sensitivity, positive predictive value, negative predictive value, specificity, diagnosis of disease, ability to function on samples undergoing genetic drift, ability to measure response to treatment, measurement of therapeutic agents Ability to be effective, or otherwise improve performance.

In one example, the Molecular Net can be fabricated in a manner that captures and localizes the bound analyte in a manner that enhances the intensity of the detectable signal or can enhance the detection of the bound analyte, such as when used in an assay utilizing optical detection . Placing the captured analytes in a layered manner by prepositioned layered capture molecules enables rapid detection of analytes through signal enhancement. Examples of signal enhancement achieved by Molecular Nets may involve fluorescence, fluorescence resonance energy transfer, absorbance, luminescence, light scattering, surface plasmon resonance, optical heterodyne detection, or others.

The molecular nets can be designed and fabricated to replace the need for expensive and time-consuming methods for ultrasensitive detection, such as PCR, branched DNA, or the multi-step detection methods required for signal amplification. The Molecular Nets can also be designed and fabricated to replace the need for expensive and complex analytical devices.

Typically, the number of capture molecules incorporated into the 3D Molecular Net matrix is less than or equal to the number of capture molecules conjugated to the surface in two dimensions using conventional methods. Two-dimensional capture molecule surface conjugates may rely on the use of a single type of linker or may rely on the sequential use of 2 linkers to conjugate the capture molecule to the solid surface. In the process of fabricating the layers of a Molecular Net, multiple types of linkers are used simultaneously to attach capture molecules to capture molecules of a new layer, and to attach attached capture molecules of a new layer to spacers or capture molecules of previous layers. molecularly. Molecular Nets can be fabricated in solution prior to placement on a solid surface. Prefabricated molecular nets can be adsorbed or covalently attached to solid surfaces. Molecular Nets can also be fabricated layer-by-layer directly onto solid surfaces. The Molecular Net can be placed on a solid surface using non-covalent (electrostatic, van der Waals or other) or covalent methods. In some examples, polystyrene, polyurethane, polyethylene, or treated surfaces such as poly-L-lysine coated surfaces, modified surfaces containing -COOH, NHS, amines, or others can be purchased from commercial sources (supplier's Examples may include Thermo, Millipore, Luminex, and others) and are used as solid phase surfaces for molecular net placement. In other examples, the solid surface can be pretreated with chemicals such as acids in order to activate surface moieties and thus create attachment points between the solid surface and the reactive moieties of the Molecular Net. In some examples, the solid phase can be pretreated with linkers to covalently attach the solid phase surface to the Molecular Net.

The design and fabrication of Molecular Nets for use on solid surfaces can produce a covalently linked multilayered three-dimensional matrix of capture molecules immobilized by covalent linkers in each layer. Design and fabrication can occur in a sequential manner, where a first layer is fabricated and subsequent layers are fabricated in a sequential manner, whereby each layer can be covalently interconnected to enhance structural integrity, topology, porosity and/or stability. Individual capture molecules, linkers, and spacers can be selected to contribute to one or more properties of the molecular net. Properties may include analyte specificity, thermal stability, layer thickness, pore diameter, absorbance, spectrum, emission spectrum, solid phase compatibility, or others.

The use of capture and linker molecules of known length and width and spacers can be used to generate different topologies on the surface of the Molecular Net. Topological features that can be imparted to molecular nets can include, but are not limited to, ripples, pits, dots, pores, mounds, branches, filaments, fibers, cracks, raised segments, or others, and can be in molecular nets Arranged randomly, pseudo-randomly, or irregularly.

Topological features of molecular nets can be generated through the use of capture molecules and linkers; capture molecules, linkers, and spacers; or linkers and spacers. In some cases, capture molecules can be used to generate topological features of molecular nets. In these examples, the capture molecules can be pre-linked to each other prior to covalent incorporation of the capture molecules into the Molecular Net layer. In some cases, linkers can be selected based on the Angstrom length of the spacer. In some examples, a spacer can be used to join a first linker to a second linker to create a long multifunctional linker. In some cases, spacers can be used to generate topological structures in molecular nets. The spacers may also be pre-attached to each other or to capture molecules prior to their incorporation into the Molecular Net layer.

Molecular Nets can be designed and fabricated to impart features such as affinity, size exclusion, filtering, fluorescence, and others into each layer of the Molecular Net. Specific capture molecules, linker molecules, and spacer molecules can be selected based on size, length, diameter, thickness, optical properties, chemical properties, or otherwise to impart characteristics into the molecular net during fabrication.

Molecular Nets can be fabricated in a manner whereby one or more capture molecules in a covalently linked multilayer three-dimensional matrix can provide a structural role, both structural and analyte capture can be provided. Some examples of capture molecules can be used in Molecular Nets for structural and/or analyte capture.

The distance between the capture molecules in each layer of the Molecular Net can be determined in part by the diameter, width and/or length of the capture molecules, linkers and spacers used in the fabrication process for each layer, whereby The molar relationship between each link-capture-spacer molecule may be similar or may be different, and the choice of the molecule may depend on the size and/or shape of the analyte to be captured, the The method and/or intended use of the analyte.

Molecular Nets can be designed and fabricated in a manner whereby each capture molecule, linker and spacer component can have an equivalent or non-equivalent molar ratio in one layer of the Molecular Net. The molar ratio between the components can be varied from time to time to create porosity or other topological features in each layer. The pores and topological features may have a diameter range and may have an associated depth range. Variations in the molar ratios between the Molecular Net components may occur in a single layer of the Molecular Net, or may occur in more than one layer of the Molecular Net, and depend on the intended use of the Molecular Net.

Table 1. Examples of molecular net structural components with analyte capture capabilities

Some examples of analyte sizes that may be considered during design and fabrication are provided in Table 2. Molecular Net surface chemistry, pore diameter, topology, layering, or other design and fabrication can be based on analyte shape; analyte structure, analyte subtype, analyte loading, analyte complex formation with other molecules, and others form. Furthermore, Molecular Nets can be designed and fabricated to bind and capture the analyte, or can be designed and fabricated to exclude the analyte. Examples of analytes and analyte sizes can be found in Table 2.

Table 2. Examples of analytes and their sizes

Molecular nets comprising structural and capture components can be arranged in covalently linked three-dimensional (3D) multilayer matrices and can be involved in the capture of one or more analytes that are associated with the following features One or more of: surface chemistry; analyte shape; analyte structure; analyte subtype; analyte loading; post-translational modification; chemical modification; activity; or others.

Molecular Nets can contain structural components that also function in ways related to the capture of analytes and can be arranged in the interconnected 3D multilayer matrix of Molecular Nets through covalent linkers. Molecular Nets may also contain spacers to interconnect the structure molecules/capture molecules in a manner that maximizes structural reinforcement, stability, and/or specific analyte capture capabilities. Examples of Molecular Nets comprising capture components/structural components, linkers and spacers are presented in Table 3.

The fabrication of Molecular Nets is unique because the capture molecules are immobilized in a 3D matrix by covalently linking the molecules. In numerous studies, Molecular Nets have been demonstrated to have improved thermal stability and extended shelf life over traditional capture technologies.

Table 3. Examples of molecular nets and their uses

In some examples, the solid phase can be particles ranging in diameter from about 2 nm to about 200 mm, and molecular nets can be attached to the surface of the particles. Particles may include: polystyrene, polyethylene, silica, composites, nylon, PVDF, nitrocellulose, cellulosic materials, carbon, or may be magnetic, paramagnetic, fluorescent, barcoded, or otherwise.

Molecular nets can be adsorbed or covalently attached to the surface of particles in such a way that pseudorandom or ordered pores are created in a single layer or in all layers of the molecular net. In its most basic form, particles can be initially coated with a molecular net layer, which can be attached to a second layer, which can be attached to a third layer. The Molecular Net layers may include the same capture molecule in each layer at the same concentration or at different concentrations. Molecular Net particles can also include different capture molecules in each layer and can be fabricated in such a way that they incorporate the same or different concentrations of capture molecules compared to the previous multiple layers.

In some examples, Molecular Nets can be attached to particle surfaces in such a way that an asymmetric particle with a predetermined polarity is created. Such particles can be designed and fabricated with an initial layer comprising structural molecules of large diameter, width and/or length, and can be attached to the particle in an asymmetric manner to create polarity. A second layer can be connected to a first layer, and a third layer can be connected to a second layer, and so on. The number of layers in a Molecular Net particle can vary depending on the use.

In some examples, a molecular net can be attached to a segment of a particle to create an asymmetric particle with a predetermined polarity. The particle is constructed whereby the initial layer is applied to a section of the particle, and whereby a second layer is connected to the initial layer on the same section of the particle, and whereby a first Three layers are connected to the second layer on the same section of the particle, and thereby a fourth layer is connected to the third layer on the same section of the particle.

In some examples, Molecular Nets can be passively adsorbed onto a non-functionalized particle surface. In other examples, particle surfaces may be functionalized and may require activation prior to attachment. In other examples, particle surfaces can be activated prior to functionalization, at which time molecular nets can be attached. In other examples, however, molecular nets can be built directly on particle surfaces. Attaching Molecular Nets to particles can alter the physical and/or chemical characteristics of the particles. In some examples, a molecular net can include a plurality of pseudorandom topological features disposed on the particle surface. In some examples, Molecular Net particles can include topological features including capture molecules and linkers, and can also include spacers. Examples of different topological features may include: appendages, peaks, plateaus, planes, mounds, fissures, skins, dots, grooves, pores, and others, and may be formed by direct connections in one or more layers of the Molecular Net and and/or directly attached to the capture component composition on one or more layers of the Molecular Net.

Other examples of topological features may include pockets, posts, protrusions, branches, protrusions, ridges, cracks, lattice-like structures, flakes, globules, spheres, or others. Topological features can be pre-shaped in solution and attached to the molecular net, or can be formed as each layer is built.

Molecular Nets on particles may include non-uniform capture molecules in one or more layers of the Molecular Net. The benefit of a heterogeneous design can be related to the capture of multiple analytes with multiple surface chemistries on a single particle. The heterogeneous capture molecules incorporated into the molecular net during fabrication can be randomly distributed throughout each layer; can be layered throughout each layer; or otherwise, depending on the application.

Molecular Nets can be attached to particles in order to increase the surface area of the particles. Molecular Nets can also be used to increase particle diameter. In addition to analyte capture capability, topological characteristics of molecular nets on particles can also be related to increased particle size.

In some examples, a first Molecular Net layer can be attached to the particle surface in order to alter the physical and/or chemical properties of the particle. In many commercial particles, the "bead effect" or "surface effect" can hamper results and is still not well understood. Traditional conjugation techniques to generate 2D conjugates and 2D conjugated surfaces are often limited by surface effects. Molecular Nets can be used to minimize or counteract bead effects in order to minimize non-specific binding to bead surfaces, bead autofluorescence, bead interference, or others in the assay. In some examples, Molecular Net particles can confer increased analyte binding capacity and can also confer blocking of undesired analyte nonspecific binding in order to increase signal-to-noise ratio, yield, and efficiency of purified analytes in assays. purity, or otherwise.

In some instances, in yet another aspect, the invention features molecular nets on particles comprising more than one layer, wherein each layer comprises capture molecules for an analyte, wherein each layer comprises a different capture molecule for a different analyte. Molecules where different layers can be directed against different analytes to enable capture of one analyte or multiple analytes.

In yet another aspect, molecular nets placed on a solid surface can be used to increase the purity of one or more analytes recovered from a sample. Compared with commercial 2D functionalized surfaces, Molecular Net-coated surfaces can reduce the non-specific binding of undesired analytes.

In some instances, molecular nets placed on particles can significantly increase the analyte capture capacity of the particles. Additional stratification of the molecular net can further increase the number of bound analytes per particle, and can be used to increase the recovery or yield of analytes from a sample, and can be used to deplete a sample of one or more analytes. Benefits of Using Molecular Net

Molecular and cellular testing strategies employ singleplex or multiplex immunoassays, PCR assays, next generation sequencing techniques, or others to identify the presence or measure the amount of one or more analytes in a sample.

In a multiplex assay, multiple reactions can be spatially separated, or can be combined into a single test reaction, and a solid phase including unique identifiers can be employed to provide information. Some examples of unique identifiers may include different barcodes, different fluorescent emissions, different chemicals, different ordered nucleotide tags, or other uses.

Solid phases can be used in singleplex and multiplex assays, can rely on specific binding of the analyte of interest in order to produce a measurable signal or a measurable change in signal, and can be used in direct assays or can be used in indirect used in the measurement. A measurable signal can result from a positive test, and can include electrical, thermal, magnetic, optical, vibrational, isotopic, or other measurable characteristics.

Many difficulties in achieving sensitive and reproducible measurements using current strategies result in high non-specific binding, low sensitivity, low signal-to-noise ratio, and thus require upstream sample processing steps to remove as much as possible from the sample The nonspecific components, combined with the use of highly sensitive readout techniques and complex algorithms required to determine real signal from noise, make their translation into near real-time, easy-to-use tools for molecular diagnostics and analyte measurement difficult.

Molecular Nets can be used to replace current commercial methods and can achieve specific and sensitive analyte capture, detection and measurement from samples. Examples of results obtained using Molecular Nets instead of current methods for analyte capture are presented in Figures 1-6. Improvements in assay sensitivity, minimal levels of analyte detection, and other features can be achieved by replacing current 2D methods with Molecular Nets for analyte capture and measurement. Reduction of background noise can be achieved by replacing current 2D methods with Molecular Nets and can be used to improve analyte purification, analyte purity, and assay sensitivity.

The benefits of Molecular Nets are presented in Table 4 and may include: rapid capture of one, several or more molecular and cellular analytes in the original sample; generation of sensitivity when used in tests involving indirect and direct detection methods; Ability to generate a signal with enhanced fluorescence intensity; ability to concentrate bound analytes; spatially separate binding in a manner that reduces steric hindrance between analytes and/or between detection molecules Analyte capacity; enhanced stability; reduced background and others.

Table 4. Proven Benefits of Molecular Nets

Molecular Nets and Their Uses

Molecular Nets can be used in a variety of applications where analyte binding efficiency, analyte binding kinetics, analyte binding capacity, analyte detection, analyte measurement, analyte enrichment, analyte purification, and analyte delivery may be important. Molecular Nets can be used in a liquid phase or can be attached to a solid phase.

Molecular Nets can be attached to a receptive surface by adsorption or covalent processes. Examples of solid phases include, but are not limited to: nanotubes, metals, particles, microtiter plates, glass slides, cartridges, probes, lateral flow test vessels, stents, catheters, valves, blood vessels, needles, solid phase devices, or others. Examples of chemistries of different solid phases that may be compatible for Molecular Net attachment include, but are not limited to: plastics, other polymers, thin films, colloidal metals, silica, carbon nanotubes, proteins, carbohydrates, lipids, Nucleic acid, cells, tissues, or others.

Molecular Nets can be attached to the surface of a solid-phase device to capture, purify, or deplete one or more analytes from a sample. An example of the use of Molecular Nets to capture and/or purify an analyte from a sample is presented in FIG. 1 . Some other examples of Molecular Nets that can be used to capture and purify analytes from samples are: Protein A, Protein G, or Protein L mesh-coated microspheres for immunoglobulin-like capture; Streptavidin mesh-coated microspheres; TNF mesh-coated microspheres for anti-TNF biotrap; IL6 mesh-coated microspheres for anti-IL6 biotrap; RNA virus capture IgM mesh-coated microspheres; IgFc mesh-coated microspheres for complement capture; antigen-net-coated microspheres for antigen-specific immunoglobulins; antigen-specific immune cell capture; and others. Molecular Nets can be used in chromatography to capture and purify one or more analytes from a sample.

Molecular Nets can be used to capture analytes from a sample for downstream analyte measurement by a separate method, referred to herein as sample preparation. Examples of independent methods may include: mass spectrometry, immunoassay, PCR, next generation sequencing, qRT-PCR, digital PCR, microscopy, fluorescence, flow cytometry, bead cytometry, or others.

In another aspect, the invention improves the signal-to-noise ratio when used in an assay.

Molecular Nets can be attached to the surface of a solid-phase device in order to measure the presence, absence, modification, or concentration of one or more analytes. Examples of the use of Molecular Nets for analyte detection and/or measurement are presented in FIGS. 3 and 4 . In some other examples, Molecular Nets can be used to simultaneously detect and measure two or more specific analytes in a direct or indirect manner. Indirect capture by Molecular Net may involve capture of a primary analyte by specific capture molecules of Molecular Net, which enables detection of one or more related secondary analytes associated with the captured primary analyte. Molecular Nets can be used as a discovery tool to capture primary analytes from samples and enable identification, detection, or measurement of secondary analytes captured by association. Molecular Nets can be used in this way for drug discovery, pathway mapping, and in proteomics, transcriptomics, glycomics, lipidomics, metabolomics, functional genomics, foodomics, nutrition, pharmacology, toxicology, and others.

In some examples, Molecular Nets can be used to detect drug resistance in cells. Cells can be tumor cells, immune cells, microbial cells, or other cells. Molecular Nets for these applications may include capture molecules targeting one or more unique characteristics of a cell type. Molecular Nets can additionally be fabricated in a manner that imparts surface topology relevant to the capture of intact cells.

In other examples, Molecular Nets can be fabricated and used for: immune cell reactivity measurement; immune response monitoring; immune response classification; immunoglobulin titration; biotinylated molecular capture; multiplex immunoassays; Sequencing reactions; PCR; microbial capture; microbial discovery; mRNA and encoded protein measurement; SNP (single nucleotide polymorphism) mapping; SNP detection; disease marker sample preparation; miRNA capture and/or measurement; post-translational modification discovery and /or capture and/or measurement; kinase activity measurement; or other.

Molecular Nets can have a number of measurable characteristics imparted during fabrication and can be used as sensors in a direct or indirect manner. Commercial methods may be used to detect measurable changes in one or more characteristics of molecular net sensors employing optical sensing, electrochemical sensing, electromagnetic sensing, electrical impedance, or others. In one example, molecular net sensors can be used to capture and bind analytes. Analyte binding can cause measurable changes in the characteristics of the Molecular Net sensor. Binding events or modification events involving the relevant Molecular Net sensor can be monitored over a period of time, and changes in characteristics of the Molecular Net sensor can be detected, relayed, and collected by the device. Other examples of using Molecular Nets as sensors may include analyte binding events, enzymatic reactions, analyte modification events, cell differentiation, cell-cell interactions, or others.

Examples of measurable characteristics include, but are not limited to: physical shape, height, density, fluorescence intensity, wavelength shift (FRET or FRAP), vibrational frequency, absorbance, flexibility, refractivity, conductance, impedance, electrical resistance, melting temperature, denaturation temperature , freezing temperatures, and others.

Measurement devices that may be compatible for use with Molecular Net sensors may include: photon multichannel analyzers, spectrometers, magnetic resonance imagers, magnetic field detectors, fiber optics, glass pipettes, electrical circuits, fluorometers, spectroscopic analysis instruments, flow cytometers, CCD cameras, microscopes, sound chambers, loudspeakers, photometers, and others. These measurement devices can be used to measure changes in thickness, topology, loading, insulation, capacitance, voltage, color, sound quality, vibration, magnetism, enzymatic activity, or other characteristics of molecular nets used as sensors.

Furthermore, Molecular Nets can be used in flexible circuits, whereby capture molecules and/or structural molecules can be attached to conductive molecules. Molecular Net circuits can be used on single-sided flex circuits, double-contact (bare-back flex circuits), engraved flex circuits, double-sided flex circuits, multi-layer flex circuits, ridge flex circuits, ridge flex circuits, polymer thick film flex circuits, or other use. Most flex circuits are passive wiring structures used to interconnect multiple electronic components such as integrated circuits, resistors, capacitors, etc., however some flex circuits are only used to connect between other electronic components directly or by means of device manufacturing interconnects. Molecular nets for use in or as circuit components may be composed of synthetic components, or may be composed of biochemical capture molecules and/or cells, or may be formed in a manner to be used in flexible circuits manufacture. Molecular Net circuits can also be used as sensors.

In some examples, Molecular Net circuits can have specific electrochemical properties and can be used to monitor different parameters in electrochemical cells/electrolyte cells, such as pH, current, voltage, impedance, or others. Binding events and modification events that may occur in molecular nets can be measurable and can be reflected by changes in conductance, current, or voltage. Rather, the introduction of a sample containing an analyte that has a specific binding affinity for, or is reactive to, a component in a Molecular Net circuit can be monitored by changes in the electrochemical properties of the Molecular Net and/or the surrounding environment. .

Examples of binding events that occur on molecular nets used in circuits may include: antibody-antigen interactions, nucleic acid-nucleic acid interactions, enzyme-substrate interactions, drug-target interactions, enzyme-cofactor interactions, ligand- Cellular interactions, or any other non-covalent interactions driven by specific surface chemistry. Analyte capture by Molecular Nets can be determined by changes in pH, current or voltage in an electrochemical cell/electrolyte cell. Measurements of changes in Molecular Net characteristics can also be derived from one or more modification events, or a plurality of modification events, that occur at one or more components in the Molecular Net or at the capture analyte. Examples of modification events may include: enzymatic cleavage; post-translational modification (such as phosphorylation, sulfonation, glycosylation, methylation, or others); removal of post-translational modification (such as dephosphorylation); or other similar modifications. Modification events can be determined by changes in pH, current or voltage in an electrochemical cell/electrolyte cell due to changes in the Molecular Net characteristics or in the surrounding buffer system.

Methods for determining changes in the electrochemical properties of molecular nets used in circuits may include the use of scanning ion current microscopy, nanofluidic diodes, nanopores or nanochannels exhibiting voltage-gated ionic currents, ion nanogating, nanopore-based sensing platforms, and other methods for measuring the flow or change in charge flow through a medium. Rather, an inherent sensitivity of many solid-state nanopore sensors is the selective permeability to electrolyte, or ionic current, when a bias voltage is applied across the nanopore. Molecular Nets can be coated onto the surface of the nanopores and changes in current, voltage, and impedance can be monitored.

Molecular Nets can also be coated onto the surface of carbon nanotubes, and thereby the Molecular Nets can be constructed in a way to create the size exclusion and affinity requirements for analyte sensing.

Biolayer interferometry (BLI) is a label-free technique for measuring biomolecular interactions. This is an optical analysis technique that analyzes the interference pattern of white light reflected from two surfaces: an immobilized molecular net layer on the biosensor tip, and an internal reflection layer. Any change in the amount of molecules bound to the biosensor tip causes a shift in the interference pattern, which can be measured in real time. The binding between the ligand immobilized on the Molecular Net-coated biosensor tip and the analyte in solution results in an increase in the optical thickness at the biosensor tip, which produces a wavelength shift Δλ, which is the thickness of the biolayer A direct measure of change. Interactions can be measured in real time, providing the ability to precisely and accurately monitor binding specificity, association and dissociation rates, or concentration. Only molecules bound to or dissociated from the Molecular Net biosensor can shift the interference pattern and generate a response curve. Unbound molecules may change the refractive index of the surrounding medium, or may change the flow rate, but will not affect the interference pattern. This is a unique feature of BLI and extends its ability to perform in crude samples used in applications for analyte-capture molecule binding, quantification, affinity and kinetics.

Molecular Net particles can also be used to deliver active agents. Active agents may be preloaded onto capture molecules located in one or more layers of the molecular net. Active agents may include: drugs, therapeutics, toxins, viruses, allergens, vaccine components, antigens, immunomodulators, surfactants, microorganisms, oligonucleotides, nutrients, or others. Molecular Net particles can be used for drug or therapeutic agent delivery, vaccine delivery, biofermentation, or others. The Molecular Net can include one or more targeting agents on the surface exposed layer to facilitate specificity in targeting the Molecular Net particle to a specific cell type, tissue type, organ type, or otherwise. The targeting agent may be a capture molecule of a molecular net. Targeting agents may include: antibodies, receptors, ligands, anti-ligands, or others. Targeting agents in the Molecular Net can be covalently linked to capture molecules, linkers and spacers in the surface exposed layer. Targeting agents can also contribute to the topological characteristics of molecular nets.

example

Example 1. Comparison of conventional 2D and 3D Molecular Net microparticles for analyte purification.

Molecular nets consisting of monomeric protein G and connexin G and crosslinkers BS3, EMCS, EGS, ^BMPH and others were used in the fabrication. Molecular nets were fabricated in real time on the surface of 0.8-10um magnetic polystyrene microparticles and 45um nitrocellulose microparticles. In some instances, a capture molecule (Protein G) was used as the only source of structural support. In some examples, pre-attached protein G and monomeric protein G were mixed to serve as additional structural support for making some layers of the molecular net. In some other examples, however, a first layer of a molecular net comprising protein G and an IgFc region is used as a structural support for making additional layers of the molecular net. In some examples, the Protein G Molecular Net includes 2 layers, and in other examples, the Protein G Molecular Net includes 3 layers. The final layer of the Molecular Net includes several topological features in order to enhance the binding and recovery of the analyte (IgG in this case) from the sample. Figure 1 is an example of data obtained using Protein G Molecular Net microparticles compared to commercial Protein G microparticles. Briefly, IgG-Alexa647 was added to human serum (lug/tube). Uncoated microparticles, commercial Protein G microparticles, and Protein G Molecular Net particles were incubated with spiked samples for 15-60 minutes at room temperature (100,000 particles (uncoated control), 100,000 particles (commercial) and 25,000 particles (molecular nets)). Particles were separated from the sample using magnetic separation and washed three times in PBST. The pellet was suspended in 2xLSB, boiled and loaded on SDS-PAGE. Recovered IgG was measured by Coomassie-stained band densitometry compared to input controls. Depicted in Figure 1 is the recovery of the input for each purification type. Use of Optimized Molecular Nets can reduce background noise and increase visible signal in assays.

Example 2. Effectiveness of conventional 2D conjugates for analyte detection.

The traditional method of covalently attaching capture antibodies to a surface is a two-dimensional approach (X and Y planes) because there is no additional layer added to the surface of the attached antibody on a surface. Traditional methods for covalently attaching capture antibodies to a surface involve a single type of linker, such as EDC, NHS, sulfo-NHS or others. Occasionally, a second linker is used to immobilize the capture antibody to a surface, but involves removal of the first linker and does not add additional height or layering to the antibody-conjugated surface. In one example, an anti-human neuroserin antibody was coupled to Luminex particles (bead zone #54) via a linker according to the manufacturer's instructions. The particles were then quenched, blocked and washed prior to use. Incubate the pellet in pre-cleaned serum + neurofilin at a concentration range (0-1 ng/mL) for 15 min. Bound particles were washed and incubated with biotin-anti-neurofilin (10 ng/mL) for 15 minutes. Neurofilin detection was visualized by avidin-PE (30 ng/mL) for 15 min. The washed particles were subsequently analyzed on a Luminex 100, collecting 100 particles per sample. Presented in Figure 2 is the median fluorescence intensity (FI) above background fluorescence intensity for each dilution.

Figure 3. Effect effectiveness of Molecular Net microparticles in measuring analytes.

Fabricate molecular nets consisting of the same anti-human neurofilin antibody (as shown in Figure 2) and linkers sulfo-NHS, EMCS, EGS, BMPH, and others to provide a three-dimensional multilayer (X, Y, and Z plane ) matrix. These molecular nets can then be covalently attached to Luminex microparticles (bead block #54). The assay performance utilizing 4-layer Molecular Nets is presented in FIG. 3 . Improved assay MFI was observed using three-dimensional multilayer molecular nets.

Figure 4. Effectiveness of molecular nets with topology in measuring analytes.

Fabricate molecular nets consisting of the same anti-human neurofilin antibody (as shown in Figure 2 and Figure 3) and linkers sulfo-NHS, EMCS, EGS, BMPH, and others to provide a three-dimensional multilayer (X, Y and Z plane) substrate. These molecular nets can then be covalently attached to Luminex microparticles (bead block #54). The assay performance utilizing 5-layer Molecular Nets is presented in FIG. 4 . Improved assay MFI was observed using three-dimensional multilayer molecular nets with enhanced topology in the outer layer.

Figure 5. Comparison of conventional conjugated microparticles and Molecular Net microparticles in ELISA.

Molecular Nets consisting of monoclonal antibodies against human-TauF and cross-linkers Sulfo-NHS, EMCS, EGS, BMPH and others were used in the fabrication. Molecular nets were fabricated in real time on the surface of 0.5um, 6.3um and 10um magnetic microparticles. In some examples, a capture molecule, an anti-Tau monoclonal antibody, was used as the only source of structural support. In some examples, spacer albumin is mixed with anti-Tau monoclonal antibody in a molar ratio of 1.5:1.0 (albumin:anti-Tau antibody) in a first layer to be used as the first layer for making the first layer. additional structural support. In some examples, a second capture molecule, human tubulin, is used, which provides both structural support and capture in the Molecular Net. Figure 5 is an example of data obtained using anti-Tau Molecular Net (LV.P6Cap-TECH) compared to a commercial Tau microparticle (partner LV) ELISA (same assay conditions, same antibody pair, etc.). Figure 5 is an example of using Molecular Nets to reduce background noise in an assay and increase visible signal in an ELISA.

Figure 6. Comparison of traditionally conjugated microparticles and Molecular Net microparticles using Luminex.

Molecular nets consisting of monoclonal antibodies against human-thyrotropin and cross-linkers EDC, BS(PEG) ₉ , EMCS, EGS, BMPH and others were used in the manufacture. Molecular Net fabrication is performed in real time on the surface of Luminex magnetic microparticles. In some examples, capture molecules, anti-TSH monoclonal antibodies, were used as the only source of structural support. In some examples, spacers (PEG, heat-denatured lysozyme, and others) are mixed with anti-TSH monoclonal antibodies in a molar ratio of 1.0:2.0 (spacer:anti-TSH antibody) in a first layer so that Serves as an additional structural support for making this first layer. In some examples, an anti-TSH molecular net includes 4 layers, and in other examples, an anti-TSH molecular net includes 6 layers, where the last layer includes multiple topologies to enhance the analyte in Luminex combination and performance. Figure 6A is an example of the use of Molecular Nets to increase overall MFI in a Luminex assay. Figure 6B is exemplary data obtained in the Luminex assay for increasing the minimum level detected in the Luminex assay.

Figure 7. Exemplary molecular nets on particles.

Figure 7 depicts some examples where Molecular Nets can be placed on the surface of a particle. In some examples, molecular nets are placed on a particle surface in a circumferential fashion (FIG. 7A, 1001), whereby the molecular nets with X, Y, and Z spatial orientations can be fairly symmetrical, and where each layer ( Instance of 3 layers, 1002, 1003 and 1004) are added on the Z plane of the particle. In some examples, Molecular Nets can be placed on a particle surface in an asymmetric manner (FIG. 7B, 1005), whereby Molecular Nets with X, Y, and Z spatial orientations can be placed in a manner that creates the polarity of the particle. on the surface of the particle, and where each layer (example of 3 layers, 1006, 1007, and 1008) is added in a layer-independent manner (e.g., some layers may have a smaller height and other layers may have a higher height) to the Z plane of the particle. In other examples, molecular nets can be placed in an asymmetric manner on a portion of the particle surface (FIG. 7C, 1009), whereby molecular nets with X, Y, and Z spatial orientations can be placed in a manner that creates the polarity of the particle on the surface of the particle, and where each layer (example of 3 layers, 1010, 1011, and 1012) is layer-independent (for example, some layers may have specificity for one analyte and others may have specificity for Specificity for other analytes) are added to the Z-plane of the particle.

Figure 8. Exemplary Molecular Net topological features on particles.

Figure 8 depicts some examples where molecular nets can be placed on the surface of a particle. In some examples, molecular nets are placed on a particle surface in a circumferential manner (FIG. 8A, 2001), whereby molecular nets with X, Y, and Z spatial orientations can be fairly symmetrical within a layer (2002) , and where each layer (example of 3 layers, 2002, 2003, and 2004) is added to the particle's Z-plane in a different and asymmetric manner (eg, creating topology). In some examples, molecular nets can be placed on the surface of a particle in an asymmetric manner (Figure 8B, 2005), whereby molecular nets with spatial orientations in X, Y, and Z can be distributed throughout the particle's layer 1 (2006) , 2 (2007) and 3 (2008) to generate structural features (2008) are placed on the surface of the particle, and where each layer is layer-independent (for example, some layers may have a smaller height and other layers can have a higher height) added to the Z-plane of the particle. In addition, the structural elements in each layer can also function as analyte capture in the molecular net. In other examples, molecular nets can be placed in an asymmetric manner on a portion of the particle surface (FIG. 8C, 1009), whereby molecular nets with X, Y, and Z spatial orientations can be placed in a manner that creates the polarity of the particle on the surface of the particle, and where each layer (example of 4 layers, 2010, 2011, 2012, and 2013) is added in a layer-independent manner to the Z-plane of the particle, and whereby each layer can function as a structure and The role of analyte capture. For example, some layers can be specific for one analyte based on size (eg, FIG. 8C, 2010), and outer layers (eg, FIG. 8C, 2013) can be specific for analytes of larger sizes.

Figure 9. Exemplary molecular nets for analyte delivery.

Figure 9 depicts examples where Molecular Nets can be placed on a particle surface for analyte capture or targeted analyte delivery. In some examples, molecular nets are placed on a particle surface in a circumferential manner (FIG. 9A, 3001), whereby molecular nets with X, Y, and Z spatial orientations can be fairly symmetrical within a layer (3002) , and where each layer (example of 3 layers, 3002, 3003, and 3004) is added to the particle's Z-plane in a different and asymmetric manner (eg, creating topology). In some examples, the analyte cargo (3003) can be preloaded onto capture molecules in one or more layers of the Molecular Net. In the outer layer, different capture molecules can be linked into one molecular net in order to generate topologies and/or affinities for different target analytes. In some examples, preloaded analytes can include drugs, therapeutics, siRNA, miRNA, dsRNA, viruses, toxins, immunogens, or others. The preloaded cargo may be non-covalently associated with one or more types of capture molecules in a layer of the Molecular Net. In some examples, the different capture molecules (3004) of the Molecular Net can be arranged in the outer layer of the Molecular Net and can function as a topology and analyte capture. Different capture molecules can have specificity for one or more different analytes, which can include antibodies, anti-ligands, ligands, receptors, antigens, or others, and can act as one or more Structure and/or affinity and/or targeting.

In some examples, the Molecular Net can be placed on the surface of the particle in a manner (FIG. 9B, 3005) whereby the analyte cargo (3006) can be preloaded into all layers of the Molecular Net. In some layers, capture molecules can be used to generate topological features (3008) that function as particle targeting. In some examples, the outer layer of a Molecular Net particle can target the particle to a specific cell, tissue, organ, or otherwise.

Figure 10. Molecular Nets used to purify analytes from samples

Molecular Nets can be used during sample purification (examples are provided in Figure 10). In some examples, Molecular Nets designed and fabricated to deplete one or more analytes can be used to process samples for analyte depletion. Exemplary methods can include incubating the Molecular Net with the sample in a batch slurry or in a chromatography column for about 15 minutes to about 24 hours. Sample supernatant or flow-through can be collected according to preferred methods. Molecular nets can be collected and analyzed for the presence and amount of captured analytes using different methods. Molecular Net processed samples can be collected and analyzed using different methods to determine the residual presence of an analyte in the sample, or the sample can be analyzed for other analytes.

Figure 11. Molecular Nets for detection and measurement of analytes from samples

Molecular Nets can be used in analyte measurement tools or diagnostic tools (examples are provided in Figure 11). In some examples, Molecular Nets designed and fabricated to capture one or more analytes can be used to process samples for analyte detection and measurement. Exemplary methods can include incubating the Molecular Net with the sample in a batch slurry, cassette, slide, microtiter plate, or otherwise for about 15 minutes to about 2 hours. Sample supernatant or flow-through can be collected according to preferred methods. Molecular nets can be collected and analyzed for the presence and amount of one or more captured analytes using different methods. Molecular Net processed samples can also be collected and analyzed using different methods to measure other analytes. Methods for measuring changes in molecular net characteristics may include optical, electrophoretic, electrical, magnetic, chemical, thermal, or other methods.

While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps in order to realize the benefits provided by the invention without departing from the scope of the invention. All such modifications are intended to come within the scope of the appended claims.

All publications and patent documents cited herein are hereby incorporated by reference as if each such publication or document were specifically and individually indicated to be incorporated by reference. Citation of publications and patent documents is not intended to indicate that any such document is pertinent prior art, nor does it constitute any admission as to its content or date.

Claims (52)

1. A device for capturing an analyte, the device comprising: a solid phase; and a molecular net coupled to at least a portion of the surface of the solid phase, the molecular net comprising at least one type of capture molecule coupled to each other via multiple types of linker molecules so as to form a covalent A multilayered three-dimensional matrix of linked, capture molecules configured to bind the analyte. 2. The device of claim 1, wherein the solid phase is made of plastics, polymers, films, colloidal metals, silica, carbon nanotubes, proteins, carbohydrates, lipids, nucleic acids, cells, and tissues Made of one or more. 3. The device of claim 1, wherein the solid phase comprises nanomaterials, modified metal surfaces, nanospheres, microspheres, microtiter plates, glass slides, pipettes, boxes, cartridges, disks, probes One or more of needles, lateral flow devices, microfluidic devices, and optical fibers. 4. The device of claim 1, wherein the molecular net is prefabricated and adsorbed onto the surface of the solid phase. 5. The device of claim 1, wherein the molecular net is covalently attached to the surface of the solid phase. 6. The device of claim 1, wherein the molecular net is built directly on the surface of the solid phase. 7. The device of claim 1, wherein the capture molecules comprise one or more of antibodies, nucleic acid probes, enzymes, recombinant proteins and peptides. 8. The device of claim 1, wherein the capture molecules comprise monoclonal antibodies for binding epitopes of the analyte. 9. The device of claim 1, wherein the capture molecules are for a plurality of mutually identified analytes. 10. The device of claim 1, wherein the linker molecules comprise one or more of homobifunctional, heterobifunctional, trifunctional and multifunctional types. 11. The device of claim 1, wherein the molecular net has at least one measurable characteristic that undergoes a change when the capture molecules bind the analyte. 12. The device of claim 11, wherein the measurable characteristic comprises physical shape, height, density, fluorescence intensity, wavelength shift, vibrational frequency, absorbance, flexibility, refractivity, conductance, impedance, resistance, melting temperature, denaturation temperature and one or more of freezing temperature. 13. The device of claim 1, wherein the capture molecules are also coupled to each other through types of spacer molecules. 14. The device of claim 13, wherein the spacer molecules comprise one or more of PEG, polymers, nucleic acids, albumin, Fc regions, and peptides. 15. The device of claim 13, wherein the spacer molecules and an amount of spacer molecules are selected to impart one or more desired physical properties to the molecular net. 16. The device of claim 15, wherein the desired physical properties include one or more of porosity, charge distribution, and topological characteristics. 17. A method of making a device for capturing an analyte, the method comprising: provide a solid phase; and placing a molecular net on at least a portion of the surface of the solid phase, the molecular net comprising at least one type of capture molecule linked to each other by various types of linker molecules so as to form a covalently linked multiple Layering the three-dimensional matrix, the capture molecules are configured to bind the analyte. 18. The method of claim 17, wherein placing a molecular net comprises prefabricating the molecular net and adsorbing the molecular net onto the surface of the solid phase. 19. The method of claim 17, wherein placing a molecular net comprises covalently attaching the molecular net to the surface of the solid phase. 20. The method of claim 17, wherein placing a molecular net comprises building the molecular net directly on the surface of the solid phase. 21. A method of measuring the amount of an analyte in a sample, the method comprising: One or more devices are provided, each device comprising a solid phase and a molecular net covering at least a portion of the surface of the solid phase, the molecular net comprising at least one type of capture molecule passed through a plurality of Linker molecules of the type linked to each other so as to form a covalently linked multilayer three-dimensional matrix, these capture molecules are configured to bind the analyte; exposing the devices to the sample; and The capture molecules of the molecular nets of the devices are allowed to bind at least a portion of the analyte. 22. The method of claim 21, further comprising measuring changes in the sample. 23. The method of claim 22, wherein measuring the change comprises using a photon multichannel analyzer, a spectrometer, a magnetic resonance imager, a magnetic field detector, an optical fiber, a glass pipette, an electrical circuit, a fluorometer, a spectral analysis One or more of detectors, potentiostats, calorimeters, electrophoresis, flow cytometers, CCD cameras, microscopes, sound chambers, loudspeakers, and photometers. 24. The method of claim 21, wherein the molecular net has at least one measurable characteristic that undergoes a change when the capture molecules bind the analyte. 25. The method of claim 24, further comprising measuring changes in the measurable characteristic of the molecular nets. 26. The method of claim 25, wherein measuring the change comprises using a photon multichannel analyzer, a spectrometer, a magnetic resonance imager, a magnetic field detector, an optical fiber, a glass pipette, an electrical circuit, a fluorometer, a spectral analysis One or more of detectors, potentiostats, calorimeters, electrophoresis, flow cytometers, CCD cameras, microscopes, sound chambers, loudspeakers, and photometers. 27. A device for capturing an analyte comprising: a solid phase; and layers coupled to at least a portion of the surface of the solid phase, the layers comprising a molecular net comprising at least one type of capture molecule coupled by types of linker molecules to each other so as to form a covalently linked multilayer three-dimensional matrix, the capture molecules are configured to bind the analyte. 28. The device of claim 27, wherein the solid phase is composed of plastics, polymers, films, colloidal metals, silica, carbon nanotubes, proteins, carbohydrates, lipids, nucleic acids, cells, and tissues Made of one or more. 29. The device of claim 27, wherein the solid phase comprises nanomaterials, modified metal surfaces, nanospheres, microspheres, microtiter plates, glass slides, pipettes, boxes, cartridges, disks, probes One or more of needles, lateral flow devices, microfluidic devices, and optical fibers. 30. The device of claim 27, wherein the molecular net is prefabricated and adsorbed onto the surface of the solid phase. 31. The device of claim 27, wherein the Molecular Net is covalently attached to the surface of the solid phase. 32. The device of claim 27, wherein the molecular net is built directly on the surface of the solid phase. 33. The device of claim 27, wherein the capture molecules comprise one or more of antibodies, nucleic acid probes, enzymes, recombinant proteins, and peptides. 34. The device of claim 27, wherein the capture molecules comprise monoclonal antibodies for binding epitopes of the analyte. 35. The device of claim 27, wherein the capture molecules are for a plurality of mutually identified analytes. 36. The device of claim 27, wherein the linker molecules comprise one or more of homobifunctional, heterobifunctional, trifunctional and multifunctional types. 37. The device of claim 27, wherein the molecular net has at least one measurable characteristic that undergoes a change when the capture molecules bind the analyte. 38. The device of claim 37, wherein the measurable characteristic comprises physical shape, height, density, fluorescence intensity, wavelength shift, vibrational frequency, absorbance, flexibility, refractivity, conductance, impedance, resistance, melting temperature, denaturation temperature and freezing temperature. 39. The device of claim 27, wherein the capture molecules are also coupled to each other by types of spacer molecules. 40. The device of claim 39, wherein the spacer molecules comprise one or more of PEG, polymers, nucleic acids, albumin, Fc regions, and peptides. 41. The device of claim 39, wherein the spacer molecules and an amount of spacer molecules are selected to impart one or more desired physical properties to the molecular net. 42. The device of claim 41, wherein the desired physical properties include one or more of porosity, charge distribution, and topological characteristics. 43. A method of making a device for capturing an analyte, the method comprising: provide a solid phase; and placing layers on at least a portion of the surface of the solid phase, the layers comprising a molecular net comprising types of capture molecules linked to each other by types of linker molecules so that Forming a covalently linked multilayered three-dimensional matrix, the capture molecules are configured to bind the analyte. 44. The method of claim 43, wherein placing layers comprises prefabricating the layers and adsorbing the layers onto the surface of the solid phase. 45. The method of claim 43, wherein placing layers comprises covalently attaching the layers to the surface of the solid phase. 46. The method of claim 43, wherein placing layers comprises building the layers directly on the surface of the solid phase. 47. A method of measuring the amount of an analyte in a sample, the method comprising: providing one or more devices, each device comprising a solid phase and layers covering at least a portion of the surface of the solid phase, the layers comprising a molecular net comprising at least one type of capture molecule, The capture molecules are linked to each other by various types of linker molecules to form a covalently linked multilayered three-dimensional matrix, the capture molecules are configured to bind the analyte; exposing the devices to the sample; and The capture molecules of the molecular nets of the devices are allowed to bind at least a portion of the analyte. 48. The method of claim 47, further comprising measuring changes in the sample. 49. The method of claim 48, wherein measuring the change comprises using a photon multichannel analyzer, a spectrometer, a magnetic resonance imager, a magnetic field detector, an optical fiber, a glass pipette, an electrical circuit, a fluorometer, a spectral analysis One or more of detectors, potentiostats, calorimeters, electrophoresis, flow cytometers, CCD cameras, microscopes, sound chambers, loudspeakers, and photometers. 50. The method of claim 47, wherein the molecular net has at least one measurable characteristic that undergoes a change when the capture molecules bind the analyte. 51. The method of claim 50, further comprising measuring changes in the measurable characteristic of the molecular nets. 52. The method of claim 51 , wherein measuring the change comprises using a photon multichannel analyzer, a spectrometer, a magnetic resonance imager, a magnetic field detector, an optical fiber, a glass pipette, an electrical circuit, a fluorometer, a spectral analysis One or more of detectors, potentiostats, calorimeters, electrophoresis, flow cytometers, CCD cameras, microscopes, sound chambers, loudspeakers, and photometers.