Biosensors comprising charged biopolymers and uses thereof

Abstract

A biosensor for detecting the presence or amount of a charged analyte in a sample is provided. The biosensor may include a core component and a charged biopolymer component, the charged biopolymer component including one or more layers of a charged biopolymer. An outermost layer of the charged biopolymer component is oppositely charged relative to the charged analyte and is capable of binding to the charged analyte. The biosensor may comprise two or more alternating layers of oppositely charged biopolymers, each layer being oppositely charged relative to an adjacent layer. Also provided are devices coupled to the biosensor for quantifying a signal resulting from binding of the biosensor to the charged analyte, and methods of using the biosensor to detect the presence or amount of a charged analyte in a sample.

Description

Technical Field

[0001] The present disclosure relates to biosensors and related methods for identifying and quantifying compounds of interest. Background Art

[0002] Biosensors can be used to detect the presence or amount of an analyte in a sample and facilitate, for example, the diagnosis and treatment of a disease or scientific research and development. Biosensors can employ solid surfaces with immobilized anti-analyte molecules to which the sample analyte molecules bind specifically and with high affinity in a defined detection zone. The binding event can be detected directly, for example, by a change in mass, reflectivity, thickness, color, or other characteristics indicative of a binding event. The analyte can be pre-labeled, for example, with a chromophore, fluorophore, or radioactive marker. Alternatively, the analyte can be labeled after binding to the detection zone, for example, with a labeled anti-analyte secondary antibody.

[0003] Current biosensors are primarily based on antibody-antigen or receptor-ligand interactions and typically involve labeling the analyte for detection. For example, typical current analyte-antianalyte pairs include antigen-antibody pairs or ligand-receptor pairs, where the analyte can be either member of the pair and the biosensor is coated with an anti-analyte that is the opposite member of the pair. The analyte, such as an antigen, antibody, ligand, or receptor, can be detected using a detectable signal, such as a chromophore, fluorophore, radiolabel, or labeled anti-analyte secondary antibody attached to the analyte before or after binding to the biosensor. In view of the foregoing, there is a need for effective biosensors based on coating chemistries that are based on molecular or biological interactions other than antigen-antibody or ligand-receptor interactions for sensing and quantifying various compounds. Summary of the Invention

[0004] The present disclosure generally relates to devices and methods that address these shortcomings in the art and provide other additional or alternative advantages. The present disclosure provides embodiments of biosensors for detecting the presence or amount of a charged analyte in a sample. The biosensor can be coated with one or more layers of charged biopolymers, wherein the outermost layer of the charged biopolymer is oppositely charged relative to the charged analyte and is capable of binding to the charged analyte. The biosensor can comprise two or more alternating layers of oppositely charged biopolymers, wherein each layer is oppositely charged relative to the adjacent layers. In some cases, the biosensor can directly detect the binding of the analyte without the use of a detectable label attached to the analyte. Also provided are devices coupled to the biosensor for quantifying the signal generated by the binding of the biosensor to the charged analyte, as well as methods for using the biosensor to detect the presence or amount of a charged analyte in a sample.

[0005] In certain aspects of the present disclosure, a biosensor for detecting the presence or amount of a charged analyte in a sample is provided. The biosensor comprises a core component and a charged biopolymer component. The charged biopolymer component comprises one or more layers of a charged biopolymer. The outermost layer of the charged biopolymer component is oppositely charged relative to the charged analyte to promote binding of the charged biopolymer component to the charged analyte in the sample and generate a signal indicative of the amount of charged analyte bound to the biosensor.

[0006] In embodiments, the biosensor comprises two or more layers of charged biopolymer. The first layer of the charged biopolymer (the innermost layer adjacent to the core component) is bound to the outer surface of the core component. Each layer of the charged biopolymer is oppositely charged relative to and bound to an adjacent layer (the inner adjacent layer and, when present, the outer adjacent layer). The outermost layer of the charged biopolymer constitutes the outer surface of the charged biopolymer component of the biosensor. Thus, the outermost layer is oppositely charged relative to the charged analyte to facilitate binding of the charged analyte to the outermost layer of the charged biopolymer and generate a signal indicative of the amount of charged analyte bound to the biosensor.

[0007] In some embodiments, the core component of the biosensor is a surface that may contain aminopropylsilane (APS)-functionalized silica (SiO2) or epoxypropylsilane (EPS)-functionalized SiO2. The negatively charged biopolymer of the biosensor's negatively charged layer (if present) may contain dextran, DNA, carboxylic acid (COOH)-functionalized beads, and / or other polysaccharide compounds. The positively charged biopolymer of the biosensor's positively charged layer (if present) may contain polyethyleneimine (PEI), chitosan, poly-L-lysine, polyallylamine, polyethylenimine, and / or other amine-functionalized compounds. The charged analyte may include a protein, viral vector, or polynucleotide.

[0008] In certain embodiments, the outermost layer of the charged biopolymer is negatively charged, allowing the outermost layer of the charged biopolymer to bind to a positively charged analyte. The positively charged analyte may include a positively charged protein, such as bovine serum albumin (BSA), neutravidin, or ovalbumin.

[0009] In certain other embodiments, the outermost layer of the charged biopolymer is negatively charged, allowing the outermost layer of the charged biopolymer to bind to a negatively charged analyte. The negatively charged analyte may include a negatively charged protein, polynucleotide, or viral vector. The viral vector may include an adeno-associated virus (AAV) vector.

[0010] In certain embodiments, different amounts, concentrations, and / or charges of charged analytes in the sample result in the biosensor having different binding kinetics for the charged analytes contained in the sample. For example, the charged analytes may comprise a population of viral capsids, which may include intact viral capsids containing polynucleotides and / or empty viral capsids not containing polynucleotides, and different intact / empty viral capsid ratios in the sample may result in the biosensor having different binding kinetics for the viral capsids contained in the sample. The population of viral capsids may include capsids of adeno-associated viruses (AAV).

[0011] In embodiments, the biosensor is coupled to a signal analyzer capable of converting and quantifying a signal generated by binding of the biosensor to the charged analyte. The signal analyzer may quantify the signal using one or more label-free techniques, including but not limited to biolayer interferometry (BLI), quartz crystal microbalance (QCM), surface plasmon resonance (SPR), surface acoustic wave (SAW), atomic force microscopy, and reflectance interferometry spectroscopy (RIfS).

[0012] In certain aspects of the present disclosure, a device for quantifying a charged analyte in a sample is provided. The biosensor comprises a core component and a charged biopolymer component. The charged biopolymer component comprises one or more layers of a charged biopolymer. The outermost layer of the charged biopolymer component is oppositely charged relative to the charged analyte to promote binding of the charged biopolymer component to the charged analyte in the sample and generate a signal indicative of the amount of charged analyte bound to the biosensor. The signal analyzer is capable of converting and quantifying the signal generated by the binding of the biosensor to the charged analyte.

[0013] In embodiments, the charged biopolymer component of the biosensor comprises two or more layers of charged biopolymer. The first layer of the charged biopolymer (the innermost layer adjacent to the core component) may be bound to the outer surface of the core component. Each layer of the charged biopolymer is oppositely charged relative to and bound to an adjacent layer (the inner adjacent layer and, when present, the outer adjacent layer). The outermost layer of the charged biopolymer constitutes the outer surface of the charged biopolymer component of the biosensor and is oppositely charged relative to the charged analyte to facilitate binding of the charged analyte to the outermost layer of the charged biopolymer and generate a signal indicative of the amount of charged analyte bound to the biosensor.

[0014] In embodiments, the signal is measured by biolayer interferometry (BLI), quartz crystal microbalance (QCM), surface plasmon resonance (SPR), surface acoustic wave (SAW), atomic force microscopy, or reflectance interferometry spectroscopy (RIfS). The core component of the biosensor is a surface that may contain aminopropylsilane (APS)-functionalized silica (SiO2) or epoxypropylsilane (EPS)-functionalized silica (SiO2). The negatively charged biopolymer of the negatively charged layer of the biosensor (if present) may contain dextran, DNA, carboxylic acid (COOH)-functionalized beads, and / or other polysaccharide compounds. The positively charged biopolymer of the positively charged layer of the biosensor (if present) may contain polyethyleneimine (PEI), chitosan, poly-L-lysine, polyallylamine, polyethylenimine, and / or other amine-functionalized compounds.

[0015] In certain aspects of the present disclosure, a method for detecting the presence or absence of a charged analyte in a sample is provided. The method includes contacting the sample with a biosensor, promoting binding of the charged analyte in the sample to a charged biopolymer component of the biosensor, and detecting the presence or absence of the charged analyte. The biosensor includes a core component and a charged biopolymer component. The outermost layer of the charged biopolymer component is oppositely charged relative to the charged analyte and allows the biosensor to bind to the charged analyte. The charged analyte is quantified at least in part based on a signal generated by the binding of the charged analyte to the biosensor and indicating the amount of charged analyte bound to the biosensor and / or the binding kinetics of the biosensor for the charged analyte. Different amounts, concentrations, and / or charges of the charged analytes in the sample result in different binding kinetics of the biosensor for the charged analyte in the sample.

[0016] The charged biopolymer component of the biosensor can include one or more layers of charged biopolymer. If the biosensor includes two or more layers of charged biopolymer, the first layer of the charged biopolymer (the innermost layer adjacent to the core component) can be bound to the outer surface of the core component. Each layer of the charged biopolymer is oppositely charged relative to and bound to an adjacent layer (the inner adjacent layer and, if present, the outer adjacent layer). The outermost layer of the charged biopolymer constitutes the outer surface of the charged biopolymer component of the biosensor and is oppositely charged relative to the charged analyte, enabling the charged analyte to bind to the outermost layer of the charged biopolymer.

[0017] In an embodiment, said signal and / or said binding kinetics are measured by bio-layer interferometry (BLI), quartz crystal microbalance (QCM), surface plasmon resonance (SPR), surface acoustic wave (SAW), atomic force microscopy or reflection interferometry spectroscopy (RIfS).

[0018] In embodiments, the charged analyte comprises one or more viral vectors. In such embodiments, the viral titer of the sample is determined at least in part based on a signal generated by binding of the one or more viral vectors to the biosensor (e.g., based at least in part on a binding rate). Additionally or alternatively, the empty / intact viral capsid ratio of the sample is determined at least in part based on the binding kinetics of the one or more viral vectors to the biosensor (e.g., based at least in part on a saturation analysis of binding of the one or more viral vectors to the biosensor).

[0019] Furthermore, any embodiment or aspect described herein may be used in combination with any or all other embodiments or aspects described herein, to the extent consistent. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Various aspects of the disclosure are described below with reference to the accompanying drawings, which are incorporated into and constitute a part of this specification.

[0021] Figures 1A-1C Schematic diagram of a biosensor coated with a charged biopolymer. Figure 1A Depicted is a biosensor coated with a layer of a negatively charged biopolymer (eg, a polysaccharide such as dextran). Figure 1B Depicted is a biosensor coated with a layer of COOH-functionalized beads. Figure 1C Depicted are biosensors having two layers of oppositely charged biopolymers, such as a first layer of a negatively charged biopolymer (e.g., dextran) and a second layer of a positively charged biopolymer (e.g., polyethyleneimine). Figure 1D Depicted is a biosensor having three layers of charged biopolymers, each layer having an opposite charge compared to the adjacent layers, eg, first and third layers of negatively charged biopolymers, and a second layer of positively charged biopolymers.

[0022] Figure 2A A schematic depicts an exemplary process for alternating biosensor coating with oppositely charged biopolymers. Sequentially, the stages include equilibration (biosensor coated with negatively charged biopolymer), coating with polyethyleneimine (PEI) in HO, washing, coating with negatively charged biopolymer, washing, and coating with PEI. The signal change (nm shift) reflects biopolymer binding.

[0023] Figure 2B A schematic depicts an exemplary process for alternating biosensor coatings with oppositely charged biopolymers. By adjusting the loading concentration of the charged biopolymers on the biosensor, the biosensor's thickness can be customized. Signal changes (nm shifts) reflect biopolymer binding.

[0024] Figure 3 Graph illustrating the direct binding of adeno-associated virus type 8 (AAV8) capsids of varying titers but identical empty / intact ratios to a biolayer interferometry (BLI) biosensor coated with alternating layers of oppositely charged biopolymers. The BLI biosensor, coated with oppositely charged biopolymers, was contacted with samples containing AAV8 capsids of varying titers, and the signal change (nm shift) was measured as an indicator of the amount of AAV capsids bound to the BLI biosensor during an exemplary process that included a pre-loaded negatively charged biopolymer equilibration phase, a positively charged biopolymer loading phase, and a capsid capture phase.

[0025] Figure 4 Schematic representation of direct binding of intact AAV2 capsids to a BLI biosensor coated with alternating layers of oppositely charged biopolymers. The BLI biosensor coated with charged biopolymers was exposed to AAV2 samples of equal titer but varying empty / intact capsid ratios, and the signal change (nm shift) was measured as an indicator of the amount of AAV2 capsids bound to the BLI biosensor during an exemplary process that included a pre-loaded negatively charged biopolymer equilibration phase, a positively charged biopolymer loading phase, and a capsid capture phase.

[0026] Figure 5 is a flow chart of a method for detecting, characterizing, and / or quantifying a charged analyte in a sample using a biosensor, according to an embodiment of the present disclosure. DETAILED DESCRIPTION

[0027] The present disclosure will now be described more fully hereinafter with reference to exemplary embodiments and accompanying drawings of the present disclosure. These exemplary embodiments are described so that this disclosure will be thorough and complete and will fully convey the scope of the disclosure to those skilled in the art. Features from one embodiment or aspect may be combined with features from any other embodiment or aspect in any appropriate combination. For example, any individual or collective feature of a method aspect or embodiment may be applied to an apparatus, product, or component aspect or embodiment, and vice versa. The present disclosure may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

[0028] As used herein, a "biosensor" refers to a sensing and / or analytical device that uses biomolecules (e.g., enzymes, antibodies, or biopolymers) or living organisms to detect the presence, characteristics, or amount of a substance (analyte). A biosensor can employ a solid surface with immobilized anti-analyte molecules to which sample analyte molecules bind specifically and with high affinity in a defined detection zone. In this type of assay, known as a solid-phase assay, the solid surface is exposed to the sample under conditions that promote binding of the analyte to the immobilized anti-analyte molecules. The binding event can be detected directly, for example, by a change in mass, reflectivity, thickness, color, or other characteristic indicative of a binding event. If the analyte is pre-labeled, for example, with a dye, chromophore, fluorophore, or radioactive label, the binding event can be detected by the presence and / or amount of a detectable label at the detection zone. Alternatively, the analyte can be labeled after binding to the detection zone, for example, with a fluorescently labeled secondary anti-analyte antibody.

[0029] As used herein, "analyte" refers to a substance whose presence, characteristics, or amount is analyzed by the biosensors provided herein. As used herein, "analyte-binding" molecules or "anti-analytes" refer to molecules or compounds that are capable of participating in a specific binding reaction with an analyte molecule. Analyte-anti-analyte pairs can include antigen-antibody pairs, ligand-receptor pairs, complementary polynucleotide pairs, or pairs of positively and negatively charged molecules, wherein the analyte can be either member of the pair and the anti-analyte is the opposite member of the pair. Anti-analytes can be immobilized or coated on a biosensor for binding and detection of analytes.

[0030] "Binding" or "binding reaction" refers to an attractive interaction between two molecules that results in a stable association of the molecules in close proximity. Binding is saturable, can be reversible, and can be competed with excess amounts of one reactant. Binding reactions are characterized by complementarity in shape, charge, and other binding determinants between the participants in a specific binding reaction.

[0031] As used herein, "biopolymer" refers to a polymer produced or derived from a living organism (e.g., plants and microorganisms) or chemically synthesized from biological materials. Biopolymers are composed of monomeric units that are covalently bonded into chains to form larger molecules. Biopolymers include polynucleotides (e.g., RNA and DNA), polypeptides (proteins and amino acid chains, such as collagen, actin, and fibrin), and polysaccharides (straight or branched chains of sugar carbohydrates, such as starch, cellulose, and alginate). Biopolymers also include natural rubber (a polymer of isoprene), suberin and lignin (complex polyphenol polymers), cutin and keratin (complex polymers of long-chain fatty acids), and melanin.

[0032] As used herein, "charged" when referring to an analyte, biosensor, or biopolymer means that the analyte, biosensor, or biopolymer possesses an electric charge due to a functional group. The charge can be positive or negative. As used herein, "oppositely charged" means that one material is positively charged and the other is negatively charged. Oppositely charged materials can bind to each other through electrostatic forces (Coulomb forces).

[0033] In the context of coating or loading a structure with a biopolymer, the term "coating" or "loading" refers to the process of bringing an equilibrated sample (e.g., a biopolymer) into contact with an equilibrated solid phase (e.g., to assemble a biosensor). Coating or loading can be performed, for example, using a chromatography device by passing the sample through the device using an external force (e.g., gravity), by pumping, or by immersing a well plate containing the sample.

[0034] "Antibody" refers to an immunoglobulin molecule having two heavy chains and two light chains produced by any method known in the art or later developed, and includes polyclonal antibodies, such as those produced by immunizing a mammal, such as a goat, mouse, rabbit, etc., with an immunogen, as well as monoclonal antibodies produced using the well-known Kohler Milstein hybridoma fusion technique. The term includes antibodies produced using genetic engineering methods, such as those produced using SCID mice reconstituted with human immunoglobulin genes, as well as antibodies humanized using surface reconstitution techniques known in the art. Antibody also refers to antibody fragments. "Antibody fragments" refer to fragments of antibody molecules produced by chemical cleavage or genetic engineering techniques, as well as single chain variable fragments (SCFv), such as those produced using combinatorial genetic libraries and phage display techniques. Antibody fragments used in accordance with the present disclosure generally retain the ability to bind their cognate antigen, and thus include variable sequences and antigen binding sites, which are within the scope of antibodies.

[0035] "Vector," as used herein, refers to a recombinant plasmid or virus which comprises a nucleic acid to be delivered to a host cell in vitro or in vivo.

[0036] "Polynucleotide," as used herein, refers to a biopolymer comprising multiple nucleotide monomers covalently bonded in a chain, including DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugar and phosphate groups (as can typically be found in RNA or DNA) or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a synthetic subunit such as a phosphoramidate, and thus can be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidatc-phosphodiester oligomer. Furthermore, a double-stranded polynucleotide can be obtained from a chemically synthesized single-stranded polynucleotide product by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase and appropriate primers.

[0037] Viral titer can be expressed in a variety of ways, and those skilled in the art can choose a way that is appropriate to the context. For example, the term "viral genomes (vg)" when used in reference to viral titer refers to the number of viral genomes, without regard to infectivity or function. The terms "genomic particles (gp)," "genome equivalents," "capsid particles," or "genome copies," when used in reference to viral titer, refer to the number of viral particles or viral capsids containing the recombinant viral DNA genome or RNA genome, without regard to infectivity or function. The number of capsid particles or genome particles in a particular vector preparation can be measured by standard methods, such as using fluorescent dyes or electron microscopy.

[0038] As used herein, "biolayer interferometry (BLI)" refers to an optical technique that measures macromolecular interactions by analyzing the interference pattern of white light reflected from the surface of a biosensor tip. BLI can be used to determine the kinetics and affinity of molecular interactions. For example, an anti-analyte can be loaded (immobilized) onto a biosensor, and the binding of the analyte to the biosensor can be measured using BLI. Changes in the number of molecules bound to the biosensor tip can result in a shift in the interference pattern, which is measured in real time. A "BLI biosensor" refers to a biosensor coupled to a biolayer interferometer, or a biosensor whose signal is measured using BLI.

[0039] As used herein, "quartz crystal microbalance (QCM)", "quartz microbalance (QMB)" or "quartz crystal nanobalance (QCN)" refers to a measurement method that evaluates the change in mass per unit area via the frequency change of a quartz crystal resonator. The resonance is perturbed by the addition or removal of small amounts of mass due to the growth / decay of oxides at the surface of the acoustic resonator or the deposition of thin films. The QCM can be used in a vacuum, gas phase or liquid environment. The QCM can be used to monitor deposition rates in thin film deposition systems under vacuum, determine the affinity of molecules (proteins, viruses, polymers) in a liquid to a functionalized surface with recognition sites, or study interactions between biomolecules. A "QCM biosensor" refers to a biosensor coupled to a QCM or a biosensor whose signal is measured by a QCM.

[0040] As used herein, "surface plasmon resonance (SPR)" refers to the phenomenon in which electrons in a thin metal sheet are excited by light directed at the sheet at a specific angle of incidence and propagate parallel to the sheet. At a constant light source wavelength and thin metal sheet, the angle of incidence that triggers SPR is related to the refractive index of the material, and even small changes in the refractive index will prevent the SPR from being observed. SPR biosensors can be used to detect a variety of analytes. "SPR biosensor" refers to a biosensor coupled to an SPR system, or a biosensor whose signal is measured by SPR.

[0041] As used herein, the term "proximal" refers to the portion of a device or component thereof that is closer to a user or machine using the device. The term "distal" refers to the portion of a device or component thereof that is further away from a user or machine using the device.

[0042] Unless the context clearly dictates otherwise, references to numbers without a specific number as used herein include plural references. Furthermore, although quantitative measurements, values, geometric relationships, and the like may be mentioned herein, any one or more (if not all) of these may be absolute or approximate, unless otherwise indicated, to account for acceptable variations that may occur, such as those due to manufacturing or engineering tolerances. References to values ​​or parameters herein include (and describe) embodiments for that value or parameter itself, within a range appropriate to the context, such as ±10%. For example, a reference to "X" includes a description of "X" and extends to the appropriate range. 1. Biosensors containing a charged biopolymer layer

[0043] The disclosed biosensor for detecting the presence or amount of a charged analyte in a sample comprises a core component and a charged biopolymer component. The charged biopolymer component comprises one or more layers of a charged biopolymer. The outermost layer of the charged biopolymer component is oppositely charged relative to the charged analyte, thereby promoting binding of the charged biopolymer component to the charged analyte in the sample and generating a signal indicative of the identity and / or amount of the charged analyte bound to the biosensor.

[0044] The biosensor can have two or more layers of charged biopolymers. The first layer of the charged biopolymer (the innermost layer adjacent to the core component) can be bound to the outer surface of the core component. Each layer of the charged biopolymer is oppositely charged relative to an adjacent layer (an inner adjacent layer and, when an outer adjacent layer is present, an outer adjacent layer) and is bound to the adjacent layer. For example, the first layer is an inner adjacent layer of the second layer, and the third layer is an outer adjacent layer of the second layer. The first and second layers are oppositely charged and bound to each other. The second and third layers are oppositely charged and bound to each other. The outermost layer of the charged biopolymer constitutes the outer surface of the charged biopolymer component of the biosensor. The outermost layer is oppositely charged relative to the charged analyte, thereby promoting binding of the charged analyte to the outermost layer of the charged biopolymer and promoting the generation of a signal indicative of the amount of charged analyte bound to the biosensor.

[0045] Any suitable materials can be used for the core component, negatively charged biopolymer layer, and positively charged biopolymer layer. The core component of the biosensor is the central portion to which the outer layers, such as biopolymers, are attached. The core component may contain an aminopropylsilane (APS)-functionalized silica (SiO2) or epoxypropylsilane (EPS)-functionalized silica (SiO2) surface, which facilitates the attachment of the outer layers. Without wishing to be bound by theory, the activation of APS and EPS on silica substrates is widely used in industrial, biomaterial, and medical applications to modify polymers / biomolecules. The negatively charged biopolymer of the negatively charged layer of the biosensor may contain dextran, DNA, carboxylic acid (COOH)-functionalized beads, and / or other polysaccharide compounds. The positively charged biopolymer of the positively charged layer of the biosensor may contain polyethyleneimine (PEI), chitosan, poly-L-lysine, polyallylamine, polyethylenimine, and / or other amine-functionalized compounds. In some embodiments, components of the biosensor, such as the core component, the negatively charged biopolymer layer, and the positively charged biopolymer layer, are non-toxic.

[0046] Figures 1A-1D Embodiments of the biosensors provided herein are depicted. Figure 1A Depicted is a biosensor 102 having a core component 104 (such as an APS or EPS) coated with a layer of negatively charged biopolymer 106 (such as dextran or DNA). Figure 1B Depicted is a biosensor 108 having a core component 104 (e.g., APS or EPS) coated with a layer of negatively charged biopolymer 110, which are carboxylic acid (COOH)-functionalized beads. As used herein, surface "functionalization" refers to modifying the surface properties of a material or device to introduce desired properties. As used herein, "COOH-functionalized beads" refer to beads that have -COOH groups on their surface. Figure 1C A biosensor 112 is depicted having a core component 104 (e.g., APS or EPS) coated with a layer of negatively charged biopolymer 106 (e.g., dextran, DNA, or COOH-functionalized beads) coated with a positively charged biopolymer 114 (e.g., PEI, chitosan, poly-L-lysine, polyallylamine, or polyaziridine). Figure 1D A biosensor 116 is depicted having a core component 104 (eg, APS or EPS) coated with a plurality of alternating layers of negatively charged biopolymers 106 and 118 and positively charged biopolymer 114 .

[0047] The biosensors provided herein can include other configurations of a core component and one or more layers of charged biopolymers. For example, the biosensors provided herein can include the following configurations: C – (-) – [(+) – (-)] n (Formula I); or C – (+) – [(-) – (+)] n (Formula II), Where C is a core component, (-) is a negatively charged biopolymer layer, (+) is a positively charged biopolymer layer, and n is the number of pairs of layers repeated in order from innermost to outermost, with C being the innermost layer. For example, a biosensor provided herein can comprise a core component coated with a layer of a positively charged biopolymer. A biosensor provided herein can comprise a core component coated with two or more alternating layers of oppositely charged biopolymers.

[0048] The biosensors provided herein can include any number of layers of alternating charged biopolymers. For example, the biosensors provided herein can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more layers of alternating charged biopolymers. In some embodiments, the biosensor includes two or more layers of charged biopolymers. Without wishing to be bound by theory, the multilayer structure (comprising at least a core component, a negatively charged biopolymer layer, and a positively charged biopolymer layer) provides stability to the biosensor. The combination, configuration, number of layers, and density of biopolymers can be customized to achieve optimal surface capacity and stability of the biosensor. For example, the number of charged polymer layers can be adjusted to optimize thickness, or the concentration of charged polymer loaded into the biosensor can be adjusted to optimize the surface capacity of the biosensor. In addition, the surface of the core component (e.g., glass or silica) can be etched or otherwise treated to increase the density of hydroxyl groups, which can bind to analyte binding molecules (anti-analytes). The biosensor surface can be densely coated with charged biopolymers such that binding of analyte molecules to the layer forces a change in the thickness of the layer rather than filling the layer.

[0049] The biosensors presented herein can be assembled using a layer-by-layer (LbL) assembly method. The LbL assembly technique assembles thin films by depositing (or loading) alternating layers of oppositely charged materials, followed by washing steps. The LbL assembly technique can produce ultrathin films with nanometer-scale thicknesses and orientationally controlled molecular order and stability. This involves the alternating adsorption of oppositely charged polyion and biopolymer layers, and can be applied to a variety of organic, inorganic, and biological matrices, including carbon nanotubes (CNTs), proteins, antigens, lysozyme, DNA, nanoparticles, metallophthalocyanines, and dendrimers. The LbL assembly technique is also a versatile method for fabricating controlled hierarchical structures from CNTs / enzymes using a simple, rapid, and inexpensive procedure. It can be used to immobilize biopolymers, biocatalysts, and biomaterials in well-defined hierarchical structures under mild conditions for biosensor and biofuel cell applications. LbL nanostructures, uniform and stable CNT-based assemblies of multilayered biopolymer (or enzyme) interfaces with desired architectures, offer greater control over the placement of analyte-binding molecules (e.g., polyelectrolyte or enzyme molecules) compared to random hydrogels. Furthermore, close proximity of spatially organized multilayers can be advantageous for the stability and surface capacity of biosensors or for sequential enzymatic reactions using single-enzyme and dual-enzyme cascade systems.

[0050] Figure 2A A schematic depicts an embodiment of a process for alternating coating of a biosensor with oppositely charged biopolymers using a layer-by-layer assembly technique. The exemplary stages, in chronological order, include equilibration 202, loading of a positively charged biopolymer (e.g., polyethyleneimine (PEI) in HO) 204, washing 206, loading of a negatively charged biopolymer 208, washing 210, loading of a positively charged biopolymer (e.g., PEI) 212, washing 214, and loading of a negatively charged biopolymer 216. Signal changes (nm shifts) reflect biopolymer binding.

[0051] Figure 2A The first stage 202 depicts one embodiment of the equilibration 202. The core component 218 of the biosensor is coated with a layer of negatively charged biopolymer 220.

[0052] Figure 2A The second stage 204 depicts one embodiment of the positively charged biopolymer loading stage. A positively charged biopolymer 222 (eg, PEI) is bound to the first layer of negatively charged biopolymer 220. Figure 2A The third stage 206 depicts washing with an appropriate buffer.

[0053] Figure 2AThe fourth stage 208 depicts one embodiment of a negatively charged biopolymer loading stage. A negatively charged biopolymer 224 is bound to the second layer 222, which is a positively charged biopolymer. Figure 2A The fifth stage 210 depicts a wash with an appropriate buffer.

[0054] Figure 2A The sixth stage 212 depicts one embodiment of a positively charged biopolymer loading stage. A positively charged biopolymer 226, such as PEI, is bound to the third layer 224, which is a negatively charged biopolymer. Figure 2A The seventh stage 214 depicts a wash with an appropriate buffer.

[0055] Figure 2A The eighth stage 216 depicts one embodiment of a negatively charged biopolymer loading stage. A negatively charged biopolymer 228 is bound to the fourth layer 226, which is a positively charged biopolymer.

[0056] The loading stages can be repeated many times to achieve optimal stability and binding capacity of the biosensor. At each loading stage, the density (concentration) of biopolymer to be loaded onto the biosensor can be adjusted to tailor the surface capacity and / or thickness of the biosensor. For example, PEI can be loaded at a concentration of 0.01-10 mg / ml, such as 0.01-0.1, 0.1-1, 1-5, 5-10 mg / ml, such as 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / ml, or greater than 10 mg / ml, to achieve a desired surface capacity of the biosensor. Any aqueous medium can be used for loading, including H2O, buffered solutions, and non-buffered solutions. In particular embodiments, PEI is loaded in H2O at a concentration of 10, 1, 0.5, or 0.1 mg / ml to achieve a desired surface capacity. Negatively charged biopolymers can be loaded at a concentration of 0.001 mg / ml and above, such as 0.001-0.01, 0.01-0.1, 0.1-1, 1-5, 5-10 mg / ml, such as 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / ml, or greater than 10 mg / ml, to achieve a desired surface capacity of the biosensor. Any aqueous medium can be used for loading, including H2O, buffered solutions, and non-buffered solutions.

[0057] Figure 2BThis schematic depicts an embodiment of a process for alternating coating of a biosensor with oppositely charged biopolymers. The exemplary stages, in chronological order, include equilibration 230, a loading phase 232 of a positively charged biopolymer (e.g., polyethyleneimine (PEI) in HO), and a wash 234. Signal changes (nm shifts) reflect biopolymer binding.

[0058] Figure 2B The first stage 230 depicts one embodiment of the equilibration 202. The core component 218 of the biosensor is coated with a layer 220 of a negatively charged biopolymer.

[0059] Figure 2B The second stage 232 depicts one embodiment of the positively charged biopolymer loading stage. A positively charged biopolymer 222, such as PEI, is bound to the first layer of negatively charged biopolymer 220. As shown in stage 232 of FIG. 2 , loading with a higher concentration of PEI results in a greater signal change, indicating an increase in the thickness of the biosensor. Thus, the thickness and / or surface capacity of the biosensor can be customized by adjusting the loading concentration of the charged biopolymer on the biosensor. Figure 2B The third stage 234 depicts washing with an appropriate buffer.

[0060] The outermost layer of the charged biopolymer of the biosensor is oppositely charged relative to the charged analyte in the sample and serves as a 3D structure to bind (precipitate) the charged analyte. The charged analyte may include a protein, a viral vector, or a polynucleotide. In certain embodiments, the outermost layer of the charged biopolymer is negatively charged, allowing the outermost layer of the charged biopolymer to bind to a positively charged analyte. The positively charged analyte may include a positively charged protein, such as bovine serum albumin (BSA), neutravidin, or ovalbumin. In certain other embodiments, the outermost layer of the charged biopolymer is positively charged, allowing the outermost layer of the charged biopolymer to bind to a negatively charged analyte. The negatively charged analyte may include a negatively charged protein, polynucleotide, or viral vector.

[0061] Non-limiting examples of viral vectors (viral capsids) that can be assessed using the methods and systems disclosed herein include viral vectors of retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex viruses. In some embodiments, the negatively charged analyte comprises a capsid of an adeno-associated virus (AAV), including a capsid of a recombinant AAV. As used herein, a "recombinant" virus or "recombinant" viral vector refers to a virus or polynucleotide vector comprising one or more heterologous sequences (in other words, nucleic acid sequences that are not of viral origin). As used herein, a "viral capsid" includes a protein shell of viral proteins surrounding a nucleic acid, including a recombinant viral capsid. A full viral capsid contains a polynucleotide, such as a therapeutic polynucleotide. An empty viral capsid does not contain a polynucleotide. As used herein, "AAV" includes recombinant AAV. AAV is a single-stranded DNA (ssDNA) nonenveloped virus belonging to the Parvoviridae family, with a diameter of 25 nm. There are at least 11 serotypes of AAV (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11), each with slightly different tropism, including retinal, lung, muscle, liver, and brain cells. The virus is composed of only protein and DNA and has three repeated capsid proteins, VP1, VP2, and VP3. In some embodiments, the AAV provided herein is AAV serotype 2 (AAV2) or AAV serotype 8 (AAV8).

[0062] Binding of the charged analyte to the biosensor produces a signal indicative of the amount of charged analyte bound to the biosensor. Further, different amounts, concentrations, and / or charges of the charged analyte in the sample can result in different binding kinetics of the biosensor to the charged analyte contained in the sample over time. Without wishing to be bound by theory, binding of the charged analyte to the biosensor can reach a higher signal in a sample containing a higher amount or concentration of the charged analyte compared to a sample containing a lower amount or concentration of the charged analyte. In embodiments, the charged analyte comprises a viral capsid (e.g., an AAV capsid), which can include a full viral capsid containing a polynucleotide and / or an empty viral capsid not containing a polynucleotide, and the ratio of full / empty viral capsids in the sample is the same. For example, as shown in Figure 3 binding of the viral capsid to the biosensor can reach a higher signal in a sample with a higher titer compared to a sample with a lower titer.

[0063] Binding of the charged analyte to the biosensor generates a signal indicative of a characteristic of the charged analyte bound to the biosensor. Moreover, different full / empty virus capsid ratios in the sample result in different binding kinetics of the charged analyte contained in the sample over time by the biosensor. As shown in Figure 4 Binding of the virus capsids (empty or full) to the biosensor reaches saturation faster in samples with a lower percentage of empty capsids (based on total capsids) compared to samples with a higher percentage of empty capsids (based on total capsids). This can be explained by the higher negative charge of full capsids with DNA core compared to empty capsids without DNA core, resulting in a more abundant negative charge from virus capsids in samples with a lower percentage of empty capsids.

[0064] Thus, the presence or amount of charged analyte in a sample or the empty / full ratio of virus capsids in a sample can be detected and measured based at least in part on the signal generated by the binding of the charged analyte to the biosensor and / or the binding kinetics of the charged analyte to the biosensor.

[0065] In embodiments, the biosensor is coupled to a signal analyzer (e.g., interferometer). The signal analyzer is capable of converting and quantifying the signal generated by the binding of the biosensor to the charged analyte or the binding kinetics of the charged analyte to the biosensor. The signal analyzer can quantify the signal by bio-layer interferometry (BLI), quartz crystal microbalance (QCM), surface plasmon resonance (SPR), surface acoustic wave (SAW), atomic force microscopy, or reflectance interferometric spectroscopy (RIfS). The biosensor and / or signal analyzer can detect the presence or amount of charged analyte without a detectable label directly or indirectly attached to the analyte and can be used to detect a variety of charged analytes. 2. Analyte Quantification Device Including Biosensor

[0066] A device for quantifying a charged analyte in a sample is provided. The device includes a biosensor and a signal analyzer coupled to the biosensor. The biosensor includes a core component and a charged biopolymer component. The charged biopolymer component comprises one or more layers of charged biopolymers. An outermost layer of the charged biopolymer component carries an opposite charge relative to the charged analyte, thereby facilitating binding of the charged biopolymer component to the charged analyte in the sample and facilitating generation of a signal indicative of an amount of the charged analyte bound to the biosensor.

[0067] In the case where the biosensor comprises two or more layers of charged biopolymers, each layer of the charged biopolymers is oppositely charged relative to an adjacent layer (an inner adjacent layer and, if present, an outer adjacent layer) and binds to the adjacent layer. For example, the first layer is an inner adjacent layer of the second layer. The first and second layers are oppositely charged and bind to each other. The outermost layer of the charged biopolymers constitutes the outer surface of the charged biopolymer component of the biosensor. The outermost layer of the charged biopolymers is oppositely charged relative to the charged analyte, allowing the charged analyte to bind to the outermost layer of the charged biopolymers and generate a signal indicative of the amount of charged analyte bound to the biosensor. The signal analyzer is capable of quantifying the signal generated by binding of the charged analyte to the biosensor and / or monitoring and analyzing the binding kinetics of the charged analyte to the biosensor over time.

[0068] In an embodiment, the signal analyzer quantifies the signal by bio-layer interferometry (BLI), quartz crystal microbalance (QCM), surface plasmon resonance (SPR), surface acoustic wave (SAW), reflection interferometry spectroscopy (RIfS), or atomic force microscopy.

[0069] In some embodiments, the biosensor may include an optical fiber having a proximal portion and a distal portion. The proximal portion may be configured to receive light from a light source and configured to transmit reflected light to a detector. The optical fiber may be a circular optical fiber having a circular cross-section. The distal portion may be configured as a biosensor having an analyte bound thereto, such that light reflected from the distal portion is phase-shifted based on the thickness of the analyte bound to the distal portion. The biosensor may also include an optical resonator located at the distal portion of the optical fiber, the optical resonator comprising a first reflective surface and a second reflective surface, the first reflective surface being configured to reflect light having a first phase, the second reflective surface being configured to reflect light having a second phase, the second phase light being phase-shifted based on the thickness of the analyte bound to the optical resonator.

[0070] The BLI sensor or optical assembly serves as a sensing element or detector tip to detect an analyte attached to its distal end. The detector unit detects the interference signal generated by interfering light waves reflected from the optical assembly. The detector can be a spectrometer, such as a charge-coupled device (CCD), capable of recording the spectrum of the reflected interference light from the optical assembly. The light source directs light into the optical assembly, where it is reflected back to the detector unit through an optical coupling assembly. The coupling assembly includes a first optical waveguide or optical fiber extending from the light source to the optical assembly, a second optical waveguide or optical fiber that carries the reflected light from the optical assembly to the detector, and an optical coupler that optically couples the first and second optical waveguides. The first and / or second optical waveguides can take the form of a fiber optic bundle (FOB). In some embodiments, the coupling assembly includes a lens system configured to focus a light beam onto the upper surface of the optical assembly and direct the reflected interference light from the optical assembly to the detector.

[0071] By interfering the reflected light beams from the two reflective surfaces in the optical assembly, the presence, concentration and / or binding rate of the analyte on the optical assembly can be measured. Specifically, as the analyte molecules attach to or detach from the surface, the average thickness of the first reflective layer changes accordingly. Because the thickness of all other layers remains unchanged, the interference waves formed by the light waves reflected from the two surfaces are phase-shifted according to this thickness change. Assuming there are two reflected light beams, the first light beam is reflected from the first reflective surface, and the second light beam is reflected from the analyte binding molecules and the bound analyte and the surrounding medium at the second reflective surface. The conversion of the phase shift into a thickness change of the bound analyte is well known in the art.

[0072] In some embodiments, the biosensor is coupled to a quartz crystal microbalance (QCM). A QCM measures mass by the frequency change of a piezoelectric quartz crystal when perturbed by the mass change caused by the product being measured. Furthermore, the QCM can provide information about the viscoelastic properties of the deposited material, determined by recording the dissipation factor. The biosensor can be connected to the QCM and a processing module that allows for the measurement of three parameters: frequency change (Af), dissipation factor change (AD), and the Af / AD ratio. Binding of a charged analyte to the biosensor results in changes in the resonant frequency and dissipation factor of the microbalance. Binding of a specific analyte should provide an Af / AD ratio that is constant over time.

[0073] In some embodiments, the biosensor is a surface plasmon resonance (SPR) biosensor. The SPR biosensor may include probe molecules immobilized on the proximal surface of a sensor chip having a metal (e.g., gold) coating and / or a functional coating (e.g., a biopolymer), and a prism attached to the distal surface of the sensor chip. The proximal surface of the sensor chip may contact a fluid medium containing an analyte, which may bind to the probe molecules immobilized on the proximal surface of the sensor chip. When a solution of target molecules flows into contact with the surface, probe-target binding occurs through affinity interactions, resulting in an increase in the refractive index at the surface of the SPR sensor. In SPR measurements, resonance or response units (RU) are used to describe signal changes, where 1 RU is equivalent to 10 -4 The critical angle shift is Δn. At the beginning of the measurement, the probe-target interaction has not yet occurred and the initial RU value corresponds to the starting critical angle. The change in refractive index Δn in a layer of thickness h is d can be calculated as Δn d = (dn / dc) vol ΔΓ / h Where (dn / dc) vol is the increase in refractive index n with the analyte bulk concentration c, and ΔΓ is the concentration of target bound to the surface. The change in refractive index is tracked in real time by coupling incident light into propagating surface plasmons (PSPs) on the sensor chip surface (e.g., a gold film). 3. Quantification of Charged Analytes

[0074] The methods provided herein for detecting the presence or absence of a charged analyte in a sample can include contacting the sample with a biosensor, allowing the charged analyte contained in the sample to bind to the biosensor, and detecting the presence or absence of the charged analyte. The biosensor includes a core component and a charged biopolymer component and can include any of the features provided herein. The charged biopolymer component can include one or more layers of charged biopolymer and an outer surface. A first layer of the charged biopolymer (the innermost layer adjacent to the core component) is bound to the outer surface of the core component. In the case where the biosensor has two or more layers of charged biopolymer, each layer of the charged biopolymer is oppositely charged relative to an adjacent layer (an inner adjacent layer and, if an outer adjacent layer is present, an outer adjacent layer) and is bound to the adjacent layer. The outermost layer of the charged biopolymer is oppositely charged relative to the charged analyte and constitutes the outer surface of the charged biopolymer component of the biosensor, such that the charged analyte binds to the outer surface of the charged biopolymer component (the outermost layer of the charged biopolymer) and generates a signal indicative of the amount and / or characteristics of the charged analyte bound to the biosensor. The presence, amount, or characteristic of the charged analyte is detected and / or quantified based at least in part on: a signal generated by the binding of the charged analyte to the biosensor and indicative of the amount of charged analyte bound to the biosensor; and / or the binding kinetics of the charged analyte to the biosensor. Different amounts, concentrations, and / or charges of the charged analyte in the sample may result in different binding kinetics to the biosensor for the charged analyte in the sample. For example, the charged analyte may comprise a population of viral capsids, which may include intact viral capsids containing polynucleotides and / or empty viral capsids that do not contain polynucleotides, and different intact / empty viral capsid ratios in the sample may result in different binding kinetics to the biosensor for the viral capsids contained in the sample. The population of viral capsids may include capsids of adeno-associated viruses (AAV).

[0075] Figure 5An exemplary method 500 for detecting the presence, amount, and / or characteristics of a charged analyte in a sample according to an embodiment of the present disclosure is depicted. Method 500 begins at step 502 by providing a biosensor having one or more layers of a charged biopolymer. Method 500 continues to step 504 by contacting the sample with the biosensor, wherein the outermost layer of the biosensor is oppositely charged relative to the charged analyte. Method 500 continues to step 506 by allowing the biosensor to bind to the charged analyte in the sample. Method 500 continues to step 508 by detecting and / or quantifying the presence, amount, and / or characteristics of the charged analyte in the sample based on one or more of the following parameters: a signal generated by binding of the charged analyte to the biosensor, which is indicative of the amount of charged analyte bound to the biosensor; and the binding kinetics of the biosensor to the charged analyte.

[0076] The outermost layer of the charged biopolymer of the biosensor is oppositely charged relative to the charged analyte in the sample and acts as a 3D structure to bind (precipitate) the charged analyte. The charged analyte may include a protein, a viral vector, or a polynucleotide. In certain embodiments, the outermost layer of the charged biopolymer is negatively charged, allowing the outermost layer of the charged biopolymer to bind to a positively charged analyte. The positively charged analyte may include a positively charged protein, such as bovine serum albumin (BSA), neutravidin, or ovalbumin. In certain other embodiments, the outermost layer of the charged biopolymer is positively charged, allowing the outermost layer of the charged biopolymer to bind to a negatively charged analyte. The negatively charged analyte may include a negatively charged protein, polynucleotide, or viral vector (e.g., an AAV vector).

[0077] The signal generated by the binding of the biosensor to the charged analyte and / or the binding kinetics of the biosensor to the charged analyte can be measured by biolayer interferometry (BLI), quartz crystal microbalance (QCM), surface acoustic wave (SAW), atomic force microscopy, reflectance interferometry spectroscopy (RIfS), or surface plasmon resonance (SPR). The methods provided herein can be used to detect the presence or amount of a charged analyte without the need for a detectable label attached directly or indirectly to the analyte, and can be used to detect a variety of charged analytes.

[0078] The charged analyte can be one or more viral vectors. In some such embodiments, the viral titer of the sample can be determined based at least in part on the signal generated by binding of the viral vector to the biosensor, e.g., Figure 5In addition or alternatively, the empty / complete viral capsid ratio of the sample can be determined based at least in part on the binding kinetics of the biosensor to the viral vector, e.g., Figure 5 As shown in step 508.

[0079] As described elsewhere in this disclosure, binding of the viral vector to the biosensor generates a signal indicative of the amount of viral vector bound to the biosensor. Binding of the viral vector to the biosensor can achieve a higher signal in samples with a higher viral titer (i.e., containing a higher amount or concentration of viral vector) than in samples with a lower viral titer (i.e., containing a lower amount or concentration of viral vector). For example, Figure 3 As shown in , the binding of viral capsids to the biosensor can achieve higher signals in samples with higher titers compared to samples with lower titers.

[0080] In addition, different amounts, concentrations, and / or charges of the viral vector in the sample may result in the biosensor having different binding kinetics for the viral vector contained in the sample over time. The viral vector may include a viral capsid (e.g., an AAV capsid) containing a complete or empty viral capsid. Different complete / empty viral capsid ratios in the sample may result in the biosensor having different binding kinetics for the viral vector contained in the sample over time. For example, Figure 4 As shown, because intact capsids with a DNA core have a higher negative charge than empty capsids without a DNA core, the binding of viral capsids (empty or intact) to the biosensor reaches saturation faster in samples with a lower percentage of empty capsids (and therefore more abundant negative charge) than in samples with a higher percentage of empty capsids. Therefore, using the methods provided herein, the viral titer and empty / intact viral capsid ratio in a sample can be determined based at least in part on the signal generated by the binding of the viral vector to the biosensor (binding rate analysis) and / or the binding kinetics of the viral vector to the biosensor (saturation analysis). Example Example 1: Binding kinetics and signal levels of adeno-associated virus type 8 (AAV8) capsids with different titers but the same empty / intact capsid ratio to a biolayer interferometry (BLI) biosensor and the associated interferometric signal

[0081] This example relates to the binding kinetics and signal levels of AAV8 capsid (negatively charged analyte) to a biolayer interferometry (BLI) biosensor in samples with varying titers.

[0082] The BLI biosensor is constructed by activating epoxypropylsilane (EPS) and coating it with a negatively charged biopolymer, which is then coated with polyethyleneimine (PEI). The biosensor thus contains two layers of oppositely charged biopolymers: an inner negatively charged layer and an outer positively charged PEI layer. BLI is used to measure the signal shift (nm) over time for samples containing 5 x 10 11 viral genomes (vg) / ml, 2.5 x 10 11 cp / ml, 1.25 x10 11 cp / ml, 6.25 x 10 10 and 3.125 x 10 10 vg / ml of AAV8 capsid, wherein a positive shift indicates that the viral capsid binds to the biosensor. Figure 3 As shown in Figure 3, binding of AAV8 capsids to the biosensor within the loading time frame showed a positive correlation between the binding signal and the capsid titer. This can be explained by the fact that samples with higher viral titers contain more AAV8 particles and bind faster and more highly to the positively charged biopolymers on the biosensor. The viral titer can be determined using a calibration curve generated based on the signal of a reference sample obtained using the methods provided herein. Example 2. Binding Kinetics and Signal Levels of Adeno-Associated Virus Type 2 (AAV2) Capsids of Equal Titer but Different Empty / Intact Capsid Ratios to a Biolayer Interferometry (BLI) Biosensor and Correlated Interferometer Signals

[0083] The signal shift (nm) over time was measured for each AAV2-containing sample using BLI, and the ratio of empty capsid to intact capsid in the sample was 10%, 17%, 26%, 36%, 50%, 63%, 70%, and 82.7%, respectively, where the shift indicated that the viral capsid bound to the biosensor. Figure 4 As shown in Figure 2, binding of AAV2 capsids (empty or intact) to the biosensor reached saturation more quickly in samples with a lower percentage of empty capsids than in samples with a higher percentage of empty capsids. This can be explained by the higher negative charge of intact capsids with a DNA core compared to empty capsids without a DNA core, resulting in a greater negative charge derived from AAV2 capsids in samples with a lower percentage of empty capsids. The empty / intact viral capsid ratio can be determined using a calibration curve generated based on the binding kinetics of a reference sample obtained using the methods provided herein.

[0084] The different binding kinetics of samples with different virus titers or different empty / intact capsid ratios to the biosensor as shown above can be used to determine the virus titer or empty / intact capsid ratio in the sample.

[0085] Although several embodiments of the present disclosure have been shown in the accompanying drawings, this is not intended to be limited thereto, as the scope of the present disclosure is intended to be as broad as the art will allow, and this specification should be read accordingly. Any combination of the above embodiments is also conceivable and within the scope of the appended claims. Therefore, the above description should not be interpreted as limiting, but merely as examples of specific embodiments. Those skilled in the art will envision other modifications within the scope of the appended claims.

Claims

1. A biosensor for detecting the presence or amount of a charged analyte in a sample, the biosensor comprising: core components; and A charged biopolymer component comprising one or more layers of a charged biopolymer, wherein the outermost layer of the charged biopolymer component is oppositely charged relative to the charged analyte to promote binding of the charged biopolymer component to the charged analyte in the sample and generate one or more signals indicative of the amount of charged analyte bound to the biosensor.

2. The biosensor according to claim 1, wherein the charged biopolymer component comprises two or more layers of charged biopolymer, wherein: A first layer of the two or more layers of charged biopolymer is bonded to an outer surface of the core component; and Each remaining layer of the two or more layers of charged biopolymers is oppositely charged relative to and bonded to an adjacent layer of the one or more layers of charged biopolymers. 3 . The biosensor according to claim 1 , wherein the core component comprises aminopropylsilane (APS) or epoxypropylsilane (EPS). 4 . The biosensor of claim 1 , wherein at least one of the one or more layers of charged biopolymer comprises one or more of dextran, DNA, and carboxylic acid (COOH) functionalized beads. 5 . The biosensor of claim 1 , wherein at least one of the one or more layers of charged biopolymer comprises one or more of polyethyleneimine (PEI), chitosan, poly-L-lysine, polyallylamine, and polyethylenimine.

6. The biosensor of claim 1, wherein the charged analyte comprises a protein, a viral vector, or a polynucleotide. 7 . The biosensor according to claim 1 , wherein the outermost layer of the charged biopolymer component is negatively charged to promote binding of the charged biopolymer component to positively charged proteins contained in the sample. 8 . The biosensor according to claim 7 , wherein the positively charged protein comprises bovine serum albumin (BSA), neutravidin, or ovalbumin.

9. The biosensor according to claim 1, wherein the outermost layer of the charged biopolymer is positively charged to facilitate binding of the charged biopolymer component to negatively charged proteins, polynucleotides or viral vectors.

10. The biosensor of claim 9, wherein the viral vector comprises adeno-associated virus (AAV).

11. The biosensor of claim 1, wherein different amounts, concentrations and / or charges of the charged analytes in the sample result in different binding kinetics of the biosensor to the charged analytes contained in the sample.

12. The biosensor of claim 11 , wherein the charged analyte comprises a population of viral capsids comprising intact viral capsids containing polynucleotides and / or empty viral capsids not containing polynucleotides, wherein different intact / empty viral capsid ratios in the sample result in the biosensor having different binding kinetics for the viral capsids contained in the sample.

13. The biosensor of claim 12, wherein the population of viral capsids comprises capsids of adeno-associated viruses (AAV).

14. The biosensor of claim 1 coupled to a signal analyzer, wherein the signal analyzer is capable of converting and quantifying a signal generated by binding of the biosensor to the charged analyte.

15. The biosensor of claim 14, wherein the signal analyzer quantifies the signal using bio-layer interferometry (BLI), quartz crystal microbalance (QCM), or surface plasmon resonance (SPR).

16. A device for quantifying a charged analyte in a sample, the device comprising: a biosensor comprising a core component and a charged biopolymer component, the charged biopolymer component comprising one or more layers of a charged biopolymer, the outermost layer of the charged biopolymer component being oppositely charged relative to the charged analyte to promote binding of the charged biopolymer component to the charged analyte in the sample and to generate a signal indicative of the amount of charged analyte bound to the biosensor; and A signal analyzer is coupled to the biosensor, the signal analyzer being capable of converting and quantifying a signal generated by binding of the biosensor to the charged analyte.

17. The device of claim 16, wherein the charged biopolymer component comprises two or more layers of charged biopolymer, wherein: A first layer of the two or more layers of charged biopolymer is bonded to an outer surface of the core component; and Each remaining layer of the two or more layers of charged biopolymers is oppositely charged relative to and bonded to an adjacent layer of the two or more layers of charged biopolymers.

18. The apparatus of claim 16, wherein: The core component comprises aminopropylsilane (APS) or epoxypropylsilane (EPS); At least one of the one or more layers of charged biopolymer comprises one or more of dextran, DNA, and carboxylic acid (COOH) functionalized beads; and / or At least one of the one or more layers of charged biopolymer comprises one or more of polyethyleneimine (PEI), chitosan, poly-L-lysine, polyallylamine, and polyaziridine.

19. A method for detecting the presence or amount of a charged analyte in a sample, the method comprising: contacting the sample with a biosensor comprising a core component and a charged biopolymer component, the charged biopolymer component comprising one or more layers of a charged biopolymer, the outermost layer of the charged biopolymer component being oppositely charged relative to the charged analyte; allowing a charged analyte in the sample to bind to the charged biopolymer component; and The presence or amount of the charged analyte is detected based at least in part on: a signal generated by binding of the charged analyte to the biosensor, wherein the signal is indicative of the amount of charged analyte bound to the biosensor; and / or The biosensor has binding kinetics for the charged analyte, wherein different amounts, concentrations, and / or charges of the charged analyte in the sample result in the biosensor having different binding kinetics for the charged analyte in the sample.

20. The method of claim 19, wherein the signal and / or the binding kinetics are measured by biolayer interferometry (BLI), quartz crystal microbalance (QCM) or surface plasmon resonance (SPR).

21. The method of claim 20, wherein the charged analyte comprises one or more viral vectors, and wherein the viral titer of the sample is determined at least in part based on a signal generated by binding of the one or more viral vectors to the biosensor.

22. The method of claim 20, wherein the charged analyte comprises one or more viral vectors, and wherein the empty / intact viral capsid ratio of the sample is determined at least in part based on the binding kinetics of the one or more viral vectors by the biosensor.

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