WO2024186540A1

WO2024186540A1 - Biosensors comprising charged biopolymers and use thereof

Info

Publication number: WO2024186540A1
Application number: PCT/US2024/017679
Authority: WO
Inventors: Yuanyuan Zhang; Ling Zhang
Original assignee: Sartorius Bioanalytical Instruments Inc
Current assignee: Sartorius Bioanalytical Instruments Inc
Priority date: 2023-03-03
Filing date: 2024-02-28
Publication date: 2024-09-12
Anticipated expiration: 2025-09-03
Also published as: CN120769984A

Abstract

Biosensors for detecting presence or amount of a charged analyte in a sample are provided. The biosensor can include a core component and a charged biopolymer component that includes one or more layers of charged biopolymer. The outermost layer of the charged biopolymer component is oppositely charged relative to the charged analyte and capable of binding to the charged analyte. The biosensor can comprise two or more layers of alternating oppositely-charged biopolymers, with each layer being oppositely charged relative to the adjacent layer. Apparatuses coupled to the biosensor for quantitating the signals generated by binding of the biosensor to the charged analyte, as well as methods for detecting presence or amount of a charged analyte in a sample using the biosensor are also provided.

Description

BIOSENSORS COMPRISING CHARGED BIOPOLYMERS AND USE THEREOF

Background

[0001] The present disclosure relates to biosensors for detection and quantitation of compounds of interest and related methods.

[0002] Biosensors can be used to detect the presence or amount of analytes in a sample and facilitate, for instance, the diagnosis and treatment of disease or scientific research and development. Biosensors can employ a solid surface having immobilized anti-analyte molecules to which sample analyte molecules bind specifically and with high affinity at a defined detection zone. The binding event can be detected directly, for example by a change in the mass, reflectivity, thickness, color, or other characteristic indicative of a binding event. The analyte can be prelabeled, for example with a chromophore, a fluorophore, or a radiolabel. Alternatively, the analyte can be labeled after it is bound at the detection zone, for example with a secondary, labeled antianalyte antibody.

[0003] Current biosensors are mainly based on antibody-antigen or receptor-ligand interactions and usually involve labeling of the analyte for detection. For example, typical current analyte-anti-analyte 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 the anti-analyte that is the opposite member of the pair. The analyte, such as an antigen, an antibody, a ligand, or a receptor can be detected by using a detectable signal, such as a chromophore, a fluorophore, a radiolabel, or a secondary, labeled anti-analyte antibody attached to the analyte before or after the binding of the analyte to the biosensor. In view of the foregoing, there is a need for effective biosensors based on coating chemistries based on molecular or biological interactions other than antigen-antibody or ligand-receptor interactions for sensing and quantitating a variety of compounds.

Summary

[0004] This disclosure is directed generally to apparatus and methods to address these shortcomings of the art and provide other additional or alternative advantages. The disclosure herein provides embodiments of biosensors for detecting the presence or amount of a charged analyte in a sample. A biosensor can be coated with one or more layers of charged biopolymer, in which the outermost layer of 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 layers of alternating oppositely-charged biopolymers, in which each layer is oppositely charged relative to the adjacent layer. In some cases, the biosensor can detect the binding of an analyte directly without use of a detectable label attached to the analyte. Apparatuses coupled to the biosensor for quantitating the signals generated by binding of the biosensor to the charged analyte, as well as methods for detecting presence or amount of a charged analyte in a sample using the biosensor are also provided.

[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 has a core component and a charged biopolymer component. The charged biopolymer component comprises one or more layers of charged biopolymer. The outermost layer of the charged biopolymer component is oppositely charged relative to the charged analyte, to facilitate binding of the charged biopolymer component to the charged analyte in the sample and generation of signals indicative of an amount of the charged analyte bound to the biosensor.

[0006] In embodiments, the biosensor has two or more layers of charged biopolymer. The first layer of charged biopolymer (the innermost layer that is adjacent to the core component) is bound to an external surface of the core component. Each layer of the charged biopolymer is oppositely charged relative to the adjacent layer(s) (the inner adjacent layer and, when the outer adjacent layer is present, the outer adjacent layer) and bound to the adjacent layer(s). The outermost layer of charged biopolymer constitutes the outer surface of the charged biopolymer component of the biosensor. Accordingly, the outermost layer is oppositely charged relative to the charged analyte, to facilitate binding of the charged analyte to the outmost layer of charged biopolymer and generation of signals indicative of an amount of the 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 silicon oxide (SiCh) or epoxypropylsilane (EPS) functionalized SiO2. The negatively charged biopolymer of a 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 a positively charged layer of the biosensor, if present, may contain polyethylenimine (PEI), chitosan, poly-L- lysine, polyallylamine, polyaziridine, and/or other amine-functionalized compounds. The charged analyte may include a protein, a viral vector, or a polynucleotide.

[0008] In certain embodiments, the outermost layer of charged biopolymer is negatively charged, allowing binding of the outmost layer of charged biopolymer 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 charged biopolymer is positively charged, allowing binding of the outmost layer of charged biopolymer to a negatively charged analyte. The negatively charged analyte may include a negatively charged protein, a polynucleotide, or a viral vector. The viral vector may include an adeno-associated virus (AAV) vector.

[0010] In particular embodiments, different amounts, concentrations, and/or charges of the charged analyte in the sample result in different binding kinetics of the biosensor towards the charged analyte contained in the sample. For example, the charged analyte may comprise a population of viral capsids, which may include a full viral capsid containing polynucleotides and/or an empty viral capsid not containing polynucleotides, and different full/empty viral capsid ratios in the sample may result in different binding kinetics of the biosensor towards the viral capsids contained in the sample. The population of viral capsids may include capsids of adeno- associated virus (AAV).

[0011] In embodiments, the biosensor is coupled to a signal analyzer. The signal analyzer is capable of transforming and quantifying the signals generated by binding of the biosensor to the charged analyte. The signal analyzer may quantify the signals using one or more label-free technologies, including, but not limited to, biolayer interferometry (BLI), quartz crystal microbalance (QCM), surface plasmon resonance (SPR), surface acoustic wave (SAW), atomic force microscopy, and reflectometric interference spectroscopy (RIfS). [0012] In certain aspects of the present disclosure, an apparatus for quantitating a charged analyte in a sample is provided. The biosensor has a core component and a charged biopolymer component. The charged biopolymer component comprises one or more layers of charged biopolymer. The outermost layer of the charged biopolymer component is oppositely charged relative to the charged analyte, to facilitate binding of the charged biopolymer component to the charged analyte in the sample and generation of signals indicative of an amount of the charged analyte bound to the biosensor. The signal analyzer is capable of transforming and quantifying the signals generated by binding of the biosensor to the charged analyte.

[0013] In embodiments, the charged biopolymer component of the biosensor has two or more layers of charged biopolymer. The first layer of charged biopolymer (the innermost layer that is adjacent to the core component) may be bound to an external surface of the core component. Each layer of the charged biopolymer is oppositely charged relative to the adjacent layer(s) (the inner adjacent layer and, when the outer adjacent layer is present, the outer adjacent layer) and bound to the adjacent layer(s). The outermost layer of 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 outmost layer of charged biopolymer and generation of signals indicative of an amount of the charged analyte bound to the biosensor.

[0014] In embodiments, the signals are measured via biolayer interferometry (BLI), quartz crystal microbalance (QCM), surface plasmon resonance (SPR), surface acoustic wave (SAW), atomic force microscopy, or reflectometric interference spectroscopy (RIfS). The core component of the biosensor is a surface that may contain aminopropylsilane (APS) functionalized silicon oxide (SiCh) or epoxypropylsilane (EPS) functionalized silicon oxide (SiC ). The negatively charged biopolymer of a 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 a positively charged layer of the biosensor, if present, may contain polyethylenimine (PEI), chitosan, poly-L-lysine, polyallylamine, polyaziridine, 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, facilitating binding of the charged analyte in the sample to the charged biopolymer component of the biosensor, and detecting the presence of 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 binding of the biosensor to the charged analyte. The charged analyte is quantitated based at least partially on signals that are generated by binding of the charged analyte to the biosensor and indicative of an amount of the charged analyte bound to the biosensor; and/or binding kinetics of the biosensor towards the charged analyte. Different amounts, concentrations, and/or charges of the charged analyte in the sample result in different binding kinetics of the biosensor towards the charged analyte in the sample.

[0016] The charged biopolymer component of the biosensor can include one or more layers of charged biopolymer. Where the biosensor includes two or more layers of charged biopolymer, the first layer of charged biopolymer (the innermost layer that is adjacent to the core component) may be bound to an external surface of the core component. Each layer of the charged biopolymer is oppositely charged relative to the adjacent layer(s) (the inner adjacent layer and, when the outer adjacent layer is present, the outer adjacent layer) and bound to the adjacent layer(s). The outermost layer of charged biopolymer constitutes the outer surface of the charged biopolymer component of the biosensor and is oppositely charged relative to the charged analyte, to enable binding of the charged analyte to the outmost layer of charged biopolymer.

[0017] In embodiments, the signals and/or the binding kinetics are measured by biolayer interferometry (BLI), quartz crystal microbalance (QCM), surface plasmon resonance (SPR), surface acoustic wave (SAW), atomic force microscopy, or reflectometric interference 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 based at least partially on the signals generated by binding of one or more viral vectors to the biosensor (such as based at least partially on binding rates). Additionally or alternatively, the empty/full viral capsid ratio of the sample is determined based at least partially on the binding kinetics of the biosensor towards the one or more viral vectors (such as based at least partially on saturation analysis from the one or more viral vectors binding to the biosensor).

[0019] Further, to the extent consistent, any of the embodiments or aspects described herein may be used in conjunction with any or all of the other embodiments or aspects described herein.

Brief Description of the Drawings

[0020] Various aspects of the present disclosure are described hereinbelow with reference to the drawings, which are incorporated in and constitute a part of this specification. [0021] FIGs. 1A-1C are diagrammatic representations of a biosensor coated with charged biopolymers. FIG. 1A depicts a biosensor coated with one layer of a negatively charged biopolymer, such as polysaccharides, such as dextran. FIG. IB depicts a biosensor coated with one layer of COOH-functionalized beads. FIG. 1C depicts a biosensor with two layers of oppositely-charged biopolymers, such as a first layer of a negatively charged biopolymer (such as dextran) and a second layer of positively charged biopolymer (such as polyethylenimine). FIG. ID depicts a biosensor with three layers of charged biopolymers, each layer oppositely charged as compared to an adjacent layer, such as first and third layers of a negatively charged biopolymer, and a second layer of positively charged biopolymer.

[0022] FIG. 2A schematically depicts an example process of coating a biosensor alternately with oppositely-charged biopolymers. The phases include, in sequential order, equilibrium (biosensor coated with a negatively charged biopolymer), coating with polyethylenimine (PEI) in H2O, wash, coating with a negatively charged biopolymer, wash, and coating with PEI. The signal change (nm shift) reflects binding of biopolymers.

[0023] FIG. 2B schematically depicts an example process of coating a biosensor alternately with oppositely-charged biopolymers. The thickness of the biosensor can be customized by adjusting the loading concentration of a charged biopolymer on the biosensor. The signal change (nm shift) reflects binding of biopolymers.

[0024] FIG. 3 is a graphical representation of the direct binding of different titers but same empty/full ratio of adeno-associated virus type 8 (AAV8) capsids 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 having different titer of AAV8 capsids, and the signal change (nm shift) was measured as an indicator of the amount of AAV capsids bound to the BLI biosensor over an example process comprising the preloaded negatively charged biopolymer equilibrium phase, the positively charged biopolymer loading phase, and the capsid capture phase.

[0025] FIG. 4 is a graphical representation of the direct binding of AAV2 full capsids to a BLI biosensor coated with alternating layers of oppositely-charged biopolymers. The BLI biosensor coated with charged biopolymers was contacted with AAV2 samples having the same titer, but different empty/ full capsid ratios, and the signal change (nm shift) was measured as an indicator of the amount of AAV2 capsids bound to the BLI biosensor over an example process comprising the preloaded negatively charged biopolymer equilibrium phase, the positively charged biopolymer loading phase, and the capsid capture phase.

[0026] FIG. 5 is a flowchart of a method of detecting, characterizing and/or quantitating a charged analyte in a sample using a biosensor, according to embodiments of the present disclosure.

Detailed Description

[0027] The present disclosure will now be described more fully hereinafter with reference to example embodiments thereof and with reference to the drawings. These example 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 can be combined with features from any other embodiment or aspect in any appropriate combination. For example, any individual or collective features of method aspects or embodiments can be applied to apparatus, product, or component aspects or embodiments and vice versa. The 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] A “biosensor” as used herein refers to a sensing and/or analytical device that detects the presence, characteristics, or amount of substances (analytes) using a biological molecule (such as an enzyme, an antibody, or a biopolymer) or a living organism. Biosensors can employ a solid surface having immobilized anti-analyte molecules to which sample analyte molecules bind specifically and with high affinity at 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 analyte binding to immobilized anti-analyte molecules. The binding event can be detected directly, e.g., by a change in the mass, reflectivity, thickness, color, or other characteristic indicative of a binding event. Where the analyte is pre-labeled, for example with a dye, a chromophore, a fluorophore, or radiolabel, the binding event is detectable by the presence and/or amount of detectable label at the detection zone. Alternatively, the analyte can be labeled after it is bound at the detection zone, for example with a secondary, fluorescent-labeled anti-analyte antibody.

[0029] An “analyte” as used herein refers to a substance the presence, characteristics, or amount of which is analyzed by the biosensor provided herein. An “analyte-binding” molecule or an “anti-analyte” as used herein refers to a molecule or a compound capable of participating in a specific binding reaction with an analyte molecule. Analyte-anti-analyte pairs can include antigenantibody pairs, ligand-receptor pairs, complementary polynucleotide pairs, or positively- and negatively charged molecule pairs, where the analyte can be either member of the pair, and the anti-analyte is the opposite member of the pair. An anti-analyte can be immobilized or coated onto the biosensor for binding and detection of an analyte. [0030] A “binding” or “binding reaction” refers to an attractive interaction between two molecules resulting in a stable association in which the molecules are in close proximity to each other. Binding is saturable, can be reversible, and can be competed with an excess of one of the reactants. Binding reactions are characterized by complementarity of shape, charge, and other binding determinants as between the participants in the specific binding reaction.

[0031] A “biopolymer” as used herein refers to a polymer that is produced by or derived from living organisms, such as plants and microbes, or chemically synthesized from biological materials. Biopolymers consist of monomeric units that are covalently bonded in chains to form larger molecules. Biopolymers include polynucleotides (such as RNA and DNA), polypeptides (proteins and chains of amino acids, such as collagen, actin, and fibrin), and polysaccharides (linear or branched chains of sugar carbohydrates, such as starch, cellulose, and alginate). Biopolymers also include natural rubbers (polymers of isoprene), suberin and lignin (complex polyphenolic polymers), cutin and cutan (complex polymers of long-chain fatty acids), and melanin.

[0032] “Charged” as used herein with reference to an analyte, a biosensor, or a biopolymer refers to the analyte, the biosensor, or the biopolymer having an electric charge due to functional groups. An electric charge can be positive or negative. “Oppositely charged” as used herein refers to one material charged positively and the other material charged negatively. Oppositely charged materials can bind to each other by electrostatic force (Coulomb force).

[0033] The term “coat” or “load” in the context of coating or loading a structure with a biopolymer to a structure refers to the process of bringing the equilibrated sample (such as biopolymer) into contact with the equilibrated solid phase (such as to assemble a biosensor). Coating or loading can be done for example with chromatography devices by causing the sample to pass through the device by means of an external force, such as by gravity .by pumping, or by dipping into a well plate that contains the sample.

[0034] An “antibody” refers to an immunoglobulin molecule having two heavy chains and two light chains prepared by any method known in the art or later developed and includes polyclonal antibodies such as those produced by inoculating 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 employing, e.g., SCID mice reconstituted with human immunoglobulin genes, as well as antibodies that have been humanized using art-known resurfacing techniques. An antibody also refers to an antibody fragment. An “antibody fragment” refers to a fragment of an antibody molecule produced by chemical cleavage or genetic engineering techniques, as well as to single chain variable fragments (SCFvs) such as those produced using combinatorial genetic libraries and phage display technologies. Antibody fragments used in accordance with the present disclosure usually retain the ability to bind their cognate antigen and so include variable sequences and antigen combining sites, which are within the scope of antibodies.

[0035] A “vector,” as used herein, refers to a recombinant plasmid or virus that comprises a nucleic acid to be delivered into a host cell, either in vitro or in vivo.

[0036] A “polynucleotide” as used herein refers to a biopolymer comprising a plurality of 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 multistranded 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 sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphorami dates and thus can be an oligodeoxynucleoside phosphoramidate (P-NH₂) or a mixed phosphoramidate- phosphodiester oligomer. In addition, a double-stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.

[0037] Viral titers can be expressed in a number of ways, from which one skilled in the art can select a way to express viral titers suitable in the context. For example, the term “viral genome (vg)” as used in reference to a viral titer refers to the number of viral genomes, regardless of infectivity or functionality. The term “genome particles (gp),” “genome equivalents,” “capsid particles” or “genome copies” as used in reference to a viral titer, refers to the number of virions containing the recombinant viral DNA genome or RNA genome or number of viral capsids, regardless of infectivity or functionality. The number of capsid particles or genome particles in a particular vector preparation can be measured by standard methods such as using a fluorescent dye or electron microscopy.

[0038] “Biolayer interferometry (BLI)” as used herein refers to an optical technique for measuring macromolecular interactions by analyzing interference patterns 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) on a biosensor and binding thereto of an analyte can be measured by BLI. A change in the number of molecules bound to the end of the biosensor tip can cause a shift in the interference pattern that is measured in real-time. A “BLI biosensor” refers to a biosensor coupled to a biolayer interferometer, or biosensor signals generated by which are measured by BLI.

[0039] “Quartz crystal microbalance (QCM),” “quartz microbalance (QMB),” or “quartz crystal nanobalance (QCN)” as used herein refers to measurement of a mass variation per unit area assessed by the change in frequency of a quartz crystal resonator. The resonance is disturbed by the addition or removal of a small mass due to oxide growth/decay or film deposition at the surface of the acoustic resonator. QCM can be used under vacuum, in gas phase, or in liquid environments. QCM can be used for monitoring the rate of deposition in thin film deposition systems under vacuum, determining the affinity of molecules (proteins, viruses, polymers) to surfaces functionalized with recognition sites in liquid, or investigating interactions between biomolecules. A “QCM biosensor” refers to a biosensor coupled to a QCM, or a biosensor signals generated by which are measured by QCM.

[0040] “Surface plasmon resonance (SPR)” as used herein refers to a phenomenon in which electrons in a thin metal sheet become excited by light that is directed to the sheet with a particular angle of incidence, and travel parallel to the sheet. Under a constant light source wavelength and a thin metal sheet, the angle of incidence that triggers SPR is related to the refractive index of the material and even a small change in the refractive index will cause SPR to not be observed. SPR biosensors can be used to detect various analytes. A “SPR biosensor” refers to a biosensor coupled to a SPR system, or a biosensor signals generated by which are measured by SPR. [0041] As used herein, the term “proximal” refers to the portion of the device or component thereof that is closer to the user or machine using the device. The term “distal” refers to the portion of the device or component thereof that is farther from the user or machine using the device.

[0042] As used herein, the singular forms “a,” “an,” “the,” and the like include plural referents unless the context clearly dictates otherwise. In addition, while reference may be made herein to quantitative measures, values, geometric relationships or the like, unless otherwise stated, any one or more if not all of these may be absolute or approximate to account for acceptable variations that may occur, such as those due to manufacturing or engineering tolerances or the like. Reference to a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se with in a range suitable in the context, for example ± 10%. For example, description referring to “X” includes description of “X” and extends to a suitable range.

1. Biosensor Comprising a Layer of Charged Biopolymer

[0043] A biosensor of the present disclosure, for detecting presence or amount of a charged analyte in a sample, has a core component and a charged biopolymer component. The charged biopolymer component comprises one or more layers of charged biopolymer. The outermost layer of the charged biopolymer component is oppositely charged relative to the charged analyte, such that binding of the charged biopolymer component to the charged analyte in the sample is facilitated and generation of signals indicative of characteristics and/or an amount of the charged analyte bound to the biosensor is facilitated.

[0044] The biosensor can have two or more layers of charged biopolymer. The first layer of charged biopolymer (the innermost layer that is adjacent to the core component) may be bound to an external surface of the core component. Each layer of the charged biopolymer is oppositely charged relative to the adjacent layer(s) (the inner adjacent layer and, when the outer adjacent layer is present, the outer adjacent layer) and bound to the adjacent layer(s). For example, the first layer is the inner adjacent layer of the second layer and the third layer is the 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 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, such that binding of the charged analyte to the outmost layer of charged biopolymer is facilitated and generation of signals indicative of an amount of the charged analyte bound to the biosensor is facilitated.

[0045] Any suitable material can be used for the core component, the negatively charged biopolymer layer, and the positively charged biopolymer layer. The core component of the biosensor is a central portion that binds outer layers such as biopolymers. The core component is a surface that may contain aminopropylsilane (APS) functionalized silicon oxide (SiCh) or epoxypropylsilane (EPS) functionalized silicon oxide (SiCh), that facilitates binding of an outer layer. Without wishing to be bound by theory, APS and EPS activation on silica substrates are widely used in industrial, biomaterial, and medical applications to enable modifications of polymers/biomolecules. The negatively charged biopolymer of a 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 a positively charged layer of the biosensor may contain polyethylenimine (PEI), chitosan, poly-L-lysine, polyallylamine, polyaziridine, and/or other amin-functionalized compounds. In some embodiments, the components of the biosensor, such as the core component, the negatively charged biopolymer layer, and the positively charged biopolymer layer are non-toxic.

[0046] FIGs. 1A-1D depict embodiments of a biosensor provided herein. FIG. 1A depicts a biosensor 102 having a core component 104 (such as APS or EPS), coated with one layer of negatively charged biopolymer 106 (such as, dextran or DNA). FIG. IB depicts biosensor 108 having a core component 104 (such as APS or EPS), coated with one layer of negatively charged biopolymer 110, which is carboxylic acid (COOH)-functionalized beads. Surface “functionalization” as used herein refers to alteration of the surface properties of a material or device to introduce a desired property. “COOH-functionalized beads” as used herein refer to beads having -COOH groups on the surface. FIG. 1C depicts biosensor 112 having a core component 104 (such as APS or EPS), coated with one layer of negatively charged biopolymer 106 (such as, dextran, DNA, or COOH-functionalized beads), coated with positively charged biopolymer 114 (such as PEI, chitosan, poly-L-lysine, polyallylamine, or polyaziridine). FIG. ID depicts biosensor 116 having a core component 104 (such as APS or EPS), coated with multiple alternating layers of negatively charged biopolymer 106 and 118 and positively charged biopolymer 114.

[0047] Biosensors provided herein can comprise other configurations of a core component and one or more layers of charged biopolymers. For example, a biosensor provided herein can comprise the following configuration:

C - (-) - [(+) - (-)]n (Formula I); or

C - (+) - [(-) - (+)]_n (Formula II), wherein C is a core component, (-) is a layer of negatively charged biopolymer, (+) is a layer of positively charged biopolymer, and n is a number of repeated pairs of layers, in the innermost to the outermost order where C is the innermost. For example, a biosensor provided herein can comprise a core component coated with one layer of positively charged biopolymer. A biosensor provided herein can comprise a core component coated with alternating two or more layers of oppositely-charged biopolymers.

[0048] The biosensor provided herein can comprise any number of layers of alternating charged biopolymers. For example, the biosensor provided herein can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 layers of alternating charged biopolymers. In some embodiments, the biosensor includes two or more layers of charged biopolymer. Without wishing to be bound by theory, the multi-layer structure (comprising at least a core component, a negatively charged biopolymer layer, and a positively charged biopolymer layer) provides stability to the biosensor. Combinations, configurations, the number of layers, and density of biopolymers can be tailored to achieve an optimal surface capacity and stability of the biosensor. For example, the number of layers of charged polymers can be adjusted to optimize the thickness, or concentrations of charged polymers to be loaded to the biosensor can be adjusted to optimize the surface capacity of the biosensor. Further, the surface of the core component (such as glass or silica) can be etched or otherwise treated to increase the density of hydroxyl groups by which analyte-binding molecules (anti-analytes) can be bound. The biosensor surface can be densely coated with charged biopolymer, so that binding of analyte molecules to the layer forces a change in the thickness of the layer, rather than filling in the layer.

[0049] Biosensors provided herein can be assembled using Layer-by-layer (LbL) assembly methods. The LbL assembly technique assembles a thin film by depositing (or loading) alternating layers of oppositely charged materials with wash steps in between. The LbL assembly technique can generate ultrathin films with nanoscale thickness, orientation control with molecular order and stability which involves the alternate adsorption of oppositely charged layers of polyions and biopolymers involving variety of organic, inorganic, and biological substrate materials including carbon nanotubes (CNTs), proteins, antigens, lysozyme, DNA, nanoparticles, metallophthalocyanines, and dendrimers. The LbL assembly technique is also a versatile method for fabrication of controlled layered structures from CNT/enzyme using simple, rapid and inexpensive procedures, and is useful for immobilization of biopolymers, biocatalysts, and biomaterials in well-defined layered structures under mild conditions rendering for biosensor and biofuel cell applications. LbL nanostructures, a homogenous and stable CNT -based assembly of multilayer biopolymer (or enzyme) interfaces with desired architecture provides control over the position of the analyte-binding molecules (such as polyelectrolyte or enzymes molecules) compared to random hydrogels. Further, spatially organized multilayers with close proximity can be advantageous for stability and surface capacity of the biosensors, or for sequential enzymatic reactions with single and bi-enzymatic cascade systems.

[0050] FIG. 2A schematically depicts an embodiment of a process of coating a biosensor alternately with oppositely-charged biopolymers, using a layer-by-layer assembly technique. The example phases include, in the chronological order, equilibrium 202, a positively charged biopolymer (such as polyethylenimine (PEI) in H2O) loading phase 204, wash 206, a negatively charged biopolymer loading phase 208, wash 210, a positively charged biopolymer (such as PEI) loading phase 212, wash 214, and a negatively charged biopolymer loading phase 216. The signal change (nm shift) reflects binding of biopolymers.

[0051] The first phase 202 of FIG. 2A depicts an embodiment of an equilibrium 202. The core component 218 of the biosensor is coated with one layer of a negatively charged biopolymer 220. [0052] The second phase 204 of FIG. 2A depicts an embodiment of a positively charged biopolymer loading phase. Positively charged biopolymer 222, such as PEI, is bound to the first layer of negatively charged biopolymer 220. The third phase 206 of FIG. 2A depicts wash with an appropriate buffer.

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

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

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

[0056] The loading phases can be repeated many times to achieve an optimal stability and binding capacity of the biosensor. At each loading phase, density (concentration) of the biopolymer to be loaded to 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-I0mg/ml, for example at a concentration of 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 solution, and unbuffered solution. In specific embodiments, PEI is loaded at a concentration of 10, 1, 0.5, or 0.1 mg/ml in H2O, to achieve a desired surface capacity. A negatively charged biopolymer 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, 5-10mg/ml, for example at a concentration of 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 solution, and unbuffered solution.

[0057] FIG. 2B schematically depicts an embodiment of a process of coating a biosensor alternately with oppositely-charged biopolymers. The example phases include, in the chronological order, equilibrium 230, a positively charged biopolymer (such as polyethylenimine (PEI) in H2O) loading phase 232, and wash 234. The signal change (nm shift) reflects binding of biopolymers.

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

[0059] The second phase 232 of FIG. 2B depicts an embodiment of a positively charged biopolymer loading phase. Positively charged biopolymer 222, such as PEI, is bound to the first layer of negatively charged biopolymer 220. As shown in phase 232 of FIG. 2, loading with higher concentrations of PEI results in greater signal change, indicative of increased thickness of the biosensor. Accordingly, the thickness and/or surface capacity of the biosensor can be customized by adjusting the loading concentration of a charged biopolymer on the biosensor. The third phase 234 of FIG. 2B depicts wash with an appropriate buffer. [0060] The outermost layer of charged biopolymer of the biosensor is oppositely charged relative to the charged analyte in a 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 charged biopolymer is negatively charged, allowing binding of the outmost layer of charged biopolymer 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 charged biopolymer is positively charged, allowing binding of the outmost layer of charged biopolymer to a negatively charged analyte. The negatively charged analyte may include a negatively charged protein, a polynucleotide, or a viral vector.

[0061] Non-limiting examples of viral vectors (viral capsids) that can be evaluated using the methods and systems disclosed herein include viral vectors of a retrovirus, an adenovirus, an adeno-associated viruses, a lentivirus, and a herpes simplex virus. In some embodiments, the negatively charged analyte comprise capsids of adeno-associated virus (AAV) including recombinant AAV. A “recombinant” virus or “recombinant” viral vector as used herein refers to a virus or polynucleotide vector comprising one or more heterologous sequences (in other words, nucleic acid sequence not of viral origin). A “viral capsid” as used herein includes a protein shell of virus protein that encloses the nucleic acid including a recombinant viral capsid. A full viral capsid contains polynucleotides, such as therapeutic polynucleotides. An empty viral capsid does not contain polynucleotides. “AAV” as used herein includes recombinant AAV. AAV is a singlestranded DNA (ssDNA) nonenveloped virus that belongs to the parvovirus family and measures 25 nm in diameter. There are at least eleven serotypes of AAV (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11), each with slightly different tropisms which include retina, lung, muscle, liver, and brain cells. The virus is only composed of protein and DNA and has three repeating 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 a charged analyte to the biosensor generates signals indicative of the amount of the charged analyte bound to the biosensor. In addition, different amounts, concentrations, and/or charges of the charged analyte in the sample can result in different binding kinetics over time of the biosensor towards the charged analyte contained in the sample. Without wishing to be bound by theory, binding of the charged analyte to the biosensor can reach higher signal in a sample containing a higher amount or concentration of the charged analyte, compared to a sample containing a lower amount of concentration of the charged analyte. In embodiments, the charged analyte includes viral capsids (such as AAV capsids), which may include a full viral capsid containing polynucleotides and/or an empty viral capsid not containing polynucleotides, and same full/empty viral capsid ratios in the sample. For example, as shown in FIG.3, the binding of viral capsids to the biosensor can reach higher signal in sample with higher titer as compared to samples with a lower titer.

[0063] Binding of a charged analyte to the biosensor generates signals indicative of the characteristics of the charged analyte bound to the biosensor. In addition, different full/empty viral capsid ratios in the sample can result in different binding kinetics over time of the biosensor towards the charged analyte contained in the sample. As shown in FIG. 4, in samples with a lower percentage of empty capsids (based on the total amount of capsids), the binding of viral capsids (empty or full) to the biosensor reached saturation faster as compared to samples with a higher percentage of empty capsids (based on the total amount of capsids). This can be explained by the higher negative charges of a full capsid with a DNA core as compared to an empty capsid without a DNA core, resulting in more abundancy of negative charges derived from viral capsids in samples with a lower percentage of empty capsids .

[0064] Accordingly, the presence or amount of the charged analyte in a sample, or the empty/full ratio of viral capsids in a sample can be detected and measured at least partially based on the signals 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., an interferometer). The signal analyzer is capable of transforming and quantifying the signals generated by binding of the biosensor to the charged analyte, or the binding kinetics of the charged analyte to the biosensor. The signal analyzer may quantify the signals via biolayer interferometry (BLI), quartz crystal microbalance (QCM), surface plasmon resonance (SPR), surface acoustic wave (SAW), atomic force microscopy, or refl ectometric interference spectroscopy (RIfS). The biosensor and/or the signal analyzer can detect the presence or amount of a charged analyte without a detectable label attached to the analyte directly or indirectly and can be used for detection of a variety of charged analytes.

2. Analyte Quantitation Apparatus Comprising a Biosensor

[0066] An apparatus for quantitating a charged analyte in a sample is provided. The apparatus 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 biopolymer. The outermost layer of the charged biopolymer component is oppositely charged relative to the charged analyte, such that binding of the charged biopolymer component to the charged analyte in the sample is facilitated and generation of signals indicative of an amount of the charged analyte bound to the biosensor is facilitated.

[0067] Where the biosensor has two or more layers of charged biopolymer, each layer of the charged biopolymer is oppositely charged relative to the adjacent layer(s) (the inner adjacent layer and, when the outer adjacent layer is present, the outer adjacent layer) and bound to the adjacent layer(s). For example, the first layer is the inner adjacent layer of the second layer. The first and second layers are oppositely charged and bound to each other. The outermost layer of charged biopolymer constitutes the outer surface of the charged biopolymer component of the biosensor. The outermost layer of charged biopolymer is oppositely charged relative to the charged analyte, such that the charged analyte binds to the outmost layer of charged biopolymer and generates signals indicative of an amount of the charged analyte bound to the biosensor. The signal analyzer is capable of quantifying the signals generated by binding of the biosensor to the charged analyte and/or monitoring and analyzing the binding kinetics over time of the charged analyte to the biosensor.

[0068] In embodiments, the signal analyzer quantifies signals by biolayer interferometry (BLI), quartz crystal microbalance (QCM), surface plasmon resonance (SPR), surface acoustic wave (SAW), refl ectometric interference spectroscopy (RIfS), or atomic force microscopy.

[0069] In some embodiments, the biosensor can comprise an optical fiber having a proximal end portion and a distal end portion. The proximal end portion may be configured to receive light from a light source and configured to deliver reflected light to the detector. The optical fiber can be a circular optical fiber having a round cross-section. The distal end portion can be configured the biosensor to have analytes bind thereto such that light reflected from the distal end portion is phase shifted based on a thickness of analytes bound to the distal end portion. The biosensor can further comprise an optical resonator at a distal end portion of the optical fiber, the optical resonator including a first reflective surface and a second reflective surface, the first reflective surface configured to reflect light with a first phase and the second reflective surface configured to reflect light with a second phase which is phase shifted based on a thickness of analytes bound to the optical resonator.

[0070] The BLI sensor or optical assembly functions as a sensing element or detector tip to detect analytes attached to an end thereof. The detector unit detects interference signals produced by interfering light waves reflected from the optical assembly. The detector may be a spectrometer, such as charge-coupled device (CCD), capable of recording the spectrum of the reflected interfering light from the optical assembly. The light source directs light into the optical assembly, which is reflected back to the detector unit through an optical coupling assembly. The coupling assembly includes a first optical waveguide or fiber that extends from the light source to the optical assembly, a second optical waveguide or fiber which carry reflected light from the optical assembly to the detector, and an optical coupler which optically couples the first optical waveguide and the second optical waveguide. The first optical waveguide and/or the second optical waveguide may be in the form of a fiber optic bundle (FOB). In some embodiments, the coupling assembly includes a lens system constructed to focus a light beam on an upper surface of the optical assembly and to direct reflected interfering light from the optical assembly to the detector.

[0071] The measurement of the presence, concentration, and/or binding rate of analyte to the optical assembly is enabled by the interference of reflected light beams from the two reflecting surfaces in the optical assembly. Specifically, as analyte molecules attach to or detach from the surface, the average thickness of the first reflecting layer changes accordingly. Because the thickness of all other layers remains the same, the interference wave formed by the light waves reflected from the two surfaces is phase shifted in accordance with this thickness change. Assuming that there are two reflected beams, the first beam is reflected from the first reflecting surface and the second beam is reflected from the analyte-binding molecules and bound analyte and the surrounding medium at the second reflecting surface. The conversion of the phase shifting to a thickness change of the bound analytes is well known in the art.

[0072] In some embodiments, the biosensor is coupled to a quartz crystal microbalance (QCM). QCM measures mass through the changes in frequency of a piezoelectric quartz crystal when disturbed by the change in mass due to the product that is intended to be measured. In addition, QCM is also capable of providing information on the viscoelastic properties of the deposited material, which are determined by recording the dissipation factor. The biosensor can be linked to QCM and a processing module, which allows the measurement of three parameters: frequency variation (Af), variation of the dissipation factor (AD), and the Af/AD coefficient. Binding of a charged analyte to the biosensor causes a change in the resonance frequency and the dissipation factor of the microbalance. Binding of a particular analyte should provide a constant Af/AD coefficient with time.

[0073] In some embodiments, the biosensor is a surface plasmon resonance (SPR) biosensor. A SPR biosensor can include probe molecules immobilized on to the proximal surface of a sensor chip having metal (such as gold) coating and/or functional coting (such as biopolymer), and a prism attached to the distal surface of the sensor chip. The proximal surface of a sensor chip can contact fluidic medium containing an analyte, which can bind to the probe molecules immobilized on the proximal surface of the sensor chip. When the solution of target molecules is flown into contact with the surface, a probe-target binding via affinity interaction occurs, which consequently induces an increase in the refractive index at the SPR sensor surface. In SPR measurements, resonance or response units (RU) are used to describe the signal change, where 1 RU is equivalent to a critical angle shift of 10’⁴ degree. At the start of the measurement where probe target interactions have not occurred, the initial RU value corresponds to the starting critical angle. The change in refractive index Ana arisen within a layer of thickness h can be calculated as

Ana = (dn/dc)voi AT/h where (dn/dc)voi is the increase of refractive index n with the volume concentration of analyte c, and Ar is the concentration of the bound target on the surface. The change in the refractive index is tracked by the coupling of incident light into a propagating surface plasmon (PSP) on the surface of the sensor chip (such as gold film) in real time.

3. Methods for Quantitating a Charged Analyte

[0074] A method provided herein for detecting the presence or absence of a charged analyte in a sample can include contacting the sample with a biosensor, bind to the biosensor the charged analyte contained in the sample, 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 features provided in the present disclosure. The charged biopolymer component can include one or more layers of charged biopolymer and an outer surface. The first layer of charged biopolymer (the innermost layer that is adjacent to the core component) is bound to an external surface of the core component. Where the biosensor has two or more layers of charged biopolymer, each layer of the charged biopolymer is oppositely charged relative to the adjacent layer(s) (the inner adjacent layer and, when the outer adjacent layer is present, the outer adjacent layer) and bound to the adjacent layer(s). The outermost layer of 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 (outermost layer of charged biopolymer) and generates signals indicative of an 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 quantitated based at least partially on: signals that are generated by binding of the charged analyte to the biosensor and indicative of an amount of the charged analyte bound to the biosensor; and/or binding kinetics of the biosensor towards the charged analyte. Different amounts, concentrations, and/or charges of the charged analyte in the sample can result in different binding kinetics of the biosensor towards the charged analyte in the sample. For example, the charged analyte may comprise a population of viral capsids, which may include a full viral capsid containing polynucleotides and/or an empty viral capsid not containing polynucleotides, and different full/empty viral capsid ratios in the sample may result in different binding kinetics of the biosensor towards the viral capsids contained in the sample. The population of viral capsids may include capsids of adeno-associated virus (AAV).

[0075] FIG. 5 depicts an example method 500 of detecting the presence, amount and/or characteristic of a charged analyte in a sample, according to embodiments of the present disclosure. The process 500 begins from step 502 by providing a biosensor having one or more layers of charged biopolymer. The process 500 continues to step 504, to contact the sample with the biosensor, having the outermost layer oppositely charged relative to the charged analyte. Process 500 continues to step 506, to allow binding of the biosensor to the charged analyte in the sample. Process 500 continues to step 508, to detect and/or quantitate the presence, amount, and/or characteristic of the charged analyte in the sample based on one or more of the following parameters: the signal generated by the 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 towards the charged analyte.

[0076] The outermost layer of charged biopolymer of the biosensor, oppositely charged relative to the charged analyte in a sample, 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 charged biopolymer is negatively charged, allowing binding of the outmost layer of charged biopolymer 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 charged biopolymer is positively charged, allowing binding of the outmost layer of charged biopolymer to a negatively charged analyte. The negatively charged analyte may include a negatively charged protein, a polynucleotide, or a viral vector (such as an AAV vector).

[0077] The signals generated by binding of the biosensor to the charged analyte and/or the binding kinetics of the biosensor towards the charged analyte can be measured by biolayer interferometry (BL1), quartz crystal microbalance (QCM), surface acoustic wave (SAW), atomic force microscopy, reflectometric interference spectroscopy (RIfS), or surface plasmon resonance (SPR). The method provided herein can be used to detect the presence or amount of a charged analyte without a detectable label attached to the analyte directly or indirectly and can be used for detection of 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 partially on the signals generated by binding of the viral vectors to the biosensor for example as depicted in step 508 of FIG. 5. Additionally or alternatively, the empty/full viral capsid ratio of the sample can be determined based at least partially on the binding kinetics of the biosensor towards the viral vectors for example as depicted in step 508 of FIG. 5.

[0079] As described elsewhere in the present disclosure, binding of viral vectors to the biosensor generates signals indicative of the amount of the viral vectors bound to the biosensor. Binding of the viral vectors to the biosensor can reach higher signal in a sample with a higher viral titer (that is, containing a higher amount or concentration of the viral vectors), compared to a sample with a lower viral titer (that is, containing a lower amount of concentration of the viral vectors). For example, as shown in FIG.3, the binding of viral capsids to the biosensor can reach higher signal in sample with higher titer as compared to samples with a lower titer.

[0080] In addition, different amounts, concentrations, and/or charges of the viral vectors in the sample can result in different binding kinetics over time of the biosensor towards the viral vectors contained in the sample. The viral vectors can include viral capsids (such as AAV capsids) containing full or empty viral capsids. Different full/empty viral capsid ratios in the sample can result in different binding kinetics over time of the biosensor towards the viral vectors contained in the sample. For example, because a full capsid with a DNA core has a higher negative charge as compared to an empty capsid without a DNA core, as shown in FIG. 4, in samples with a lower percentage of empty capsids (and thus with more abundancy of negative charges), the binding of viral capsids (empty or full) to the biosensor reached saturation faster as compared to samples with a higher percentage of empty capsids. Accordingly, using the method provided herein, the viral titers and the empty/full viral capsid ratio in a sample can be determined based at least partially on the signals generated by the binding of the viral vectors to the biosensor (binding rate analysis) and/or the binding kinetics of the viral vectors to the biosensor (saturation analysis).

Examples

Example 1: Binding kinetics and signal level of adeno-associated virus type 8 (AAV8) capsids with varied titers, but same empty/full capsid ratio, to a biolayer interferometer (BLI) biosensor and associated interferometry signals

[0081] This example relates to binding kinetics and signal levels of AAV8 capsids (negatively charged analytes) to the biolayer interferometer (BLI) biosensor in samples with varied titers.

[0082] The epoxypropylsilane (EPS) of a BLI biosensor was activated and coated with a negatively charged biopolymer, and then coated with polyethylenimine (PEI). Accordingly, the biosensor contained two layers of oppositely-charged biopolymers: the inner negatively charged layer, and the outer positively charged layer of PEI. Samples each containing AAV8 capsids at the concentration (titer) of 5 x 10¹¹ viral genome (vg)/ml, 2.5 x 10¹¹ cp/ml, 1.25 x 10¹¹ cp/ml, 6.25 x 10¹⁰and 3.125 x IO¹⁰ vg/ml, respectively, were measured over time using BLI for a signal shift (nm), with a positive shift indicating binding of viral capsids to the biosensor. As shown in FIG. 3, the binding of AAV8 capsids to the biosensor showed positive correlation between binding signal and capsid titer within the loading time frame. This can be explained by more AAV8 particles in samples with a higher viral titer, and faster and higher binding towards the positively charged biopolymer on the biosensor. The viral titer can be determined using a calibration curve generated based on signals of reference samples obtained using the methods provided herein. Example 2. Binding kinetics and signal level of adeno-associated virus type 2 (AAV2) capsids with same titer, but varied empty/full capsid ratio, to a biolayer interferometer (BLI) biosensor and associated interferometry signals

[0083] Signal shift (nm) was measured over time using BLI for samples each containing AAV2 with the empty to full capsid ratio of 10%, 17%, 26%, 36%, 50%, 63%, 70%, and 82.7%, respectively, with a shift indicating binding of viral capsids to the biosensor. As shown in FIG. 4, in samples with a lower percentage of empty capsids, the binding of AAV2 capsids (empty or full) to the biosensor reached saturation faster as compared to samples with a higher percentage of empty capsids. This can be explained by the higher negative charges of a full capsid with a DNA core as compared to an empty capsid without a DNA core, resulting in more abundancy of negative charges derived from AAV2 capsids in samples with a lower percentage of empty capsids. The empty/full viral capsid ratio can be determined using a calibration curve generated based on binding kinetics of reference samples obtained using the methods provided herein.

[0084] Different binding kinetics towards the biosensor of samples with different viral titers or different empty/full capsid ratios as shown above can be used to determine the viral titer or empty/full capsid ratio in a sample.

[0085] While several embodiments of the disclosure have been shown in the drawings, it is not intended that the disclosure be limited thereto, as it is intended that the disclosure be as broad in scope as the art will allow and that the specification be read likewise. Any combination of the above embodiments is also envisioned and is within the scope of the appended claims. Therefore, the above description should not be construed as limiting, but merely as exemplifications of particular embodiments. Those skilled in the art will envision other modifications within the scope of the claims appended hereto.

Claims

What is Claimed;

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

2. The biosensor of 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 bound to an external surface of the core component; and each remaining layer of the two or more layers of charged biopolymer is oppositely charged relative to an adjacent layer of the one or more layers of charged biopolymer and is bound thereto.

3. The biosensor of claim 1, wherein the core component comprises aminopropylsilane (APS) or epoxypropylsilane (EPS).

4. The biosensor of claim 1, wherein at least one layer 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 layer of the one or more layers of charged biopolymer comprises one or more of polyethylenimine (PEI), chitosan, poly-L-lysine, polyallylamine, and polyaziridine.

6. The biosensor of claim 1, wherein the charged analyte comprises a protein, a viral vector, or a polynucleotide.

7. The biosensor of claim 1, wherein the outermost layer of the charged biopolymer component is negatively charged, such that binding of the charged biopolymer component to a positively charged protein contained in the sample is facilitated.

8. The biosensor of claim 7, wherein the positively charged protein comprises bovine serum albumin (BSA), neutravidin, or ovalbumin.

9. The biosensor of claim 1, wherein the outermost layer of the charged biopolymer is positively charged, such that binding of the charged biopolymer component to a negatively charged protein, a polynucleotide, or a viral vector is facilitated.

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

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

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

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

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

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

16. An apparatus for quantitating a charged analyte in a sample, the apparatus comprising: a biosensor comprising a core component and a charged biopolymer component, the charged biopolymer component comprising one or more layers of charged biopolymer, an outermost layer of the charged biopolymer component being oppositely charged relative to the charged analyte to facilitate binding of the charged biopolymer component to the charged analyte in the sample and generating of signals indicative of an amount of the charged analyte bound to the biosensor; and a signal analyzer coupled to the biosensor, the signal analyzer capable of transforming and quantifying the signals generated by binding of the biosensor to the charged analyte.

17. The apparatus 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 bound to an external surface of the core component; and each remaining layer of the two or more layers of charged biopolymer is oppositely charged relative to an adjacent layer of the two or more layers of charged biopolymer and is bound thereto.

18. The apparatus of claim 16, wherein: the core component comprises aminopropylsilane (APS) or epoxypropylsilane (EPS); at least one layer 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 layer of the one or more layers of charged biopolymer comprises one or more of polyethylenimine (PEI), chitosan, poly-L -lysine, polyallylamine, and polyaziridine.

19. A method for detecting presence or amount of a charged analyte in a sample, the method comprising: contacting the sample with a biosensor, the biosensor comprising a core component and a charged biopolymer component, the charged biopolymer component comprising one or more layers of charged biopolymer, an outermost layer of the charged biopolymer component being oppositely charged relative to the charged analyte; binding the charged analyte in the sample to the charged biopolymer component; and detecting the presence or amount of the charged analyte based at least partially on: signals generated by binding of the charged analyte to the biosensor, wherein the signals are indicative of an amount of the charged analyte bound to the biosensor; and/or binding kinetics of the biosensor towards the charged analyte, wherein different amounts, concentrations, and/or charges of the charged analyte in the sample result in different binding kinetics of the biosensor towards the charged analyte in the sample.

20. The method of claim 19, wherein the signals 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 a viral titer of the sample is determined based at least partially on the signals 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 an empty/full viral capsid ratio of the sample is determined based at least partially on the binding kinetics of the biosensor towards the one or more viral vectors.