WO2025014978A1

WO2025014978A1 - Methods and compositions for characterizing viral particles in a biological sample

Info

Publication number: WO2025014978A1
Application number: PCT/US2024/037252
Authority: WO
Inventors: Qimin Quan; Angad GARG; Tal Raz
Original assignee: Nanomosaic Inc
Current assignee: Nanomosaic Inc
Priority date: 2023-07-09
Filing date: 2024-07-09
Publication date: 2025-01-16
Anticipated expiration: 2026-01-09

Abstract

The present disclosure provides methods and compositions for determining (i) the presence, absence, and/or amount of one or more groups of viral particles and/or (ii) the composition of viral particles, in a biological sample using a functionalized nanostructure-based sensor.

Description

METHODS AND COMPOSITIONS FOR CHARACTERIZING VIRAL PARTICLES IN A BIOLOGICAL SAMPLE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/525,716 filed on July 9, 2023, U.S. Provisional Patent Application No. 63/602,618 filed on November 26, 2023, and U.S. Provisional Patent Application No. 63/569,729 filed on March 26, 2024, the disclosures of each of which are hereby incorporated by reference in their entireties for all purposes.

FIELD

[0002] The disclosure relates to methods and compositions for characterizing viral particles and their composition in a biological sample using a nanostructure-based sensor.

BACKGROUND

[0003] The detection and quantification of viral particles and the composition of viral genomes included within the viral particles is required in a variety of applications. For example, during the manufacture of viral particles used in gene therapy it is critical to assess the presence and amount of viral particles, and the completeness of the viral genome at various stages during the upstream (cell line development and viral harvest) and downstream (viral purification and formulation) processes. The genetic characterization of the viral particles is critical for informing treatment options, such as appropriate dosages to be administered to a subject.

[0004] Existing methods for characterizing viral particles suffer from slow speed (sample- to-answer time), low throughput, low correlation across analytical platforms (conventional approaches require consolidation of multiple technologies, instruments, consumable, and workflows), large sample amount and/or purity requirement (an inability to work with a crude sample), which can become a bottleneck during process development and manufacturing. A major challenge with such approaches is based on the requirement for calibration of the results across multiple measurements from different assay workflows and various testing instruments, including, for example, enzyme-linked immunosorbent assays (ELISA), UV/visible spectroscopy, mass spectroscopy (MS), transmission electron microscopy (TEM), quantitative PCR (qPCR), digital PCR (dPCR) and AUC/MS quantifications. Such methods typically require nucleic acid purification, dilution, amplification, and an additional variety of steps before detection of genetic alteration can be accomplished. These processes are cumbersome and result in significant loss of sample and in quantification bias.

[0005] Despite the advances made to date there is still a need for new methods and compositions for characterizing viral particles present in biological samples, especially viral particles that will be administered to humans during a gene therapy protocol.

SUMMARY

[0006] The present disclosure generally relates to nanosensor-based methods and compositions that facilitate highly sensitive detection of viral particles and genomic DNA contained within the viral particles. Furthermore, the present disclosure details the utilization of nanosensors for detection and quantification of nucleic acids of a sequence of interest, directly in crude samples such as cell lysates and body fluids (e.g., plasma, serum, cerebrospinal fluid, urine, or tears) without requiring sample purification. For example, the methods and compositions described herein provide an approach for the routine analysis of viral particles within a biological sample to determine, for example, whether a viral particle contains a full transgene, an empty capsid, a partial transgene, or a transgene or partial transgene containing a nucleotide insertion, deletion or substitution. Furthermore, the methods and compositions described herein facilitate the routine analysis of biological samples to determine whether the viral capsids in the sample are empty, full, partially full and/or have the desired integrity. The methods and compositions described herein can characterize viral particle titers and genetic composition, using a single instrument, the same assay plate, and no sample purification.

[0007] In a first aspect, the disclosure provides a method of determining the presence, absence, and/or amount of one or more groups of viral particles in a biological sample, the method comprising: (a) contacting a sensor defining a plurality of functionalized nanostructures with a first test sample comprising one or more nucleic acid molecules under conditions that permit the functionalized nanostructures to bind the one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are harvested from the viral particles in the biological sample or are amplicons derived therefrom; (b) after step (a), detecting a change in a property of the nanostructures thereby to determine the presence, absence, and/or amount of the one or more nucleic acid molecules; and (c) after step (b), using the presence, absence, and/or amount of the one or more nucleic acid molecules and, optionally a concentration of viral particles in the biological sample, to determine the presence, absence, and/or amount of the one or more groups of viral particles within the first test sample.

[0008] Depending upon the circumstances, the functionalized nanostructures can each comprise a capture agent. In some embodiments, the capture agent is an antibody or an antigen-binding fragment thereof capable of binding the one or more nucleic acid molecules.

[0009] In some embodiments, the amplicons are produced by amplification of nucleic acid molecules harvested from the viral particles. Alternatively or in addition, the amplicons can comprise a detection label capable of binding to a detection agent. Depending on the circumstances, pairs of detection label and the detection agent can be selected from the group consisting of: biotin and streptavidin, fluorescein and an anti-fluorescein antibody, digoxigenin and an anti-digoxigenin antibody, 3,3',5,5'-Tetramethylbenzidine (TMB) and an antibody conjugated to horseradish peroxidase (HRP), an oligonucleotide and a complementary oligonucleotide, an oligonucleotide and an aptamer, an oligonucleotide and its binding partner, a click chemistry pair (e.g., an azide and an alkyne, an azide and a dibenzocyclooctyne, or a tetrazine and a transcyclooctene).

[0010] In some embodiments, the amplicons comprise a capture label, and optionally or in addition the capture agent for use with the capture label is an antibody or an antigen-binding fragment thereof capable of binding the one or more nucleic acid molecules. Depending on the circumstances, pairs of capture label and capture agent can be selected from the group consisting of: a target antigen and an antibody, an aptamer-binding partner and an aptamer, biotin and streptavidin, a fluorophore (e.g., fluorescein, Tamra, Cy5, or Cy3) and an antibody that binds the fluorophore, digoxigenin and an anti-digoxigenin antibody, a nucleic acid molecule and a complementary nucleic acid molecule, a nucleic acid molecule and an aptamer capable of binding the nucleic acid molecule, a poly- A nucleic acid sequence and a poly-T nucleic acid sequence, a poly-A nucleic acid sequence and an antibody that binds the poly-A nucleic acid sequence, a poly-T nucleic acid sequence and an antibody that binds the poly-T nucleic acid sequence, a poly-C nucleic acid sequence and a poly-G nucleic acid sequence, a poly-C nucleic acid sequence and an antibody that binds the poly-C nucleic acid sequence, a poly-G nucleic acid sequence and an antibody that binds the poly-G nucleic acid sequence, and a click chemistry pair (e.g., an azide and an alkyne, an azide and a dibenzocyclooctyne, or a tetrazine and a transcyclooctene). In some embodiments, the functionalized nanostructures each comprise an antibody or antigen binding fragment thereof that binds the capture label. [0011] In some embodiments, the method further comprises, prior to step (c): (i) contacting an additional substrate comprising a plurality of functionalized nanostructures with an additional test sample comprising a plurality of viral particles, wherein the functionalized nanostructures of the additional substrate are capable of binding the viral particles; and (ii) after step (i), detecting a change in a property of the nanostructures thereby to determine the concentration of the viral particles in the biological sample.

[0012] In some embodiments, the first test sample and the additional test sample are derived from a single viral culture and/or cell culture grown in a bioreactor. In some embodiments, the method further comprises, prior to step (ii), contacting the viral particles with a detection antibody capable of binding the viral particles. In some embodiments, the detection antibody is selected from the group consisting of an antibody that binds a viral protein of a specific serotype of the viral particle (e.g., an anti -VP 1 antibody, an anti-VP2 antibody, an anti-VP3 antibody), a serotype non-specific antibody, an antibody that binds a specific serotype of the viral particle (e.g., an anti-AAV9 antibody, an anti-AAV5 antibody, an anti-AAV2 antibody, or an anti-AAV8 antibody), an antibody that binds a lentiviral particle, an antibody that binds a lentiviral capsid protein, an antibody that binds to a baculovirus particle, and an antibody that binds to baculovirus capsid protein.

[0013] In certain embodiments, the detection antibody is conjugated to a second detection agent selected from the group consisting of: streptavidin, HRP, HRP and TMB, HRP and 3,3 '-Diaminobenzidine (DAB)), a nucleic acid molecule, a bead (e.g., a polystyrene bead or a magnetic bead), and a nanoparticle (e.g., a gold nanoparticle).

[0014] In another aspect, the present disclosure provides a method of determining the presence, absence, and/or amount of two or more groups of viral particles in a biological sample, the method comprising: (a) contacting a first region, a second region, and a third region of a sensor defining a plurality of functionalized nanostructures with a test sample comprising one or more nucleic acid molecules and one or more viral particles or viral capsid proteins thereof under conditions that permit the functionalized nanostructures to bind the one or more nucleic acid molecules and/or the one or more of the viral capsid proteins, wherein the first region of the sensor is configured to bind a first nucleic acid molecule, the second region of the senor is configured to bind a second, different nucleic acid molecule, and the third region of the sensor is configured to bind the viral particles or viral capsid proteins, and wherein the one or more nucleic acid molecules are harvested from the viral particles in the biological sample or are amplicons derived therefrom; (b) after step (a), detecting a change in a property of the nanostructures thereby to determine the presence, absence and/or amount of the first nucleic acid molecule, the second nucleic acid molecule, and the one or more viral particles or viral capsid proteins; and (c) after step (b), using the presence, absence, and/or amount of the one or more nucleic acid molecules of the first test sample, the one or more nucleic acid molecules of the second test sample, and the one or more viral particles or viral capsid proteins in the biological sample to determine the presence, absence, and/or amount of the two or more groups of viral particles within the biological sample.

[0015] In certain embodiments, the functionalized nanostructures comprise a capture agent. In certain embodiments, the amplicons are produced by amplification of nucleic acid molecules harvested from the viral particles. Depending upon the circumstances, the amplicons can comprise a detection label capable of binding to a detection agent. In some embodiments, pairs of the detection label and the detection agent can be selected from the group consisting of: biotin and streptavidin, fluorescein and an anti-fluorescein antibody, digoxigenin and an anti-digoxigenin antibody, 3,3',5,5'-Tetramethylbenzidine (TMB) and an antibody conjugated to horseradish peroxidase (HRP), an oligonucleotide and a complementary oligonucleotide, an oligonucleotide and an aptamer, an oligonucleotide and its binding partner, a click chemistry pair (e.g, an azide and an alkyne, an azide and a dibenzocyclooctyne, or a tetrazine and a transcyclooctene). In some embodiments, the capture agent is an antibody or an antigen-binding fragment thereof capable of binding the one or more nucleic acid molecules.

[0016] In certain embodiments, the amplicons comprise a capture label. In certain embodiments, the capture agent is an antibody or antigen binding fragment thereof that binds the capture label. In some embodiments, pairs of the capture agent and the capture label can be selected from the group consisting of: an antibody and a target antigen, an aptamer and an aptamer-binding partner, biotin and streptavidin, an antibody that binds the fluorophore and a fluorophore (e.g., fluorescein, Tamra, Cy5, or Cy3), an anti-digoxigenin antibody and digoxigenin, a nucleic acid molecule and a complementary nucleic acid molecule, a nucleic acid molecule and an aptamer capable of binding the nucleic acid molecule, a poly-A nucleic acid sequence and a poly-T nucleic acid sequence, a poly-A nucleic acid sequence and an antibody that binds the poly-A nucleic acid sequence, a poly-T nucleic acid sequence and an antibody that binds the poly-T nucleic acid sequence, a poly-C nucleic acid sequence and a poly-G nucleic acid sequence, a poly-C nucleic acid sequence and an antibody that binds the poly-C nucleic acid sequence, a poly-G nucleic acid sequence and an antibody that binds the poly-G nucleic acid sequence, and a click chemistry pair (e.g., an azide and an alkyne, an azide and a dibenzocyclooctyne, or a tetrazine and a transcyclooctene). In certain embodiments, nanostructures in the first region, the nanostructures in the second region, and the nanostructures in the third region each comprise a distinct capture agent; and amplicons bound by the first region and amplicons bound by the second region comprise different capture labels.

[0017] In certain embodiments, the one or more nucleic acid molecules each independently comprise (i) a full length transgene, (ii) a fragment of the transgene, or (iii) a full length transgene or fragment thereof comprising a nucleotide insertion, deletion or substitution. Depending upon the circumstances, the method further comprises, prior to step (b), contacting the one or more nucleic acid molecules with a detection agent, wherein binding of the detection agent to the one or more nucleic acid molecules enhances the change in the property of the nanostructures.

[0018] In some embodiments, the biological sample is a viral lysate and/or a cell lysate, for example, a viral lysate produced from viral particles grown in a bioreactor and/or a cell lysate produced from cells grown in a bioreactor. In certain embodiments, the viral particles are adenoviral particles, adeno associated viral (AAV) particles, lentiviral particles, baculovirus particles, or engineered viral particles of any one of the foregoing. In certain embodiments, the viral particles are adenoviral particles. In some embodiments, the amplicons are produced by amplification of nucleic acid molecules harvested from the viral particles. In some embodiments, the one or more nucleic acid molecules are DNA or RNA molecules. In some embodiments, the one or more nucleic acid molecules are DNA molecules.

[0019] In certain embodiments, the amplicons comprise a double stranded nucleic acid. In certain methods, a first strand of the double stranded nucleic acid comprises a detection label and the second strand of the double stranded nucleic acid comprises a capture label.

[0020] In some embodiments, prior to step (a), the amplicons are produced using a first primer optionally coupled to a detection label and a second primer optionally coupled to a capture label and a thermostable polymerase.

[0021] In certain embodiments, the first primer hybridizes to a first locus on the one or more nucleic acid molecules and the second primer hybridizes to a second locus on the one or more nucleic acid molecules. Depending upon the circumstances, the first locus is located at a 5' end of a first strand of the one or more nucleic acid molecules and the second locus is located at a 5' end of a second strand of the one or more nucleic acid molecules, wherein the first strand and second strand are complementary. Alternatively, the first locus can be located at a 5' end of a first strand of the one or more nucleic acid molecules and the second locus can be located at an interior region of a second strand of the one or more nucleic acid molecules, wherein the first strand and second strand are complementary. Alternatively, the first locus can be located at an interior region of a first strand of the one or more nucleic acid molecules and the second locus can be located at a 5' end of a second strand of the one or more nucleic acid molecules, wherein the first strand and second strand are complementary. Alternatively, the first locus is located at an interior region of a first strand of the one or more nucleic acid molecules and the second locus is located at an interior region of a second strand of the one or more nucleic acid molecules, wherein the first strand and second strand are complementary.

[0022] Depending upon the method being implemented, the first primer and the second primer are resistant to exonuclease digestion. As a result, nucleic acids not containing the first primer or the second primer are digested by exposure to an exonuclease.

[0023] In certain embodiments, the method is performed over two or more replicates, wherein each replicate comprises a distinct first primer and/or second primer, thereby detecting the presence or absence of a plurality of loci within the one or more nucleic acid molecules.

[0024] In certain embodiments, the detectable property of the nanostructures is an optical property associated with the nanostructures.

[0025] In some embodiments, the sensor comprises a plate defining a plurality of addressable wells, each well comprising a grid disposed therein, wherein the grid comprises the plurality of nanostructures. The nanostructure can comprise a variety of configurations, but in certain circumstances the nanostructure preferably is a nanoneedle. Furthermore, the nanostructures (e.g., nanoneedles) can be integral with at least one of a planar support, a spherical surface, or a flexible substrate.

[0026] In another aspect, the disclosure provides a method of determining the presence, absence, and/or amount of a plurality of regions within a target nucleic acid. The method comprises: (a) preparing a plurality of reaction mixtures comprising: (i) the target nucleic acid; (ii) a first primer that hybridizes to a first locus on the target nucleic acid, optionally wherein the first primer is coupled to a detection label; (iii) a second primer that hybridizes to a second locus on the target nucleic acid, optionally wherein the second primer is coupled to a capture label; and (iv) a thermostable polymerase, wherein each reaction mixture comprises a distinct first primer and/or second primer that hybridize to distinct loci within the target nucleic acid; (b) performing a plurality of PCR cycles on the plurality of reaction mixtures, thereby generating amplicons comprising a region of the target nucleic acid between the first and second loci; and (c) detecting the amplicons from each reaction mixture, thereby determining the presence, absence, and/or amount of each region within the target nucleic acid.

[0027] In another aspect, the disclosure provides a method of determining the presence, absence, and/or amount of a plurality of subregions within target complementary nucleic acid sequences. The method comprising: (a) preparing a first reaction mixture comprising: (i) a first target nucleic acid sequence that is in a forward orientation; (ii) a first primer that hybridizes to a first locus on the first target nucleic acid sequence; (iii) a second target nucleic acid sequence that it is in a reverse orientation to the first nucleic acid sequence; (iv) a second primer that hybridizes to a second locus on the second target nucleic acid sequence; and (v) a thermostable polymerase, (b) performing a plurality of PCR cycles on the first reaction mixture to generate a plurality of amplicons, a first amplicon comprising the first primer and a sequence complementary to the first target nucleic acid sequence and a second amplicon comprising a second primer and a sequence complementary to the second nucleic acid sequence; (c) enzymatically digesting the first target nucleic acid sequence and the second target nucleic acid sequence to produce a second reaction mixture; (d) adding to the second reaction mixture a third primer that hybridizes to a first locus of the first amplicon, the third primer coupled to a detection label and a fourth primer that hybridizes to a second locus of the second amplicon, the fourth primer coupled to a capture label, and performing a plurality of PCR cycles on the second reaction mixture thereby generating a plurality of double stranded amplicons, wherein a first strand of each double stranded amplicon corresponds a first subregion within the first target nucleic acid sequence and a second strand of each double stranded amplicon corresponds to a second subregion within the second target nucleic acid sequence; and (e) detecting the plurality of double stranded amplicons, if present, thereby to determine the presence, absence, and/or amount of the first and second subregions within the first and second target nucleic acid sequences.

[0028] In certain embodiments, prior to step (e), a double stranded amplicon is captured on the surface of a nanostructure via a capture label. In certain embodiments, a mass amplifier is bound by the detection label. Depending upon the circumstances, the detecting step comprises detecting an optical signal, e.g., light scattering.

[0029] In another aspect, the disclosure provides a sensor for characterizing a nucleic acid molecule in a sample. The sensor comprises: (i) a first region comprising a first series of nanostructures comprising capture agents attached thereto; (ii) a nucleic acid complex bound to at least one nanostructure in the first series of nanostructures, the nucleic acid complex comprising a first nucleic acid sequence comprising a capture probe affixed thereto and a second nucleic acid sequence comprising a detection probe affixed thereto, wherein the first nucleic acid sequence is hybridized to the second nucleic acid sequence, and the nucleic acid complex is bound to the at least one nanostructure via at least one of the capture agents affixed thereto, and (iii) a mass amplifier bound to the detection probe, whereupon binding of the nucleic acid complex and the mass amplifier to the at least one nanostructure produces a detectable signal in the first series of nanostructures.

[0030] In certain embodiments, the first nucleic acid sequence and/or the second nucleic acid sequence is an amplicon derived from the nucleic acid molecule.

[0031] In another aspect, the disclosure provides a sensor for characterizing a nucleic acid molecule in a sample, the sensor comprising:

(a) a first region comprising: (i) a first series of nanostructures comprising a first series of capture agents attached thereto; (ii) a first nucleic acid complex bound to at least one nanostructure in the first series of nanostructures, the nucleic acid complex comprising a first nucleic acid sequence comprising a first capture probe affixed thereto and a second nucleic acid sequence comprising a first detection probe affixed thereto, wherein the first nucleic acid sequence is hybridized to the second nucleic acid sequence, and the nucleic acid complex is bound to the at least one nanostructure in the first series of nanostructures via at least one of the capture agents affixed thereto; and (iii) a first mass amplifier bound to the first detection probe;

(b) optionally, a second region comprising: (i) a second series of nanostructures comprising a second series of capture agents attached thereto; (ii) a second nucleic acid complex bound to at least one nanostructure in the second series of nanostructures, the nucleic acid complex comprising a third nucleic acid sequence comprising a second capture probe affixed thereto and a fourth nucleic acid sequence comprising a second detection probe affixed thereto, wherein the third nucleic acid sequence is hybridized to the fourth nucleic acid sequence, and the second nucleic acid complex is bound to the at least one nanostructure in the second series of nanostructures via at least one of the capture agents affixed thereto; and (iii) a second mass amplifier bound to the second detection probe; and

(c) a third region comprising: (i) a third series of nanostructures comprising a third series of capture agents attached thereto; and (ii) a viral particle or viral capsid protein thereof bound to at least one nanostructure in the third series of nanostructures; whereupon binding of the first nucleic acid complex and the first mass amplifier to the at least one nanostructure in the first series of nanostructures produces a detectable signal in the first series of nanostructures, binding of the second nucleic acid complex and the second mass amplifier to the at least one nanostructure in the second series of nanostructures produces a detectable signal in the second series of nanostructures, and/or binding of the viral particle or viral capsid protein thereof to the at least one nanostructure in the third series of nanostructures produces a detectable signal in the third series of nanostructures. In certain embodiments, the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, and/or the fourth nucleic acid sequence is an amplicon derived from the nucleic acid molecule.

[0032] In certain embodiments, each capture probe is an antibody or antigen binding fragment thereof. Alternatively or in addition, each detection probe is an antibody or antigen binding fragment thereof. In certain embodiments, each mass amplifier is a ligand bound the antibody or antigen binding fragment thereof.

[0033] In certain embodiments, each series of nanostructures are integral with at least one of a planar support, a spherical surface, or a flexible substrate. In certain embodiments, the detectable signal is an optically detectable signal, e.g., light scattering produced by the at least one nanostructure. In certain embodiments, the nucleic acid molecule is harvested from a viral particle, e.g., an adenoviral particle, an AAV particle, a lentiviral particle, or a baculovirus particle. In certain embodiments, the viral particle is an AAV particle.

[0034] In another aspect, the disclosure provides a sensor for characterizing nucleic acid molecules in a sample. The sensor comprises: a first region and a second region, the first region comprising a first series of nanostructures each comprising capture agents attached thereto capable of binding at least a first nucleic acid complex and producing a detectable signal indicative of a concentration of the nucleic acid molecules within a first concentration range, wherein the series of nanostructures are integral with at least one of a planar support, a spherical surface, or a flexible substrate, and the second region comprising a second series of different nanostructures comprising capture agents attached thereto capable of binding a second nucleic acid complex and producing a detectable signal indicative of a concentration of the nucleic acid molecules within a second, different concentration range, wherein the series of nanostructures are integral with at least one of a planar support, a spherical surface, or a flexible substrate. In such a sensor, (i) the first and second nucleic acid complexes each comprise a first nucleic acid sequence comprising a capture probe attached thereto and a second nucleic acid sequence comprising a detection probe affixed thereto and the first nucleic acid sequence is hybridized to the second nucleic acid sequence, and each nucleic acid complex is bound to the nanostructure via at least one of the capture agents affixed thereto, and (ii) the sensor is capable of quantifying the amount of nucleic acid molecules in the sample across both the first concentration range and the second concentration range.

[0035] In certain embodiments, the nucleic acid sequences of the first and second complexes are amplicons derived from the nucleic acid molecules. In certain embodiments, the nucleic acid complexes further comprise a mass amplifier bound via each detection probe. In certain embodiments, the concentration of nucleic acid molecules in the sample, if within the first concentration range, is determined from a number of individual nanostructures in the first series that have bound molecules of analyte, and wherein the concentration of nucleic acid molecules in the sample, if within the second concentration range, is determined by analog detection of a substantially uniform change in a detectable property of the nanostructures in the second region as a function of the concentration of nucleic acid molecules in the sample. In some embodiments, the first concentration range has a lower detectable value than that of the second concentration range.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In the drawings, like reference characters generally refer to the same parts throughout the different views. Also, the drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the disclosure. In the following description, various embodiments of the present invention are described with reference to the following drawings.

[0037] FIGs. 1A and IB are directed to exemplary sensors for use in characterizing viral particles in a biological sample. FIG. 1A is a schematic diagram illustrating an exemplary sensor with a plurality of wells, each well containing a grid of nanostructure arrays. FIG. IB is a schematic illustration showing the dynamic range of the exemplary sensor of FIG. 1A.

[0038] FIG. 2A is a schematic representation of different formats of series of nanostructures in a sensor of interest. FIG. 2B is a schematic illustration depicting a series of exemplary sensors for measuring ultra-low, low, medium, and high concentrations of analytes.

[0039] FIGs. 3A - 3C show the operability of exemplary sensors in measuring analyte over a large dynamic range. FIG. 3A is a schematic illustration depicting a sensor containing both digital and analog (color shifting) nanostructure arrays. FIG. 3B is a pictorial representation depicting the quantification of Tau protein over a 6 log dynamic range by a combination of digital single molecule quantification (left hand panel) and by analog quantification (right hand panel). FIG. 3C is an image depicting the operability of a digital sensor as a function of analyte concentration.

[0040] FIG. 4 is a graph showing the digital and analog measurements of exemplary data generated by a sensor exemplified in FIG. 3B.

[0041] FIG. 5 is a pictorial representation of an exemplary silicon wafer-based sensor containing both a series of digital nanostructures (25,600) and three series of analog nanostructures (1,000 per series).

[0042] FIG. 6 is a pictorial representation of another exemplary silicon wafer-based sensor comprising a plurality of series of digital nanostructures and three series of analog nanostructures.

[0043] FIG. 7 is a schematic representation depicting cross-sectional views of exemplary nanostructures useful in the methods described herein.

[0044] FIG. 8 is a schematic representation depicting cross-sectional views of exemplary nanostructures composed of two different materials useful in the methods described herein.

[0045] FIGs. 9A - 9D are a series of cross-sectional schematic diagrams illustrating the fabrication of a series of exemplary nanostructures by photoresist patterning, development and etching processes.

[0046] FIGs. 10A - 10G are a series of cross-sectional schematic diagrams illustrating the fabrication of a series of exemplary nanostructures by deposition of a layer on a substrate, spin coating a photoresist on the deposited layer, patterning and developing the resist, evaporating metal on the resist, removal of the resist in a solution, etching the substrate, and removing the photoresist.

[0047] FIGs. 11A - 11F are a series of cross-sectional schematic diagrams illustrating the fabrication of a series of exemplary nanostructures by coating two layers on a substrate, patterning the top layer resist, developing the resist, evaporating materials on the patterned resist, lift-off and spin additional low viscosity materials to achieve a particular surface condition.

[0048] FIG. 12A - 12F are a series of cross-sectional schematic diagrams illustrating the fabrication of a series of exemplary nanostructures by patterning photoresist on an oxide substrate, developing the resist, depositing silicon on the resist, lift-off, and growth of silicon to grow additional structures on the patterned substrate.

[0049] FIGs. 13A - 13D are a series of cross-sectional schematic diagrams illustrating the patterning of photoresist with a mold.

[0050] FIG. 14A is a schematic illustration showing a silicon wafer with multiple series of nanostructures and FIG. 14B is a schematic illustration showing an enlarged image of a single series of nanostructures. FIG. 14C is a schematic diagram of an exemplary sensor for conducting a single antibody label-free assay on nanostructure needles. Antibodies coupled to the nanostructure needles capture specific analytes in a test sample to produce a quantifiable signal. FIG. 14D is a schematic diagram of an embodiment where a singleantibody is disposed on nanostructure needles. Antibodies coupled to the nanostructure needles capture specific analytes in a test sample to produce a quantifiable signal, and the resultant signal is amplified. FIG. 14E is a schematic diagram of an embodiment, where a dual antibody (sandwich) assay on nanostructure needles is used. The first antibody is coupled to the nanostructure needles to capture analytes in a test sample to produce a quantifiable signal, a second antibody is added to the reaction to form a sandwich, and the resultant signal is amplified.

[0051] FIGs. 15A - 15D are schematic depictions of a gasket-based approach sensor design. FIG. 15A depicts a four-pl ex gasket. FIG. 15B depicts a hybrid 16-plex gasket covering half the sensor and a standard 96-well plate covering the other half. FIG. 15C depicts a two gasket-layer approach, where a first layer comprises a four-plex gasket, and a second gasket is layered to cover four of the four-plex wells. FIG. 15D depicts a hybrid four-plex gasket with a second gasket layer covering four of the four-plex wells covering half the sensor and a standard 96-well plate covering the other half.

[0052] FIGs. 16A-16C depict exemplary substrates of functionalized nanostructures useful for detection of viral proteins, capsids, and nucleic acids. FIG. 16A depicts three 96-well Society of Biomolecular Screening (SBS) format substrates of functionalized nanostructures for detection of proteins, viral particles and viral genomes respectively. FIG. 16B depicts one 96-well SBS format substrates of functionalized nanostructures for detection of proteins, viral particles and viral genomes on one chip. FIG. 16C depicts one 96-SBS format substrates of functionalized nanostructures for detection of proteins, viral particles and viral genomes in the same well.

[0053] FIGs. 17A and 17B are perspective views of a nanosensor assembly (consumable) incorporating series of nanostructures useful in the methods described herein.

[0054] FIGs. 18A and 18B are schematic representations of a cartridge assembly comprising a wafer substrate, gasket and retaining base (FIG. 18A) and an exploded perspective view showing the components of the cartridge assembly (FIG. 18B).

[0055] FIG. 19 is a schematic representation of a single plex cartridge and a 1,000-plex cartridge, useful in the methods described herein.

[0056] FIG. 20 is a perspective view of a detection system for use with a sensor during the methods described herein.

[0057] FIG. 21 is a schematic illustration depicting an exemplary optical detection system for imaging an exemplary sensor useful in the methods described herein.

[0058] FIG. 22 is a schematic illustration depicting the interrogation of a sensor. The readout signal can be optical (e.g., imaging), electrical, or mechanical.

[0059] FIG. 23 is a schematic representation showing the data analysis of the output of an exemplary sensor containing digital nanostructures.

[0060] FIG. 24 is a flowchart illustrating an algorithm useful in characterizing the position of viral particles in a biological system.

[0061] FIGs. 25A and 25B are schematic illustrations depicting series of nanostructures configured to detect and/or quantify multiple analytes at the same time. [0062] FIGs. 26A and 26B show an exemplary representation of detecting oligonucleotides using nanosensors by hybridizing the oligonucleotides to complementary oligonucleotides on the nanosensors. FIG. 26A shows capture oligonucleotides attached to the surface of the nanostructures. FIG. 26B shows the hybridization process of the target oligos to the capture oligos are detected with nanostructures.

[0063] FIGs. 27A-27D show an illustration of an exemplary method of detecting a target oligonucleotide by enzymatically depositing non-soluble materials on the nanosensors. FIG. 27A shows capture oligonucleotides attached to the surface of the nanostructures. FIG. 27B shows the target nucleic acid analyte hybridized to the capture oligonucleotides. FIG. 27C shows a second biotinylated probe oligos hybridized to the analyte. FIG. 27D depicts streptavidin-HRP bound to biotin and reacting with TMB to form a non-soluble deposit on the nanostructure.

[0064] FIGs. 28A-28D is a schematic representation showing the detection of oligonucleotides using rolling circle amplification. FIG. 28A shows capture oligonucleotides complementary to the target nucleic acid analyte conjugated to the nanostructures. FIG. 28B shows a target nucleic acids hybridized to the capture oligonucleotides. FIG. 28C shows a circular DNA molecule hybridized to the probe oligonucleotide. FIG. 28D shows additional oligonucleotides deposited on the nanostructures by rolling circle amplification.

[0065] FIGs. 29A and 29B depict an exemplary approach for covalently linking a capture nucleic acid to a protein that can be adsorbed onto a nanostructure. FIG. 29A shows a nucleic acid oligonucleotide with a terminal 3’ ribonucleotide base oxidized using sodium periodate. The oxidized base is reacted with the lysine s-amines of the polypeptide (FIG. 29B)

[0066] FIGs. 30A and 30B show the immobilization of oligonucleotides to nanostructures by hydrophobic interactions.

[0067] FIGs. 31A and 31B show the immobilization of oligonucleotides to nanostructures by antigen-antibody interactions.

[0068] FIG. 32 depicts an exemplary method for measuring nucleic acids on nanostructures.

[0069] FIG. 33 depicts an exemplary method of measuring nucleic acids from viral particles (e.g., AAV particles). [0070] FIG. 34 depicts an exemplary method of detecting viral particles using antibodies specific to surface proteins of the viral particle.

[0071] FIG. 35 depicts an exemplary method of detecting viral capsids using functionalized nanostructures.

[0072] FIG. 36 depicts an exemplary method of detecting viral genomic DNA using functionalized nanostructures.

[0073] FIG. 37 depicts an exemplary nucleic acid labeling scheme that may be used during nanostructure capture and detection.

[0074] FIG. 38 depicts an exemplary method for characterizing nucleic acids by multiple capture oligonucleotides and multiple nanostructures.

[0075] FIG. 39 depicts an exemplary nano sensor well containing nanostructures functionalized for capture and detection of multiple analytes.

[0076] FIG. 40 depicts an exemplary workflow for generating a double stranded detectable PCR product from a short transgene sequence for detection using the functionalized nanostructures described herein.

[0077] FIG. 41A depicts an exemplary workflow for generating a double stranded detectable PCR product comprising affinity probes from a full-length transgene sequence for detection using the functionalized nanostructures described herein. FIG. 41B depicts an exemplary workflow for generating a double stranded detectable PCR product from a full- length transgene sequence for detection using the functionalized nanostructures described herein. FIG. 41C is a graph depicting readout (in Nano Units) as a function of viral genome (VG) concentration (VG/mL).

[0078] FIG. 42 depicts an exemplary workflow for generating a double stranded detectable PCR product from a viral genomic DNA for detection of multiple genomic regions of interest using the functionalized nanostructures described herein.

[0079] FIG. 43A and 43B depicts the structure of exemplary capture and detection primers. FIG. 43A depicts the components of an exemplary capture primer, where the 5’ end is a universal capture sequence, the middle segment is an 18 atom hexa-ethyleneglycol spacer, and the final 3’ segment is the sequence complementary to the Watson (+) strand of the target sequence and is a primer for a PCR polymerase reaction. FIG. 43B depicts the structure of an exemplary detection primer that has a 5’ biotin modification on a sequence that is complementary to Crick strand (-) of the target sequence.

[0080] FIG. 44 depicts a schematic illustration of capture and detection primers amplifying from a template, where the spacer prevents amplification into the poly(A) region of the capture oligonucleotide. The amplified product ultimately has a poly(A) overhang with a spacer on one end, and a biotin label on the other 5’ end.

[0081] FIG. 45A depicts a schematic illustration of the surface of a nanostructure coated with an antibody with specificity to an antigen (e.g., DIG, FAM, etc.) depicted by a green circle at the 3’ end of the universal oligonucleotide followed by a sequence complementary to a universal poly(A) capture sequence. FIG. 45B depicts the interaction of a DNA molecule with the universal capture sequence to the universal oligonucleotide on one end, and with the interaction of biotin and a mass amplifier on the other end.

[0082] FIG. 46 is a graph showing the quantification of a viral genome using a 6-cycle PCR amplification protocol with a capture and detection primer set spanning a promoter in the sampled genome. Identifiers - Triangle: sample; Circle: standards; Solid line: 5PL fit. The number indicates quantification from an 8-fold dilution of the sample.

[0083] FIGs. 47A and 47B are graphs showing viral genome quantification with a single stranded DNA approach. FIG. 47A is a graph showing the results of a method in which both the capture primer and detection probes are positioned proximal to the 3’ ITR and therefore quantify both full length and 3’ truncated viral genomes. FIG. 47B is a graph showing the results of a method in which the capture primer is adjacent to the 3’ ITR and the detection probe is positioned in the farthest distal region of the viral genome - adjacent to the 5’ ITR.

[0084] FIG. 48 is a graph showing a real time trace of the light scattering of nanostructures in three different scenarios, which include empty capsids (1), capsids containing partial genomes (2), and capsids containing full genomes (3).

[0085] FIG. 49 is a graph showing viral particle titer as measured using nanostructures described herein.

[0086] FIG. 50 is a graph showing the viral genome titer as measured using nanostructures described herein. The grey line depicts a plasmid standard curve, and the blue point depicts a commercial orthogonally tested AAV sample. [0087] FIGs. 51A-51B are graphs of standard curves of viral capsid titers measured via ELISA (FIG. 51A) or functionalized nanostructures (FIG. 51B).

[0088] FIG. 52 is a graph showing detected viral capsid titers as a percentage of expected values at various viral concentrations.

[0089] FIG. 53 is bar chart showing detected viral capsid titers in samples prepared with different lysis buffers.

[0090] FIG. 54 is a graph showing the detection of observed viral capsids/mL as a function of expected viral capsids/mL in samples prepared with different lysis buffers.

[0091] FIGs. 55A-55C depict exemplary approaches for detecting nucleic acids in a sample. FIG. 55A depicts an exemplary protocol for comparing analysis of viral DNA using droplet digital PCR (ddPCR) or functionalized nanostructures described herein. FIG. 55B depicts an exemplary protocol for detecting a proportion of full versus partial transgene sequences in a mixed sample. FIG. 55C depicts a schematic of expected readouts for a method of detecting full or partial transgene sequences by indicated probe sets.

[0092] FIGs. 56A-56C depict an assay to detect full-length and truncated viral transgenes. FIG. 56A depicts a schematic representation mixed populations of full-length and truncated viral DNA at indicated proportions of full-length DNA. FIG. 56B is a bar chart showing detection of the transgene as measured by probes detecting a partial region of the viral transgene. FIG. 56C is a bar chart showing detection of the transgene as measured by probes detecting the full-length viral transgene.

[0093] FIG. 57 depicts probed regions on the left using the ITR region with ddPCR or the full-length region with nanostructures. An example distribution of the sample shown on the right where identification with specific probes is shown.

[0094] FIGs. 58A-58C depict an assay for detecting regions of different lengths within a transgene. FIG. 58A depicts detection of probed regions using the ITR region with ddPCR or the full-length region with nanostructures. FIG. 58B depicts a graph showing detected concentrations of regions of indicated length within a plasmid. FIG. 58C depicts a graph showing detected concentrations of regions of indicated length within a sample.

DETAILED DESCRIPTION OF THE INVENTION

[0095] The present disclosure generally relates to nanosensor-based methods and compositions that facilitate highly sensitive detection of viral particles and genomic DNA contained within the viral particles. Furthermore, the present disclosure details the utilization of nanosensors for detection and quantification of nucleic acids of a sequence of interest, directly in crude samples such as cell lysates and body fluids (e.g., plasma, serum, cerebrospinal fluid, urine, or tears) without requiring sample purification. For example, the methods and compositions described herein provide an approach for the routine analysis of viral particles within a biological sample to determine, for example, whether a viral particle contains a full transgene, an empty capsid, a partial transgene, or a transgene or partial transgene containing a nucleotide insertion, deletion or substitution. Furthermore, the methods and compositions described herein facilitate the routine analysis of biological samples to determine whether the viral capsids in the sample are empty, full, partially full and/or have the desired integrity.

[0096] In certain embodiments, the present disclosure enables the characterization of viral particle titers and genetic composition, using a single instrument, the same assay plate, and no sample purification. Furthermore, the methods described herein can be used in the quantification of genetic alterations such as mutations, deletions, insertion, or other genetic variations, in nucleic acid samples including, but not limited to cell culture media, plasma, serum, cerebrospinal fluid, urine, or tears. Depending upon the circumstances, the detection, quantification, and/or characterization of nucleic acids can be accomplished free of bias and loss, and requiring no dilution, amplification, or purification steps.

[0097] In various embodiments, the present disclosure provides a method of determining the presence, absence, and/or amount of one or more groups of viral particles nucleic acids in a biological sample, the method comprising: (a) contacting a sensor defining a plurality of functionalized nanostructures with a first test sample comprising one or more nucleic acid molecules under conditions that permit the functionalized nanostructures to bind the one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are harvested from the viral particles in the biological sample or are amplicons derived therefrom; (b) after step (a), detecting a change in a property of the nanostructures thereby to determine the presence, absence and/or amount of the one or more nucleic acid molecules; and (c) after step (b), using the presence, absence, and/or amount of the one or more nucleic acid molecules and, optionally a concentration of viral particles in the biological sample, to determine the presence, absence, and/or amount of the one or more groups of viral particles within the first test sample. I. Definitions

[0098] As used herein, the term “subject” refers to an organism to be tested by the methods and compositions described herein. Such organisms preferably include mammals (e.g., human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon, and rhesus), and more preferably humans.

[0099] As used herein, the term “nanostructure” is understood to mean any structure, for example, a nanosensor, that has at least one dimension having a length in the range of at least 1 nm to less than 1,000 nm.

[0100] As used herein, the term “analog quantification” is understood to mean a quantification process whereby a substantially uniform change in a detectable property (for example, an optically detectable property, e.g., a color) of nanostructures in a series of nanostructures is detected, when the nanostructures bind a plurality of analytes. In certain embodiments, changes in the detectable property (e.g., color changes) occur as a function of the concentration of analyte in a sample of interest across a precalibrated concentration range of the analyte to be detected. The term “substantially uniform” is understood to mean that, at least 60%, 70%, 80%, 90% or 95% of the nanostructures share the same detectable property, for example, color. An “analog series” or “analog array” is understood to mean a respective series or array of nanostructures configured to permit analog detection.

[0101] As used herein, the term “digital quantification” is understood to mean a quantification process whereby individual nanostructures in a series of nanostructures are detected (for example, optically detected) that flip from one state to another upon binding one or more analytes. A “digital series” or “digital array” is understood to mean a respective series or array of nanostructures configured to permit digital quantification.

[0102] The term “binding agent” or “binder” as used herein refers to an agent (e.g., an antibody) that binds specifically to an analyte of interest. The terms “binds specifically” or “bind preferentially,” as used in connection with a binding agent refers to an agent that binds and/or associates (i) more stably, (ii) more rapidly, (iii) with stronger affinity, (iv) with greater duration, or (v) a combination of any two or more of (i)-(iv), with a particular target analyte than it does with a molecule other than the target analyte. For example, a binding agent that specifically or preferentially binds a target analyte is a binding domain that binds a target analyte, e.g., with stronger affinity, avidity, more readily, and/or with greater duration than it binds a different analyte. The binding agent may be an affinity for the analyte of about 100 nM, 50 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, or 0.01 nM, or stronger, as determined by surface plasmon resonance. For example, the binding agent may have an affinity for the analyte within the range from about 0.01 nM to about 100 nM, from about 0.1 nM to about 100 nM, or from about 1 nM to about 100 nM. It is understood that a binding agent that binds preferentially to a first target analyte may or may not preferentially bind to a second target analyte. As such, “preferential binding” does not necessarily require (although it can include) exclusive binding.

[0103] As used herein, unless otherwise indicated, the term “antibody” is understood to mean an intact antibody (e.g., an intact monoclonal antibody) or antigen-binding fragment of an antibody (for example, an antigen-binding fragment of a monoclonal antibody), including an intact antibody or antigen-binding fragment that has been modified, engineered, or chemically conjugated. Examples of antibodies that have been modified or engineered include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen-binding fragments include Fab, Fab’, (Fab’)2, Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies.

IL Methods of Fabricating a Sensor

[0104] The disclosure provides a sensor for detecting the determining the presence, absence, and/or amount of one or more groups of viral particles in a biological sample. The sensor can comprise a plate defining a plurality of addressable wells, each well comprising a grid disposed therein, wherein (i) the grid comprises a plurality of nanostructure arrays with each nanostructure array comprising a plurality of nanostructures (e.g., nanoneedles), and (ii) each nanostructure array is functionalized with one or more binding moieties for binding a viral protein, nucleic acid, or capsid of a set of viral particles in a biological sample.

[0105] Referring to FIG. 1A, a sensor for detecting presence or quantifying the amount of viral particles or components of viral particles includes a plate. The plate 3 (also referred to herein as a panel) include an array of addressable wells, e.g., 8 x 12 (96 plate), 16 x 24 (384 plate), 32 x 48 (1536 plate) wells. As an example, each well 4 of the 96 well plate includes a grid 5 disposed therein, e.g., a 10 x 10 grid, with each block 6 of the grid being, e.g., about 400 microns x 400 microns, and functionalized with different binding moieties, e.g., antibodies. More specifically, each block 6 of the grid 5 includes one nanostructure array 7, with each nanostructure array including a plurality of nanostructures, as discussed below. Each nanostructure array is functionalized with one or more binding moieties, such as antibodies, nanobodies, affinity probes, or aptamers, for binding one or more analytes (e.g., protein or nucleic acid). In some embodiments, all the nanostructure arrays within a well are functionalized with a binding moiety for binding a specific protein within a set of test proteins or a nucleic acid within a set of test nucleic acids. In other embodiments, a portion of the nanostructure arrays within a well are functionalized with a binding moiety for binding a specific protein within the set of test proteins or a specific nucleic acids with in set of test nucleic acids.

[0106] The sensor may include about 25, 50, 100, 150, 300, 600, or 1,200 different binding moieties for binding each member of the set of test proteins and/or nucleic acids.

III. Sensor Considerations

[0107] The sensors disclosed herein facilitate the detection and/or quantification, with high sensitivity over a large dynamic range, of the amount of an analyte (e.g., viral particle, protein or nucleic acid) in a sample of interest.

[0108] FIG. IB illustrates the dynamic range 10 achievable with a sensor described herein that can detect analytes in a sample within a concentration range between less than 0.01 pg/mL (10 fg/mL) and 1 pg/mL or greater (at least 8 logs). In general, other commercially available assay systems (for example, typical manual ELISA, special manual ELISA, microfluidic-based ELISA assays, blotting-based technologies (e.g., Western blotting and dot blotting technologies) and automated bead-based technologies) can measure analytes in samples of interest but cannot measure analytes over the entire dynamic range achievable with a sensor disclosed herein. As a result, use of the sensor described herein may facilitate the measurement of concentrations of analyte over a concentration range that heretofore could only be achieved using a combination of prior art assay systems.

Sensor Configurations

[0109] It is contemplated that the sensor may comprise nanostructures in a variety of configurations (see, e.g., PCT/US2020/020204) For example, as shown in FIG. 2A, the sensor may comprise a first series of nanostructures 20d, for example, a series of nanostructures configured for digital quantification (FIG. 2A(i)); a second series of nanostructures 20a, for example, a series of nanostructures configured for analog quantification (FIG. 2A(ii)); two series of nanostructures 20d (FIG. 2A(iii)); two series of nanostructures 20a (FIG. 2A(iv)); two series of nanostructures one of 20d and one of 20a (FIG. 2A(v)); and three series of nanostructures, one of 20d and two of 20a (FIG. 2A(vi)). It is contemplated that the sensor may comprise other series of nanostructures in different configurations depending upon the analytes (e.g., viral particles, proteins or nucleic acids) to be detected and the dynamic range desired.

[0110] In one exemplary sensor for detecting the presence, or quantifying the amount, of an analyte in a sample of interest, the sensor comprises a first region and a second region. The first region comprises a first series of nanostructures capable of binding the analyte and producing a detectable signal indicative of a concentration of the analyte in the sample within a first concentration range. The second region comprises a second series of different nanostructures capable of binding the analyte and producing a detectable signal indicative of a concentration of the analyte in the sample within a second, different concentration range, wherein the sensor is capable of quantifying the amount of analyte in a sample across both the first concentration range and the second concentration range. The first concentration range can have a lower detectable value than that of the second concentration range and/or the second concentration range can have a higher detectable value than that of the first concentration range. It is contemplated that the first concentration range can overlap the second concentration range.

[OHl] It is understood that the sensors described herein are capable of detecting the concentration of analyte in the sample across a range (also referred to as dynamic range) spanning at least 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 orders of magnitude (or 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 logs). In certain embodiments, the sensor is capable of detecting the concentration of analyte in the sample across a concentration range spanning at least 5, 6, 7, 8 or 9 orders of magnitude (or 5, 6, 7, 8 or 9 logs). The sensor maybe configured to measure the concentration of a given analyte in the range from less than 1 pg/mL to greater than 100 ng/mL, from less than 0.1 pg/mL to greater than 1 pg/mL, or from less than 0.01 pg/mL to greater than 100 pg/mL, or from less than 1 fg/mL to greater than 1 mg/mL, where, for example, the sample does not need to be diluted prior to application to the sensor.

[0112] In one exemplary sensor, the first region comprises a first series of nanostructures capable of binding the analyte and producing a detectable signal indicative of a concentration of the analyte in the sample within a first concentration range, wherein individual nanostructures of the first series that bind the analyte are detected (e.g., optically detected) upon binding the analyte, whereupon the concentration of analyte in the sample, if within the first concentration range, is determined from a number of individual nanostructures in the first series that have bound molecules of analyte. The second region comprises a second series of different nanostructures capable of binding the analyte and producing a detectable signal indicative of a concentration of the analyte in the sample within a second, different concentration range, wherein the concentration of analyte in the sample, if within the second concentration range, is determined by analog detection of a substantially uniform change in a detectable property (for example, an optically detectable property, such as color) of the nanostructures in the second region as a function of the concentration of the analyte, wherein the sensor is capable of quantifying the amount of analyte in a sample across both the first concentration range and the second concentration range.

[0113] The first concentration range has a lower detectable value than that of the second concentration range and/or the second concentration range has a higher detectable value than that of the first concentration range. It is contemplated that the first concentration range can overlap the second concentration range.

[0114] In each of the foregoing sensors, the first region of the sensor optionally comprises one or more of: (i) center-to-center spacing of adjacent nanostructures of at least 1 pm; (ii) a minimum cross-sectional dimension or diameter of each nanostructure of at least 10 nm; (iii) a maximum cross-sectional dimension or diameter of each nanostructure of no more than 200 nm; or (iv) a height of each nanostructure in a range of 50 nm to 1,000 nm, e.g., 75 nm to 500 nm or 75 nm to 250 nm. The sensor optionally further comprises one or more of a (i) a fiducial marker or (ii) a nanostructure fabrication control feature.

[0115] It is contemplated that any of the sensors may comprises one or more of the following features. For example, it is contemplated that the sensor may further comprise a third region comprising a third series of further different nanostructures capable of binding the analyte and producing a detectable signal indicative of the concentration of the analyte in the sample within a third concentration range, wherein the sensor is capable of quantifying the amount of the analyte in the sample across the first, second and/or third concentration ranges.

[0116] Similarly, the nanostructures in any second series can comprise one of more of (i) an average height, (ii) an average volume, (iii) an average surface area, (iv) an average mass, and (v) an average number of analyte binding sites, that is greater than that of the nanostructures in the first series.

[0117] Furthermore, whenever the sensor comprises a third series, the nanostructures of the third series can comprise one of more of (i) an average height, (ii) an average volume, (iii) an average surface area, (iv) an average mass, and (v) an average number of analyte binding sites, that is greater than that of the nanostructures in any second series.

[0118] The nanostructures in the first series, and where applicable, the second and third series, are functionalized with a binding agent that binds the analyte, for example, binding agent, for example, a biological binding agent, that binds the analyte. The biological binding agent can be, for example, an antibody, an aptamer, a member of a ligand-receptor pair, an enzyme, or a nucleic acid. Under certain circumstances, it may be advantageous to use a binding agent in the first series that has a higher binding affinity for the analyte than the binding agent in a second, third or subsequent series.

[0119] The sensor may be designed to detect and/or quantify any analyte of interest in a sample. For example, the analyte may be a biological molecule, for example, a protein (e.g., a glycoprotein, lipoprotein, nucleoprotein or a peptide), a nucleic acid (e.g., a double stranded or single stranded DNA or RNA), or a particle (e.g., a viral particle). Furthermore, a nanostructure or series of nanostructures in a given sensor may be configured to bind, detect and/or quantify a plurality of different analytes simultaneously or sequentially. For example, the sensor can comprise a plurality of different binding agents for detecting a corresponding plurality of different analytes in the test sample.

[0120] The sensor can be configured to detect the binding of an analyte via a change in an optical property, electrical property, or mechanical property. For example, sensor can be configured to detect the binding of an analyte via a change in an optically detectable property (for example, color, light scattering, refraction, or resonance (for example, surface plasmon resonance, electric resonance, electromagnetic resonance, and magnetic resonance)) of at least one series of nanostructures.

[0121] It is contemplated that the sensors may be configured in a variety of different ways. For example, at least one of the first, second or third series of nanostructures can comprise an array of nanostructures. Alternatively, each of the first, second and third series of nanostructures can comprise an array of nanostructures. It is contemplated that sensor may comprise a single series of nanostructures or a plurality of series of nanostructures, for example, a plurality of series of nanostructures operative to detect analyte within different concentration ranges. When the sensor comprises a plurality of series of nanostructures, the different series of nanostructures may operate (i) in the same manner (for example, via digital detection where single nanostructures are detected or quantified, or via analog detection where a cumulative change in an optical property of the nanostructures within a given series is detected as a function of concentration) or (ii) in a different manner, for example by a combination of digital detection and analog detection. Furthermore, it is contemplated that the sensor may comprise a plurality of different series that operate by digital detection and/or analog detection. For example, the sensor may comprise a plurality of series that operate to detect an analyte by digital detection within the same concentration range and/or a plurality of series that operate to detect an analyte by analog detection over different concentration ranges.

[0122] For example, during digital detection (e.g., see FIG. 3A), in the first series of nanostructures, individual nanostructures that bind the analyte are detected upon binding either a single molecule of analyte or less than a predetermined number of molecules of the analyte, whereupon the concentration of analyte in the sample, if present in the first concentration range, is determined from a number of individual nanostructures in the first series that have bound molecules of the analyte. For example, the concentration of analyte in the sample is determined by digital counting of the number of individual nanostructures in the first series that have bound the analyte relative to either (i) a remaining number of individual nanostructures that have not bound analyte or (ii) a total number of nanostructures in the first series.

[0123] In this approach, a large number of nanostructures typically are densely patterned in a region of a sensor. When the number of the nanostructures is greater than the number of analytes to be detected, each nanostructure typically captures at most a single analyte, for example, based on mass transfer and Poisson distribution effects. Each nanostructure can have one of two states (for example, denoted as 1 or 0) depending upon whether analyte is bound or not. Accordingly, the number of nanostructures with state 1 after exposure to a sample with analytes can equal to the number of analytes. In certain embodiments, each individual nanostructure may have only a limited number of binding sites to capture one or a few (for example, less than 10) analytes, e.g., proteins or peptides. Each nanostructure has a corresponding signal scale from 1 to a few (<10), and thus counting the number of molecules can be equivalent to counting the discrete signals of each nanostructure. The different signal level of the series of nanostructures forms a nanomosaic pattern, which can be detected.

[0124] Similarly, the concentration of analyte, if within the second range, as depicted in FIG. 2A(iii), or the third range, can be determined by digital counting of the number of individual nanostructures in the second and/or third series that have bound the analyte relative to either (i) a remaining number of individual nanostructures in the appropriate series that have not bound analyte or (ii) a total number of nanostructures in the corresponding second and/or third series. In other words, the concentration of analyte in a sample across both the first concentration range, the second concentration range, and the optional third (or more) concentration range is determined from a number of individual nanostructures in each of the first series, the second series, and/or the optional third (or more) series that have bound molecules of the analyte.

[0125] Alternatively or in addition, the concentration of analyte, if within the second concentration range or the optional third concentration range, can be determined by analog detection (see, e.g., FIG. 3B) of a substantially uniform change in an optically detectable property of the nanostructures in the second region and/or the third region as a function of the concentration of the analyte. For example, the change in the optically detectable property can be a substantially uniform color change created by the second series and/or the optional third series as a function of the concentration of the analyte. In other words, the concentration of analyte in a sample across both the second concentration range and optional third (or more) concentration range(s) is determined by analog detection of a substantially uniform change in an optically detectable property of the nanostructures in each of the second region and/or the third region.

[0126] Each individual series (or region) of nanostructures may comprise binding sites for up to 10,000 molecules of the analyte of interest. Each region has a precalibrated continuous signal scale (analog scale) that relates to the number of proteins captured by the region. The analog scale for each region corresponds to a gradual change of physical signal for readout. Different scales may correspond to, for example, different colors from each region under a detector (for example, an optical detector). The region defines a nanomosaic that has a continuum of a property change (for example, color change) as a function of analyte concentration. In the case of optical detection, for example, the different scales may relate to one or more of (i) a light intensity of the region under a microscope which has a continuum of intensity change as a function of concentration or (ii) an electronic measurement, e.g., a current or voltage signal of each region, which has a continuum of current or voltage signal as a function of concentration.

[0127] It is contemplated that the nanostructures in a given series can be planar-faced and/or curve-faced nanostructures. The nanostructures can be disposed upon a planar support, a spherical surface, and/or a flexible substrate, where the nanostructures can be integral with the planar support, the spherical surface, and/or the flexible substrate. The nanostructures can be fabricated from a semi-conductive material (e.g., silicon) or a metal.

[0128] It is contemplated that the sensor may further comprise a fiducial marker, e.g., a fiducial marker that is optically detectable by light field microscopy and/or dark field microscopy. The fiducial marker can be used to calibrate the location of the sensors within the field of detection by the detection system. The sensor may also contain one or more nanostructure fabrication controls that demonstrate, e.g., that the nanostructures fabricated show a change in color as a function of the diameter of the nanostructures.

[0129] In another exemplary sensor, as depicted in FIG. 2A(i),the sensor comprises a first region comprising a first series of nanostructures capable of binding the analyte and producing a detectable signal indicative of a concentration of the analyte in the sample within a first concentration range, wherein individual nanostructures of the first series that bind the analyte are optically detected upon binding the analyte, whereupon the concentration of analyte in the sample, if within the first concentration range, is determined from a number of individual nanostructures in the first series that have bound molecules of analyte. The first region of the sensor optionally comprises one or more of: (i) center-to-center spacing of adjacent nanostructures of at least 1 pm; (ii) a minimum cross-sectional dimension or diameter of each nanostructure of at least 10 nm; (iii) a maximum cross-sectional dimension or diameter of each nanostructure of no more than 200 nm; or (iv) a height of each nanostructure in a range of 50 nm to 1000 nm. The sensor optionally further comprises a second region comprising one or more of a (i) a fiducial marker or (ii) a nanostructure fabrication control feature.

[0130] In another exemplary sensor, as depicted in FIG. 2A(ii),the sensor comprises a first region comprising a first series of nanostructures capable of binding the analyte and producing a detectable signal indicative of a concentration of the analyte in the sample within a first concentration range, wherein the concentration of analyte in the sample, if within the first concentration range, is determined by analog detection of a substantially uniform change in an optically detectable property of the nanostructures in the first region as a function of the concentration of the analyte. The first region further comprises one or more of: (i) center-to- center spacing of adjacent nanostructures of at least 1 pm; (ii) a minimum cross-sectional dimension or diameter of each nanostructure of at least 100 nm; (iii) a maximum cross- sectional dimension or diameter of each nanostructure of no more than 300 nm; or (iv) a height of each nanostructure in a range of 50 nm to 1,000 nm. The sensor optionally further comprises a second region comprising one or more of (i) a fiducial marker or (ii) a nanostructure fabrication control feature.

[0131] The sensing region of the disclosed sensors is the physical spot that interacts with biological analytes. In certain embodiments, the sensing region is divided into different parts, with each part targeting a specific concentration range. At very low concentrations, an array of single molecule nanostructures can be used. If analytes are captured by the single molecule sensor, the sensor produces a digital “yes” signal, and thus, the concentration of molecules can be related to the counts of digital sensors. At low-to-medium concentration ranges, a larger nanostructure that has a certain dynamic range to produce an analog signal is used to measure the concentration of analytes. The read-out signal can be resonance spectrum associated with the nanostructure, or scattering intensity, etc. To improve the detection accuracy, an array of these sensors may be used to achieve a statistical average.

[0132] As a non-limiting example, the sensing area of a sensor may be divided into multiple regions. By way of example, FIG. 2B is a schematic illustration of a sensor 30 with four sensor regions 32, 34, 36, 38. Each region comprises a series of nanostructures 20. In one embodiment, the series of nanostructures 20d of the ultra-low concentration sensor region 32 define a single molecule sensitivity. As a result, the concentration of analytes correlates with the number of single molecule nanostructures 20d that flip to produce a detectable signal, for example, a “yes” digital signal. The nanostructures 20a of the low, medium and high concentration sensor regions 34, 36, 38 have increasing size and, therefore, lower sensitivities but increasingly larger dynamic ranges. Each of the regions 32, 34 36, 38 are optimized for a specific dynamic range. Together, the results obtained from each region can be aggregated to provide a dynamic range that results from an aggregation of the dynamic ranges achievable by regions 32, 34, 36, 38.

[0133] FIG. 3A depicts a schematic representation of an exemplary sensor and the quantification of an analyte of interested achieved using such a sensor. This sensor 30 includes a first region 50 with a series of nanostructures 20d configured for digital quantification and a second region 60 with a series of nanostructures 20a configured for analog quantification where shifts in color indicate different concentrations. In this example, digital quantification 70 is performed for analyte concentrations ranging from pg/mL to ng/mL, and analog quantification 80 is performed for analyte concentration ranging from ng/mL to pg/mL. When concentrations of analyte are in the range of pg/mL to ng/mL, the analyte concentration can be measured based on the number of nanostructures in the series in region 50 that change state (e.g., flip from one state to another). However, as the concentrations of analyte reach the upper limits of the detectable range, the sensor in region 50 becomes saturated and the sensor cannot quantify higher concentrations of analyte. Saturation of the first series may occur when at least 60%, 70%, 80%, 90%, 95%, or greater of the binding sites have bound an analyte. As a result, this sensor 30 also includes a plurality of series of nanostructures that change their optical properties (for example, detected as a color change) when the concentration of analyte in the sample falls within the range of analyte concentrations that is detectable by a given series of nanostructures. In this embodiment, the series of nanostructures in region 60 are calibrated to change their optical properties (for example, color) in adjacent or overlapping concentration ranges.

[0134] In FIG. 3B, sensor 40 includes a series of nanostructures for digital detection/quantification 70 and a series of nanostructures for analog detection/quantification 80. In particular, the series of nanostructures for digital detection 70 comprises nanostructures 20d in the form of an array. As the concentration of analyte increases from 1.2 pg/mL to 10 ng/mL, the number of nanostructures that have flipped from one state another increases, as indicated by the ration under each panel 90. At analyte concentrations at or above 10 ng/mL, the series of nanostructures saturates as all or substantially all of the nanostructures (for example, at least 60%, 70%, 80%, 90%, 95% of the binding sites have bound analytes) have flipped from one state to the other. The right-hand side box illustrates the change in optical properties (e.g., colorimetric change) in a series of nanostructures 20a configured for analog detection 80. For example, as the concentration of analyte increases up to 10 ng/mL, the change in optical property (for example, color hue) of the series of nanostructures does not shift. However, as the concentration of analyte is greater than 10 ng/mL, a change in an optical property of the series of nanostructures becomes detectable, for example, as a change in color as a function of analyte concentration. Greater dynamic ranges can be achieved by including in a sensor additional series of nanostructures (for example, digital arrays and/or analog arrays) calibrated to detect and quantify analyte in other concentration ranges.

[0135] FIG. 3C illustrates digital quantification performed by a sensor 100 described herein. As illustrated, the sensor is able to detect analyte molecules (e.g., a protein) at a concentration 50 fg/mL, with 96 out of 2046 digital nanostructures (20d) being flipped from one optical property to another that is detectable by a detector. In this particular example, the sensor 100 becomes saturated at molecule concentrations at about 50 pg/mL, when all or substantially all of the nanostructures are flipped from one optical state to the other.

[0136] FIG. 4 is a graph depicting data compiled from measurements obtained by the exemplary sensor 40 of FIG. 3B. In the analyte concentration range of 1 pg/mL to 1 ng/mL, the digital quantification mode 70 provides high sensitivity and a dynamic range of 3 logs. In the analyte concentration range of 1 ng/mL to 1 pg/mL, the analog colorimetric measurement 80 extends the detectable concentration range by an additional 3 logs. The transition between the digital quantification measurements and analog quantification measurements to form a continuous curve spanning the entire dynamic range can be automated using an algorithm of the type described herein. In this example, a 6 log dynamic range is achieved using a combination of a series of nanostructures configured for digital quantification with a series of nanostructures configured for analog quantification. It has been discovered that the sensors described herein can achieve large dynamic ranges (for example, 6 logs or more) with high sensitivity (for example, 50 fg/mL) using small volumes of sample (for example, less than 100 pL, 50 pL, 25 pL, 10 pL or 5 pL).

[0137] The nanostructure may have any suitable shape and/or size. In some cases, for example, the nanostructure may be a nanoneedle, a nanowire, a nanorod, a nanocone, or the like. Other shapes are also possible, e.g., nanoribbons, nanofilaments, nanotubes, or the like. In certain embodiments, the nanostructures are vertically aligned, although other angles or alignments are also possible. Nanostructures such as nanoneedles, nanodots, nanodisks, nanopillars, etc. have single molecule level sensitivity due to their ability to confine electromagnetic energy through coupling to surface polaritons.

[0138] The physical form of a sensor may be an array or matrix of nanostructures, for example, nanoneedles, nanowires, nanopillars, nanodots, etc., fabricated on a surface by bottom-up and/or top-down methods. The surface can be a flat surface, such as a top surface of a wafer. Alternatively, the surface may also be curved or flexible, or part of a three dimensional structure such as a fiber or a wire or the like.

[0139] The functional form of the sensor can comprise nano-optical structures, nanomechanical structures or nano-electrical structures. Accordingly, the read-out signal includes but is not limited to optical signals, electrical signals and mechanical signals. Accordingly, the concentration of the analytes may be determined by changes in optical, electrical or nanomechanical properties of the nanostructures. The optical features include, for example, surface plasmon resonance, nanophotonic resonance, electric resonance, magnetic resonance, scattering, absorption, fluorescence, color changes, or the like. The electrical features include, e.g., resistance, capacitance, current, voltage, or the like. The nanomechanical features include, for example, vibrational resonance, vibration magnitude, mechanical mass, or the like.

[0140] The foregoing structures may also be used to detect high concentration of analytes by observing changes in their optical properties, for example, surface plasmon resonances, scattering intensities, or absorptions. Sensitivity and detection ranges of these structures are closely related to the sizes of the structures. Planar fabrication technology enables scalable and flexible integration of differently sized and shaped nanostructures in one device. Different nanostructures may be used to achieve high sensitivity and a high dynamic range for the determination of molecules and analytes in a biological sample.

[0141] In certain embodiments, the surface properties of different structures can be designed such that the nanostructures in a first series of nanostructures may have higher binding affinities for binding the analyte than that of the second and/or third series of nanostructures. This can be achieved using binding agents having different binding affinities to a given analyte. As a result, at low concentrations, analytes are preferentially captured and detected by the single molecule nanostructures. As the concentration increases, the nanostructures of the first series saturate and signals from other series of nanostructures can be used to extend the dynamic range.

[0142] FIG. 5 is a pictorial representation of an exemplary sensor (for example, a nanomosaic chip) 150 which includes multiple series of nanostructures. In the column on the left hand side of sensor 150, the separate regions represent fabrication control structures 155 which demonstrate that the nanostructures change color as the diameter of the nanostructures is increased. The middle region 160 represents multiple separate arrays (z.e., 16 arrays) each defining a corresponding series of nanostructures (collectively comprising 25,600 nanostructures that each define single molecule nanostructures) configured for digital quantification for measuring ultra-low concentration levels of analytes. The region on the right hand side comprises three series of nanostructures (e.g., a second, third, and fourth series of nanostructures) depicted as regions 165, 170, 175, for analog quantification. Each of the regions 165, 170, 175 are calibrated to measure analyte concentrations within three separate adjacent or overlapping concentration ranges. In certain embodiments, the three regions may each comprise 1,000 nanostructures. [0143] In an alternative embodiment, as shown pictorially in FIG. 6, another exemplary sensor (e.g., a nanomosaic chip) 150 comprises numerous series (regions) of nanostructures. In the center, a fiducial marker 200 is located to assist in aligning the sensor with an optical detection system. The fiducial marker can be any desired design. For example, as shown in FIG. 6, the fiducial marker 200 comprises a diamond pattern and three triangular patterns arranged in a way that does not have rotational symmetry to provide location and rotational orientation information. As a result, the fiducial marker can be used to (i) locate the sensor position, and (ii) align the horizontal and vertical planes of the nanostructures. Fabrication control structures 155 are disposed around the fiducial. Arrays of digital single molecule nanostructures 20d are disposed on the left and the right regions of the sensor, and arrays of analog molecule nanostructures 20a are disposed in the center row surrounding the fiducial and fabrication control structures. The fabrication control shown in FIG. 6 comprises 8 blocks of nanostructures (e.g., nanoneedles) whose diameters range from 80 nm to 150 nm. The color of the nanostructures (nanoneedles) under dark field imaging changes as the diameter increases.

[0144] In certain embodiments, the nanostructure has a length, determined from an end or a point of attachment with a substrate, of less than about 500 nm, 450 nm, 350 nm, 300 nm, 250 nm, 200 nm, 150 nm, 100 nm, 50 nm, 30 nm, 20 nm, 10 nm, 5 nm, 3 nm, or 2 nm. In certain embodiments, the length of the nanostructure may be at least about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, or 500 nm.

[0145] The nanostructure may have any suitable cross-sectional shape, for example, square, circular, triangular, ellipsoidal, polygonal, star, irregular shape, etc. The nanostructure may maintain the same cross-sectional shape throughout its length, or may have different cross- sectional shapes in different portions of the nanostructure. In addition, the nanostructures may have any suitable cross-sectional diameter. The cross-sectional diameter may be constant (e.g., as in a nanoneedle or a nanorod), or varying (e.g., as in a nanocone). The average cross-sectional diameter may be, for example, less than about 1,000 nm, 750 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 40 nm, 30 nm, 20 nm, or 10 nm. In certain embodiments, the cross-sectional diameter may be at least about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 75 nm, 100 nm, 125 nm 150 nm, 175 nm, 200 nm, 300 nm, 400 nm, 500 nm, 750 nm, or 1,000 nm. Combinations are also possible in various embodiments. For example, the average diameter of the nanostructures may be between 50 nm and 300 nm, 75 nm and 250 nm, or 100 nm to 200 nm.

Fabrication Considerations

[0146] The nanostructure may be formed out of any suitable material, and may be the same or different from a substrate upon which it is disposed. In certain embodiments, the nanostructures (e.g., nanoneedles) can be formed from silicon and/or other suitable semi- conductive materials (e.g., germanium). Additional, non-limiting examples of materials include metals (e.g., nickel or copper), silica, glass, or the like.

[0147] In certain embodiments, the nanostructure (e.g., nanoneedle) may be disposed on a substrate can be formed from a unitary material. In other words, the nanostructure (e.g., nanoneedle) and the underlying substrate (e.g., planar substrate) maybe unitary and may be formed from the same material. In other approaches, the nanostructure (e.g., nanoneedle) maybe bonded or adhered to an underlying substrate (e.g., planar substrate), which may be formed from the same material or from different materials.

[0148] It is contemplated that the sensors described herein can be fabricated by a number of different approaches, for example, using semiconductor manufacturing approaches. As discussed above and in more detail below, any suitable method can be used to form the series of nanostructures useful in creating the sensors described herein. Examples include, but are not limited to, lithographic techniques such as e-beam lithography, photolithography, X-ray lithography, extreme ultraviolet lithography, ion projection lithography, etc. Alternatively or in addition, the nanostructure may be formed from one or more materials that are susceptible to etching with a suitable etchant.

[0149] For example, in certain embodiments, the nanostructures may be formed from one or more materials that are susceptible to etching with a suitable etchant. For instance, the nanostructures may comprise materials such as silica or glass, which can be etched using HF (hydrofluoric acid) or BOE (buffered oxide etch). As another example, the nanostructures may comprise a metal such as copper, iron, nickel, and/or steel, which can be etched using acids such as HC1 (hydrochloric acid), HNO3 (nitric acid), sulfuric acid (H2SO4), and/or other etching compounds such as such as ferric chloride (FeCh) or copper sulfate (CuSO4). As yet another example, the nanostructures may comprise silicon or other semiconductor materials, which can be etched using etchants such as EDP (a solution of ethylene diamine and pyrocatechol), KOH (potassium hydroxide), and/or TMAH (tetramethylammonium hydroxide). The nanostructures may also comprise, in some cases, a plastic or a polymer, e.g., polymethylmethacrylate, polystyrene, polyperfluorobutenylvinylether, etc., which can be etched using KOH (potassium hydroxide), and/or other acids such as those described herein. In some embodiments, the nanostructures may comprise a photoresist, where the photoresist may be patterned via lithography (such as electron beam lithography or photolithography), nanoimprinting or nanostamping, as discussed below.

Nanostructure Fabrication

[0150] It is contemplated that the sensors described herein can be fabricated by conventional semiconductor manufacturing technologies, for example, CMOS technologies, that have led to high manufacturing capacity, at high throughputs and yields in a cost- effective manner. Using such approaches it is possible to make sensors containing one of more series of nanostructures, e.g., nanoneedles, nanodots, nanodisks, nanowires, and nanopillars disposed upon or integral with a substrate. Exemplary nanostructures are depicted schematically in FIGs. 7 and 8. As non-limiting examples, FIG. 7 illustrates several nanostructures 20 that can be directly formed on a substrate with current nanofabrication technologies, including electron beam lithography, photolithography, nanoimprinting, etc. For example, the nanostructure 20 can be a nanopillar (a uniform nanoneedle), a nanodisk, a cone-shaped nanoneedle, or a nanodot. In addition, FIG. 8 depicts nanostructures 20 (e.g., nanoneedles) fabricated from two or more materials, e.g., first and second materials 300 and 305, respectively. The compositions of each material can be used to control the binding capacity of the nanostructures for binding analyte or to achieve specific optical, electrical, or magnetic properties, as discussed below.

[0151] The fabrication of nanostructures may be performed either at wafer scale or at chip scale with equivalent scaling capability. In this type of approach, a mask is first made for the designed nanostructure. In certain embodiments, an inverse to the design structure is used as the pattern on the mask. For example, a photoresist is coated onto the wafer or on the chip, for example, using a spin-coating or dip-coating process. The photoresist may then be exposed to electromagnetic radiation through the mask to the photoresist. Thereafter, the exposed photoresist is developed. In certain embodiments, the pattern on the photoresist can also be directly written by means of a laser beam or an electron beam. The pattern on the photoresist can then be transferred to the substrate by physical vapor deposition, including thermal evaporation, electron beam evaporation, sputter or chemical deposition, or atomic layer deposition of a desired material. [0152] In certain embodiments, the pattern on the photoresist can be transferred to the substrate using top down etching process, including wet etching, dry etching such as reactive ion etching, sputter etching, and/or vapor phase etching. The patterning, deposition, etching, and functionalization processes can be repeated for multiple cycles. In certain embodiments, arrays of nanoneedles, nanopillars, nanodots and/or nanowires can be fabricated using semiconductor manufacturing processes. In other embodiments, arrays of nanoneedles, nanopillars, nanodots and/or nanowires can be fabricated using mold-stamping process.

[0153] An exemplary fabrication approach is depicted in the cross-sectional views shown in FIGs. 9A - 9D. Referring to FIG. 9A, more specifically, a layer of ebeam resist or photoresist 310 is coated onto a semiconductor substrate 320, such as a silicon substrate. Referring to FIG. 9B, the resist layer is then patterned by electron beam exposure or electromagnetic radiation exposure to form resist layer features 325, for example, by using an Elionix or Raith electron beam lithography system. Referring to FIG. 9C, the resist is developed in resist developer, to remove portions thereof and leaving only the resist features 325. Referring to FIG. 9D, an etching process is then performed with the patterned resist serving as a mask. The etching process may be, e.g., a wet or a dry etch. A suitable wet etch can be, for example, a solution of ethylenediamine pyrocatechol (EDP), potassium hydroxide (KOH), or tetramethylammonium hydroxide (TMAH). As a result, silicon nanoneedles 330 are created with resist 325 disposed upon the top surface of the nanoneedles. The height of the nanoneedles can range from 2 nm to 1,000 nm. The diameter of the nanoneedles can range from 10 nm to 1,000 nm. Resist features 325 may be removed using a conventional wet etching buffer (not shown).

[0154] The surface of the etched structure can be chemically activated using chemical vapor deposition or atomic layer deposition or a hybrid of both. This activation process can also be performed in a wet solution. The chemically activated structure is then ready to bind a biological material, a binding agent described herein via, for example, chemisorption (e.g., covalent binding) or physisorption.

[0155] A suitable silicon substrate can be, for example, a round 12” silicon wafer. In order to comply with Society of Biomolecular Screening (SBS) recommended microplate specifications, the round wafer is diced into a rectangular shape. The dicing step can be performed at the end of the fabrication process as described above. Alternatively, dicing into half of the depth of the wafer can be performed in the beginning of the fabrication process; then, after completion of all fabrication steps (including spin coating, patterning, deposition and etching), the wafers can be easily cleaved into the SBS format.

[0156] In some embodiments, rather than performing an etching process as described above with reference to FIG. 9D with the resist 325 being removed after the etching process, the resist features 325 defined by lithography are themselves the nanoneedles, without the need for a subsequent etch step. A suitable resist can be a positive or negative resist, including positive or negative ultraviolet photoresist, deep ultraviolet photoresist, extreme ultraviolet photoresist, electron beam photoresist, ion beam photoresist, or X-ray photoresist. Specific suitable resists include polymethylmethacrylate (PMMA), the Shipley or SPR series available from DuPont de Nemours, Inc., SU-8 or KMPR resist from Kayaku Advanced Materials, Inc., ma-N from Microresist, etc.

[0157] Another fabrication approach is depicted in the cross-sectional views shown in FIGs. 10A - 10G. Referring to FIG. 10A, a silicon dioxide layer 335 is formed on a top surface of a silicon substrate 320 using chemical vapor deposition, atomic layer deposition or a combination of both. The thickness of the layer can range from 2 nm to 100 nm. A resist layer 310 comprising, e.g., polymethyl methacrylate, is spun coated onto the silicon dioxide layer 335. Referring to FIGs. 10B and 10C, the resist layer 310 is patterned by an electron beam or electromagnetic radiation, and then developed in resist developer to form resist features 325. Referring to FIG. 10D, an aluminum layer 340 is deposited over the patterned resist layer features 325 by, e.g., thermal evaporation (or electron evaporation) with, e.g., a Sharon thermal evaporator or Denton e-beam evaporator. The aluminum layer 340 is preferably 20 nm to 100 nm thick. Referring to FIG. 10E, a lift-off process is performed to remove the resist layer features 325, leaving behind an aluminum mask over the silicon dioxide layer 335. Referring to FIG. 10F, an etching process, such as a reactive ion etch with an STS ICP RIE system or an Oxford plasma RIE system is performed to etch silicon oxide nanoneedles 335. The RIE etching can further proceed into the silicon layer 320, resulting in a two layer SiO2-Si nanostructures. Referring to FIG. 10G, the aluminum mask 340 may be etched off the tops of silicon nanoneedles 342 in an aluminum etchant buffer, e.g., a mixtures of 1-5 % HNO3, H3PO4 and CH3COOH.

[0158] Yet another fabrication approach is depicted in the cross-sectional views shown in FIGs. 11A - 11F. Referring to FIG. 11A, a silicon dioxide layer 335 is grown on a top surface of a silicon substrate 320. A resist layer 310 is spun coated onto the silicon dioxide layer 335. Referring to FIGs. 11B and 11C, the resist layer 310 is patterned by electron beam or electromagnetic radiation, and then developed in resist developer to form resist features 325. Referring to FIG. 11D, a metal layer, such as an aluminum layer 340, is deposited over the patterned resist layer 310 by, for example, a thermal evaporation (or electron evaporation) process. Referring to FIG. HE, a lift-off process is then performed to remove the resist layer 310, leaving behind aluminum nanoneedles disposed upon the oxide layer on the substrate. Referring to FIG. HF, a coating layer 345 can be spun coated to modify the surface properties of the substrate. The coating layer can be a hydrophobic material, such as TEFLON, or a layer of polyethylene glycol molecules. The thickness of the coating layer is smaller than the height of the aluminum nanoneedles.

[0159] Another fabrication approach is depicted in the cross-sectional views shown in FIGs. 12A - 12F. Referring to FIG. 12A, a resist layer 310 is spun coated on an oxide substrate 350. The oxide layer can be a thermally grown silicon oxide, or formed by chemical vapor deposition. In some embodiments, the substrate 350 may be a glass slide. Referring to FIGs. 12B and 12C, electromagnetic radiation can be used to pattern features in the resist layer 310, which is then developed in resist developer to form resist features 325. Referring to FIG. 12D, a silicon layer 355 is deposited over the patterned resist layer 310 by, for example, using chemical vapor deposition. Referring to FIG. 12E, a lift-off process is performed to remove the patterned resist layer 310, which results in a silicon nanodot 360 structure on the oxide substrate. Referring to FIG. 12F, silicon nanoneedle structures 365 may be epitaxially grown using the silicon nanodots 360 as seeds, by, e.g., VLS (vaporliquid-solid) method.

[0160] Another fabrication approach is depicted in the cross-sectional views shown in FIGs. 13A - 13D, in which a photoresist layer may be patterned by using a mold. Referring to FIG. 13A, a mold 370 is made from e.g., Si or quartz. The mold can be made by high resolution patterning technology, such as ebeam lithography. The mold has feature sizes similar to that of the target nanostructures to be replicated. Referring to FIG. 13B, a resist layer 310 is spun coated on silicon substrate 320. Specific suitable resists include PMMA, the Shipley or SPR series available from DuPont de Nemours, Inc., SU-8 or KMPR resist from Kayaku Advanced Materials, Inc., ma-N from Microresist, etc. Referring to FIG. 13C, the features in mold 370 are then stamped into the resist by nanoimprinting or nanostamping, and then crosslinked by e.g., UV or heat. Referring to FIG. 13D, the imprinted photoresist can be used as the mask for the subsequent etching process to obtain the silicon nanostructures. In some embodiments, the imprinted photoresist features themselves can be used as the nanostructures, without the performance of the subsequent etching process of

FIG. 13D

[0161] With reference to FIGs. 14A and 14B, by replicating the fabrication steps described hereinabove it is possible to produce a plurality of sensors 375 fabricated on a wafer 320, to create, for example, a 10 x 10 array of sensors disposed on each wafer 320. As shown in FIG. 14B, each sensor comprises an array of nanostructures, e.g., nanoneedles 330 disposed upon a silicon substrate.

[0162] It should be noted that the nanostructures depicted in FIGs. 10 - 14 have at least one dimension in the range of 1-999 nm, 1-750 nm, 1-500 nm, 1-400 nm, 1-300 nm, 1-200 nm, 1-100 nm, 10-999 nm, 10-750 nm, 10-500 nm, 10-400 nm, 10-300 nm, 10-200 nm, 10- 100 nm, 20-999 nm, 20-750 nm, 20-500 nm, 20-400 nm, 20-300 nm, 20-200 nm, 20-100 nm, 30-999 nm, 30-750 nm, 30-500 nm, 30-400 nm, 30-300 nm, 30-200 nm, 30-100 nm, 40-999 nm, 40-750 nm, 40-500 nm, 40-400 nm, 40-300 nm, 40-200 nm, 40-100 nm, 50-999 nm, 50- 750 nm, 50-500 nm, 50-400 nm, 50-300 nm, 50-200 nm, or 50-100 nm. The pitch, i.e., center-to-center distance, between nanostructures, for example in FIG. 14B, is typically 1- 100 pm, for example, at least 1.5 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80 pm, or 90 pm. Other dimensions may be used for the pitches of the structures. The array of nanostructures in FIG. 14B, in its entirety, can also be arranged in an array format, as shown in FIG. 14A. For example, the pitch in between two arrays of nanostructures, shown in FIG. 14A may range from less than 100 pm to larger than a few centimeters. Furthermore, it is contemplated that the pitch and size of the nanostructures may be different in different parts of the chip, or within each series of nanostructures. Combinations of any of these are also possible in various embodiments.

[0163] Furthermore, the distance or pitch between nanostructures in a periodic structure may be controlled, for example, such that the nanostructures form a meta-surface. For example, the pitch may be set to be less than the wavelength of the incident light. For instance, the pitch may be less than 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 50 nm, 25 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm 3 nm or 2 nm, and/or greater than 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm 10 nm, 25 nm, 50 nm, 100 nm 200 nm, 300 nm, 400 nm, 500 nm, 600 nm or 700 nm. For example, under certain circumstances, the pitch may be between 400 nm and 500 nm. The nanostructures may have any of the dimensions provided herein. Under certain circumstances, the average cross- sectional diameter or minimum or maximum cross-sectional dimension of the nanostructure is less than the wavelength of the incident light. Under certain circumstances, the individual nanostructures are configured to be optically resolvable, where, for example, the pitch may be less than 100 pm, less than 10 pm, less than 5 pm, and/or greater than 1 pm, or greater than 5 pm.

[0164] Table 1 describes exemplary parameters of the nanostructures described herein for optical read-outs.

TABLE 1

[0165] Table 2 describes exemplary parameters of the nanostructures described herein for a mechanical read-out.

TABLE 2

[0166] Table 3 describes exemplary parameters of the nanostructures described herein for an electrical read-out.

TABLE 3

Nanostructure Functionalization

[0167] The nanostructures in the first series and, where applicable, the second and third series, are functionalized with a binding agent that binds the analyte, for example, binding agent, for example, a biological binding agent, that binds the analyte. The biological binding agent can be, for example, an antibody, an aptamer, a member of a ligand-receptor pair, an enzyme, or a nucleic acid. Under certain circumstances, for example, when the first series is used to measure very low concentrations of analyte, it may be advantageous to use a binding agent in the first series that has a higher binding affinity for the analyte than the binding agent in a second, third or subsequent series.

[0168] The number of binding agents applied to a given nanostructure may vary depending upon the desired assay, for example, the required dynamic range, number of analytes to be detected, etc. For example, under certain circumstances, a nanostructure may be functionalized with 1, 5, 10, 20, 25, 50, 75, 100 or more binding agents. These values may range from 1-1,000, 1-500, 1-250, 1-100, 1-50, 1-25, 1-10 or 1-5 binding agents per nanostructures.

[0169] The sensor may be designed to detect and/or quantify any analyte of interest in a sample. Furthermore, a nanostructure or series of nanostructures in a given sensor may be configured to bind, detect and/or quantify plurality of different analytes simultaneously or sequentially. For example, the sensor can comprise a plurality of different binding agents for detecting a corresponding plurality of different analytes in the test sample.

[0170] Analytes may be detected and/or quantified in a variety of biological samples. The sample can be in any form that allows for measurement of the analyte. In other words, the sample must be permit analyte extraction or processing to permit detection of the analyte. Accordingly, the sample can be fresh, preserved through suitable cryogenic techniques, or preserved through non-cryogenic techniques. In certain embodiments, the sample is a fluid sample removed from a bioreactor, e.g., viral particle containing growth media. Alternatively, the sample may be a body fluid sample, e.g., blood, serum, plasma, urine, cerebrospinal fluid, or interstitial fluid sample.

[0171] The nanostructures can be functionalized using standard chemistries known in the art. As an initial matter, the surfaces of the nanostructures may be activated for binding a binding agent using standard chemistries, including standard linker chemistries.

[0172] The binding agent may contain or be engineered to contain a functional group capable of reacting with the surface of the nanostructure (e.g., via silanol groups present on or at the surface of the nanostructure), either directly or via a chemical linker.

[0173] In one approach, the surface silanol groups of the nanostructure may be activated with one or more activating agents, such as an alkoxy silane, a chlorosilane, or an alternative silane modality, having a reactive group (e.g., a primary amine). Exemplary alkoxy silanes having a reactive group may include, for example, an aminosilane (e.g., (3-aminopropyl)- trimethoxysilane (APTMS), (3 -aminopropyl)-tri ethoxy silane (APTES), (3-aminopropyl)- diethoxy-methylsilane (APDEMS), 3-(2-aminoethyaminopropyl)trimethoxysilane (AEAPTM)), a glycidoxy silane (e.g., (3 -glycidoxypropyl)-dimethyl-ethoxy silane (GPMES)), or a mercaptosilane (e.g., (3-mercaptopropyl)-trimethoxysilane (MPTMS) or (3- mercaptopropyl)-methyl-dimethoxysilane (MPDMS). Exemplary chlorosilanes having a reactive group include 3-(trichlorosilyl)propyl methacrylate (TPM) and 10- i socy anatodecy Itri chi orosil ane .

[0174] Thereafter, a functional group on the binding agent, for example, a primary amine on the side chain on a lysine residue can be attached to the reactive group added to the surface of the nanostructure using a variety of cross-linking agents. Exemplary cross-linking agents can include, for example, homobifunctional cross-linking agents (e.g., glutaraldehyde, bismaleimidohexane, bis(2-[Succinimidooxycarbonyloxy]ethyl) sulfone (BSOCOES), [bis(sulfosuccinimidyl)suberate] (BS3), (l,4-di-(3’-[2pyridyldithio]-propionamido)butane) (DPDPB), disuccinimidyl suberate (DSS), disuccinimidyl tartrate (DST), sulfodisuccinimidyl tartrate (Sulfo DST), dithiobis(succinimidyl propionate (DSP), 3,3’- dithiobis(sulfosuccinimidyl propionate (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), bis(P-[4-azidosalicylamido]-ethyl)disulfide iodinatable (BASED), homobifunctional NHS crosslinking reagents ( .g., bis N-succinimidyl-[pentaethylene glycol] ester (Bis(NHS)PEO-5), and homobifuctional isothiocyanate derivatives of PEG or dextran polymers) and heterobifunctional cross-linking agents (e.g., succinimidyl 4-(N maleimidom ethyl) cyclohexane- 1 -carboxylate (SMCC), succinimidyl-4-(N maleimidomethyl)-cyclohexane-l-carboxy(6-amidocaproate) (LC-SMCC), N maleimidobenzoyl-N-hydroxy succinimide ester (MBS), succinimide 4-(p-maleimidophenyl) butyrate (SMPB), N-hydroxy-succinimide and N-ethyl-‘(dimethylaminopropyl)carbodiimide (NHSZEDC), (N-E-maleimido-caproic acid)hydrazide (sulfoEMCS), N-succinimidyl-S- acetylthioacetate (SATA), monofluoro cyclooctyne (MFCO), bicyclo[6.1.0]nonyne (BCN), N-succinimidyl-S-acetylthiopropionate (SATP), maleimido and dibenzocyclooctyne ester (a DBCO ester), and l-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)).

[0175] By way of example, the nanostructures described herein, may be activated via an alkoxy silane (e.g., APTMS) to modify the free hydroxyl groups of the surface silanol groups to create a reactive group (for example, primary amines). The reactive group (for example, primary amines) created on the nanostructure then may be reacted with a cross-linking agent, for example, glutaraldehyde, that forms a covalent linkage with the free amine group present, for example, in the side chain of a lysine amino acid in a protein, for example, an antibody of interest.

[0176] It is contemplated that other activation and conjugation chemistries known in the art can be used to covalently couple one or more binding agents to the surface of the nanostructures described herein.

[0177] It is contemplated that a given nanostructure or series of nanostructures may be functionalized with a binding agent that binds an analyte of interest. Exemplary binding agents include antibodies (for example, that bind antigens), aptamers, antigens (for example, that bind target antibodies), nucleic acid single-strand polymers (for example, that bind nucleic acid molecules to form, for example, DNA-DNA, RNA-RNA, or DNA-RNA double strands), and synthetic molecules that bind with target analytes. Other binding partners can be used including avidin and biotin or streptavidin and biotin pairs.

[0178] In various embodiments, a binding agent is an antibody. In certain embodiments, an antibody binds to its target with a KD of about 300 pM, 250 pM, 200 pM, 190 pM, 180 pM, 170 pM, 160 pM, 150 pM, 140 pM, 130 pM, 120 pM, 110 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM, or lower. An antibody may have a human IgGl, IgG2, IgG3, IgG4, or IgE isotype.

[0179] Methods for producing antibodies as well as other protein-based binding agents are known in the art. For example, the protein-based binding agents may be purified from natural sources or produced using recombinant DNA technologies. For example, DNA molecules encoding, for example, a protein binding agent can be synthesized chemically or by recombinant DNA methodologies. The resulting nucleic acids encoding desired proteinbased binding agents can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques. The transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the proteins of interest. Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is to be expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Trp or Tac, and a prokaryotic signal sequence. The expressed secreted protein accumulates in refractile or inclusion bodies, and can be harvested after disruption of the cells by French press or sonication. The refractile bodies then are solubilized, and the proteins refolded and cleaved by methods known in the art. If the engineered gene is to be expressed in eukaryotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, a poly A sequence, and a stop codon. The gene construct can be introduced into eukaryotic host cells using conventional techniques. Thereafter, the host cells are cultured under conditions that permit expression of the protein based binding agent. Following expression, the polypeptide can be harvested and purified or isolated using techniques known in the art including, for example, affinity tags such as glutathione-S-transferase (GST) or histidine tags.

[0180] Exemplary nucleic acid based binding agents include modified or modified nucleic acids (including, for example, DNA and RNA molecules, which can be double or single stranded), aptamers and spiegelmers. Aptamers are nucleic acid-based sequences that have strong binding activity for a specific target molecule. Spiegelmers are similar to aptamers with regard to binding affinities and functionality but have a structure that prevents enzymatic degradation, which is achieved by using nuclease resistant L-oligonucleotides rather than naturally occurring, nuclease sensitive D-oligonucleotides.

[0181] The nucleic acid-based binding agents, e.g., short (e.g., PCR primer sequences) or longer oligonucleotide sequences, can be prepared using routine synthetic procedures and/or can be purchased commercially from a number of sources.

[0182] Aptamers are specific nucleic acid sequences that bind to target molecules with high affinity and specificity and are identified by a method commonly known as Selective Evolution of Ligands by Evolution (SELEX), as described, for example, in U.S. Patent Nos. 5,475,096 and 5,270,163. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. The SELEX process is based on the observation that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets.

[0183] The SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule. Thus, this method allows for the screening of large random pools of nucleic acid molecules for a particular functionality, such as binding to a given target molecule.

[0184] The SELEX method also encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability and protease resistance. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified nucleic acid ligands containing modified nucleotides are described in U.S. Patent Nos. 5,660,985 and 5,580,737, which include highly specific nucleic acid ligands containing one or more nucleotides modified at the 2’ position with, for example, a 2’ -amino, 2’ -fluoro, and/or 2’-O-methyl moiety.

[0185] Instead of using aptamers, which may require additional modifications to become more resistant to nuclease activity, it is contemplated that spiegelmers, mirror image aptamers composed of L-ribose or L-2’ deoxyribose units (see, U.S. Patent Nos. 8,841,431, 8,691,784, 8367,629, 8,193,159 and 8,314,223) can be used in the practice of the invention. The chiral inversion in spiegelmers results in an improved plasma stability compared with natural D- oligonucleotide aptamers. L-nucleic acids are enantiomers of naturally occurring D-nucleic acids that are not very stable in aqueous solutions and in biological samples due to the widespread presence of nucleases. Naturally occurring nucleases, particularly nucleases from animal cells are not capable of degrading L-nucleic acids.

[0186] Using in vitro selection, an oligonucleotide that binds to the synthetic enantiomer of a target molecule, e.g., a D-peptide, can be selected. The resulting aptamer is then resynthesized in the L-configuration to create a spiegelmer (from the German “spiegel” for mirror) that binds the physiological target with the same affinity and specificity as the original aptamer to the mirror-image target. This approach has been used to synthesize spiegelmers that bind, for example, hepcidin (see, U.S. Patent No. 8,841,431), MCP-1 (see, U.S. Patent Nos. 8,691,784, 8367,629 and 8,193,159) and SDF-1 (see, U.S. Patent No. 8,314,223).

Cartridge

[0187] The sensors described herein, once fabricated, can be included in, or otherwise assembled into, a cartridge for use within a detection system. The cartridge may be used for detecting the presence, or quantifying the amount, of an analyte in a sample of interest. The cartridge comprises a housing defining at least one well comprising any one or more of the foregoing sensors. The housing may define a plurality of wells, each well comprising any one or more of the foregoing sensors. The wells can be defined by (e.g., integral with) the substrate or can be defined by a hole formed in a gasket disposed upon the substrate. [0188] As a non-limiting example, in FIG.16A, a sensor, e.g., a nanosensor chip, compatible with the Society for Biomolecular Screening (SBS) 96 well format is shown schematically. Each well contains one or more nanosensors, such as the sensors described herein. An illustration of exemplary nanoneedle sensors is shown in enlarged images in FIG.16A. The well format is not limited to 96 well. For example, it can be a 384-well SBS format or 1,536-well SBS format, or any other well format. The device set shown can comprise three chips, functionalized with affinity entities that can specifically bind a protein (“2”), a viral particle (“4”) and a nucleic acid (e.g., transgene disposed within a viral genome;“6”), respectively. The detection of each analyte can be accomplished in the same workstream or in separate workstreams.

[0189] As a non-limiting example, as shown in FIG.16B, three different affinity entities are used in different parts of the 96-well SBS chip. “1” for protein, “2” for viral particle and “3” for target nucleic acid disposed within a viral genome. A multi-analyte detection can be achieved with one 96-well chip. The chip format is not limited to 96 well. It can be 384-well SBS format or 1536-well SBS format, or any other well format. The detection can be finished in the same workflow or in separate workflows.

[0190] As a non-limiting example, in FIG.16C, three different binding agents are used in different parts of same well of a 96-well SBS chip. “1” for protein, “2” for viral particle and “3” for a nucleic acid disposed with the viral genome. A multi-analyte detection can be achieved with one 96-well chip. It is contemplated the chip format is not limited to 96 well, and can be a 384-well SBS format, a 1,536-well SBS format, or any other well format. The detection can be accomplished in the same work stream or in separate work streams

[0191] Alternatively or in addition, with reference to FIGs. 17A, 17B, 18A and 18B, the sensors described herein may be incorporated into a cartridge assembly (a consumable assembly) 400. The cartridge assembly may include a housing or base 410, a wafer substrate 420 upon which the series of nanostructures are disposed, and gasket 430. The gasket 430, when placed over wafer substrate 420, can define wells, wherein the base of each well can comprise one or more sensors. The wafer substrate interfits into housing or base 410, which is configured to hold the substrate and to be easily insertable into a detection system. The housing or base may be made from a variety of different materials, for example, a metal such as aluminum, as well as plastic or rubber. The housing or base may have a feature, such as an angled corner, to facilitate placement thereof into the sensor system and/or to confirm orientation. [0192] Gasket 430 can be fabricated, for example, from silicone or plastic, sized and shaped to be placed over the wafer substrate, with openings 440 dimensioned to create wells with the wafer substrate containing the sensors disposed upon or within the wafer substrate. The openings 440 that define the wells may be dimensioned to contain at least a portion of the sample, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 50 pL, to be analyzed. Typically, a well includes walls defined by the gasket 430 and a bottom portion defined by the wafer substrate 420, with a sensor being disposed on the substrate in the well. A diameter of the well may range from 600 pm to 90 mm (e.g., from 1 mm to 80 mm,) and may have a thickness of 1 mm. In some embodiments, the wells may be formed integrally with the substrate during the fabrication process.

[0193] FIG. 19 shows a perspective view of a single plex consumable cartridge 400 and a 1,000 plex consumable cartridge 400’. In these embodiments, the sensor for the single plex cartridge is configured to detect and/or quantify a single analyte, whereas the 1,000 plex cartridge is configured to simultaneously detect and/or quantify up to 1,000 different analytes. Also, the dimensions and placement of wells 440 in the gasket 430 is adjusted to accommodate the number of sensors to be included in a single well. It is understood that the technologies described herein are scalable and the cartridge may be fabricated in a wide range of shapes and sizes. In certain embodiments, the cartridge is configured to meet Society for Biomolecular Screening (SBS) dimensional standards for microplates, for example, standard 96 well microplates. Accordingly, both the wafer substrate and the base may be rectangular in shape, with the base having a length of 128 mm and a width of 86 mm, which facilitates interfacing with various liquid handling systems and ease of portability on various liquid handling platforms.

System Considerations

[0194] Also described herein is a system for detecting the presence, or quantifying the amount, of an analyte in a sample of interest. The system comprises (a) a receiving chamber for receiving any one or more of the foregoing sensors any one or more of the foregoing cartridges; (b) a light source for illuminating at least the first series and/or any second series and/or any third series of nanostructures; and (c) a detector for detecting a change in an optical property in at least the first series and/or any second series and/or any third series of nanostructures; and optionally (d) a computer processor implementing a computer algorithm that identifies an interface between the first concentration range and optionally any second concentration range and optionally an interface between any second concentration range and any third concentration range.

[0195] With reference to FIGs. 20 and 21, an exemplary sensor system 500 is configured to facilitate the detection, or quantification of the amount, of an analyte in a sample of interest. The sensor system 500 can include a system housing 510 with a touch screen interface 520 and, for example, a data port 530. A load/unload door 540 in the housing may be sized and configured to enable the introduction of a cartridge 400 into a receiving chamber 550 of the sensor system that contains, for example, an X-Y stage 560 for holding and positioning the cartridge relative to an optical detection system 570. A light source 580 is configured to transmit a light through a camera/detector 590. The camera is configured to be positioned over the cartridge during use, and to detect a change in an optical property in at least a first, a second, and/or a third series of nanostructures on the substrate 420 disposed in the cartridge. The light source 580 is configured to illuminate nanostructures, for example, nanostructures disposed on the wafer substrate of a cartridge. The system can include a computer 600 including a computer processor for implementing the algorithm for identifying an interface between first concentration ranges and/or second concentration ranges and/or third concentration ranges, and for quantifying analytes in samples. The sensor system may also include a control platform 610 for controlling the system. Accordingly, the system includes three major sub-assemblies: a control system, an imaging system, and a cartridge handling system. These sub-assemblies may employ commercially available components to minimize supply chain complexity and to reduce assembly time.

[0196] The imaging system includes the optical detection system 570, in which the light source 580 is configured to direct light through an illuminator assembly 620 and an objective 630 to impinge on a plurality of nanostructures disposed upon a substrate of the sensor. After interacting with the sensor, the reflected light passes through the objective 630 and is captured by the detector 590. A stop 640 is disposed above the objective 630. The stop is a dark field light stop, which controls illumination, including how illumination reaches the substrate and how the image is transmitted to the detector. The mechanical tube length of the microscope system is indicated as LI, and may range from 10 mm to 300 mm. A working distance of the objective is designated as L2, and may range from about 2 mm to about 5 mm. In certain embodiments, LI is greater than L2.

[0197] As illustrated in FIG. 22, the measurement can be an optical measurement. For example, light source 580 can be used to irradiate substrate 320 with nanostructures 20 and analytes 650 disposed thereon, and one or more detectors 590 is/are positioned to detect the light that impinges the substrate. The light that is deflected from the substrate can be in the same direction of the light source, in the opposite direction, at orthogonal direction or at an angle to the light source. The data present in the images obtained by use of the optical detection system can be processed to provide the concentration of analyte present in a sample.

[0198] FIG. 23 shows one approach to informatics related to various embodiments of the sensor and related system. On average, all of the nanostructures in a given region are of substantially the same configuration and statistically have a substantially similar quantity or number of analyte binding sites. Accordingly, for a given concentration of analyte in the sample, each nanostructure in that region can be expected to bind the same number of molecules. In order for the sensor to have a wide dynamic range, a plurality of digital and analog regions with nanostructures of various configurations can be provided.

[0199] As the concentration of analyte in the samples range from the lowest detectable concentration to the highest detectable concentration in the digital regions of the sensor, the system is configured to detect the quantity or number of nanostructures evidencing an isolated color change corresponding to the binding of analyte above a threshold value (e.g., by flipping from one state to another). The higher the percentage of discrete nanostructures that exhibit a detectable color change or that have flipped, the higher the number of bound analytes and, accordingly, the higher the concentration of analyte in the sample. As depicted in FIG. 23, this flipping behavior can be presented visually in a variety of formats, including scatter plots that show data clustering, histograms that show data distribution, etc.

Comparative images of each region can also be provided, showing a particular region of the sensor before exposure to the sample, as well as after exposure. A third annotated image can be provided depicting with greater clarity the results of the flipping determination.

Numerical data is also advantageously presented, indicating absolute numbers of flipped and valid nanostructures, as well as the associated ratio value of the flipped to valid nanostructures. In particular, “flipped needles” denotes the number of sensors that have exceeded the threshold and are counted as positive. “Total valid needles” denotes the number of sensors that are counted as part of the total population. Sensors that behave outside of expected parameters are discarded and not included in subsequent analysis. Only the sensors that remain are considered “valid”. The flipped ratio is the calculated value of flipped needles divided by total valid needles. The rejection rate can also be depicted, z.e., the percentage of needles that are discarded from the pre-image. This is used as a measure of sensor quality /health. Sensors with rejection rate values of around 10% or higher are considered poor quality and generally do not provide reliable data.

[0200] At some higher threshold concentration, however, all of the digital region nanostructures have bound analyte. The digital regions of the sensor have effectively become saturated. All nanostructures have flipped and no local color change is readily evident. At this point, attention is shifted to the analog regions, that generally have larger nanostructures with more numerous binding sites.

[0201] The degree of color change of a given nanostructure can be related to the ratio of the total mass of bound molecules to the total mass of that nanostructure. Smaller analog region nanostructures (e.g., nanoneedles) that may only be able to bind less than 100 molecules can evidence a cool color hue initially (e.g., in the blue/green range). Larger analog region nanostructures (e.g., nanoneedles) that may be able to bind a few hundred molecules can evidence a warmer color hue initially (e.g., in the yellow/orange range). At the higher detectable concentrations in the analog regions, as more analytes bind to a given nanostructure, the detectable color hue shifts more warmly. Accordingly, an unexposed blue nanostructure exhibits a more greenish hue after binding for a particular analyte concentration in the sample. At higher analyte concentrations in the sample, the hue can shift to be more yellowish. Similarly, in an analog region with larger nanostructures and more binding sites configured to detect higher concentrations, the initial unexposed yellow nanostructure exhibits a more orange hue after binding for a particular analyte concentration in the sample. At higher analyte concentrations in the sample, the hue can shift to be more reddish.

[0202] While the color shift is detectable with solely a single analog nanostructure, regions of a series or array of similarly sized nanostructures are advantageously employed. By providing a large distribution of similarly sized nanostructures, an average readout can be provided to more reliably detect the analog region color shift and, accordingly, the detected analyte concentration.

[0203] More specifically, FIG. 24 shows a workflow chart of one approach for aggregating, at a system level, the detected output of the various digital and analog regions of one embodiment of a sensor, to reliably detect analyte concentration across the full dynamic range of the sensor. Use of this form of hybrid informatic engine algorithm permits the use of discrete digital and analog regions to reliably reject inaccurate higher concentration data from the digital regions and inaccurate lower concentration data from the analog regions.

[0204] In Step 1 of FIG. 24, the various digital and analog regions of a clean sensor are optically imaged as part of an overall image of the sensor, to provide a reliable baseline recording of the image status of each region and its associated nanostructures (e.g., presence or absence, initial color hue, etc.) for a particular sensor. In Step 2, the sensor is exposed to the sample, any analytes in the sample bind to associated sites on the nanostructures, and the sensor is subsequently conventionally prepared for subsequent imaging. In Step 3, the system captures the post exposure image of the sensor, that will be used to compare to the image of Step 1 to detect flipping in the digital regions and any color hue change in the analog regions. In Step 4, the algorithm identifies the different detection regions of the sensor (z.e., one or more digital regions and one or more analog regions) and their layout relative to the fiducial mark of the sensor. This permits the system to correlate and align the pre and post images to identify corresponding nanostructures in each image. Steps 5 and 6 entail individual, discrete analysis of the pre and post image data on a nanostructure-by- nanostructure basis in each corresponding region. For digital regions, Step 7A quantifies and counts the number of nanostructures with bound analyte by confirming a sufficiently large shift in the local image above a threshold to identify each nanostructure that has bound analyte. For analog regions, Step 7B detects color hue changes locally and across the analog region, evidencing a sufficiently large shift in the local image above the pre image color to deem the nanostructures locally and collectively to have bound analyte. In Step 8, assuming the color change in the analog region exceeds a predetermined threshold value, the analog region is deemed to have detected a concentration of analyte within its detectable range. The actual concentration of analyte corresponding to the color change is determined by comparison of the detected color change to a standard curve stored in system memory developed with known concentration control samples. If, however, the color change in the analog region fails to exceed a predetermined threshold value, the concentration of analyte is deemed to be below that reliably detectable by that analog region. If a lower concentration- configured analog region is available, a similar analysis can be performed. Otherwise, the system relies on the digital count of flipped nanostructures in the digital regions of the sensor. The actual concentration of analyte corresponding to the quantity or number of flipped nanostructures is determined by comparison of the number of flipped digital nanostructures to a standard curve stored in system memory developed with known concentration control samples.

[0205] An exemplary algorithm for determining the transition between a digital quantification measurement and an analog comprises the steps of (a) measuring the nanostructures that have changed (flipped) from one state to another relative to the nanostructures in the first series upon application of the solution to be tested; (b) measuring the color space changes of nanostructures in the second series upon application of the solution to be tested; and (c) if the color space change of the second series is greater than a preselected threshold value then use the analog measurements identified in step (b) and if the color space changes of the second series is less than the preselected threshold value, then use the digital measurements identified in step (a).

[0206] It is contemplated that, based on the choice of nanostructure (e.g., nanoneedle) and binding agent and other reagents, it is possible to detect and/or quantify multiple analytes at the same time. For example, as shown in FIG. 25A, a sensor can comprise a substrate 420 having disposed thereon a first series of nanostructures 700 and a second series of nanostructures 710 that can bind two separate and distinct analytes. It is contemplated that the substrate can contain a number of series of nanostructures, depending upon the number of analytes to be detected. Similarly, as shown in FIG. 25B, a sensor can comprise a substrate having disposed thereon a series of two different nanostructures 700, 710 that bind two separate and distinct analytes. It is contemplated that the series of nanostructures can contain nanostructures that bind to additional analytes.

IV. Methods of Characterizing Viral Particle Composition

[0207] An analysis of the presence, absence, and/or amount of one or more groups of viral particles in a biological sample may be conducted on a portion of the biological sample or derived from the biological sample (collectively, a test sample) as described herein. The groups of viral particles can include, for example, viral particles that lack a viral genome entirety, viral particles that contain a full viral genome and optionally one or more transgenes disposed therein, viral particles that contain fragments of the viral genome and/or fragments of the one or more transgenes disposed therein, or viral particles that contain a complete genome or a fragment thereof and/or a complete transgene or a fragment thereof, but which also contain an insertion, deletion or substitution of a given nucleotide or group of nucleotides. [0208] Depending upon the circumstances, a fragment of a transgene comprises 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 65% or less, 70% or less, 75% or less, 80% or less, 85% or less, 90% or less, 95% or less, or less than 100% of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 5% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 10% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 15% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 20% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 25% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 30% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 35% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 40% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 45% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 50% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 55% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 60% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 65% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 70% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 75% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 80% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 85% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 90% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises 95% or less of the full-length transgene sequence. In certain embodiments, a fragment of a transgene comprises less than 100% of the full-length transgene sequence.

[0209] In certain embodiments, a fragment of a transgene comprises less than 50 bases, less than 100 bases, less than 150 bases, less than 200 bases, less than 250 bases, less than 300 bases, less than 350 bases, less than 400 bases, less than 450 bases, less than 500 bases, less than 600 bases, less than 700 bases, less than 800 bases, less than 900 bases, less than 1.0 kilobases, less than 1.5 kilobases, less than 2.0 kilobases, less than 2.5 kilobases, less than 3.0 kilobases, less than 3.5 kilobases, less than 4.0 kilobases, or less than 4.5 kilobases. In certain embodiments, a fragment of a transgene comprises less than 50 bases. In certain embodiments, a fragment of a transgene comprises less than 100 bases. In certain embodiments, a fragment of a transgene comprises less than 150 bases. In certain embodiments, a fragment of a transgene comprises less than 200 bases. In certain embodiments, a fragment of a transgene comprises less than 250 bases. In certain embodiments, a fragment of a transgene comprises less than 300 bases. In certain embodiments, a fragment of a transgene comprises less than 350 bases. In certain embodiments, a fragment of a transgene comprises less than 400 bases. In certain embodiments, a fragment of a transgene comprises less than 450 bases. In certain embodiments, a fragment of a transgene comprises less than 500 bases. In certain embodiments, a fragment of a transgene comprises less than 600 bases. In certain embodiments, a fragment of a transgene comprises less than 700 bases. In certain embodiments, a fragment of a transgene comprises less than 800 bases. In certain embodiments, a fragment of a transgene comprises less than 900 bases. In certain embodiments, a fragment of a transgene comprises less than 1.0 kilobases. In certain embodiments, a fragment of a transgene comprises less than 1.5 kilobases. In certain embodiments, a fragment of a transgene comprises less than 2.0 kilobases. In certain embodiments, a fragment of a transgene comprises less than 2.5 kilobases. In certain embodiments, a fragment of a transgene comprises less than 3.0 kilobases. In certain embodiments, a fragment of a transgene comprises less than 3.5 kilobases. In certain embodiments, a fragment of a transgene comprises less than 4.0 kilobases. In certain embodiments, a fragment of a transgene comprises less than 4.5 kilobases.

[0210] In the method, a first step (step a) comprises contacting a sensor defining a plurality of functionalized nanostructures with a first test sample comprising one or more nucleic acid molecules under conditions that permit the functionalized nanostructures to bind the one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are harvested from the viral particles in the biological sample or are amplicons derived therefrom. After step (a), the method comprises a second step (step b) which comprises detecting a change in a property of the nanostructures (a detectable signal) thereby to determine the presence, absence and/or amount of the one or more nucleic acid molecules (e.g., a first nucleic acid molecule and a second nucleic acid molecule) and/or the one or more viral particles or viral capsid proteins. After step (b), the method comprises a third step (step c) comprising using the presence, absence, and/or amount of the one or more nucleic acid molecules and, optionally a concentration of viral particles in the biological sample, to determine the presence, absence, and/or amount of the one or more groups of viral particles within the first test sample.

[0211] In certain embodiments, step (a) comprises contacting a first region, a second region, and a third region of a sensor defining a plurality of functionalized nanostructures with a test sample comprising one or more nucleic acid molecules and one or more viral particles or viral capsid proteins thereof under conditions that permit the functionalized nanostructures to bind the one or more nucleic acid molecules and/or the one or more of the viral capsid proteins, wherein the first region of the sensor is configured to bind a first nucleic acid molecule, the second region of the senor is configured to bind a second, different nucleic acid molecule, and the third region of the sensor is configured to bind the viral particles or viral capsid proteins, and wherein the one or more nucleic acid molecules are harvested from the viral particles in the biological sample or are amplicons derived therefrom.

[0212] In certain embodiments, during step (b), detectable signals from the nanostructures of the sensor are quantified. The detectable signals can include a change in a property, e.g., an optically detectable property, e.g., spectral properties, of at least a portion of the nanostructures may be detected. The optically detectable property can include, for example, color, light scattering, refraction, or resonance (for example, surface plasmon resonance, electric resonance, electromagnetic resonance, and magnetic resonance). In certain embodiments, electromagnetic radiation may be applied to the nanostructure or a series of nanostructures, and the applied electromagnetic radiation may be altered by the nanostructure or series of nanostructures that have bound an analyte. For example, the presence of the analyte may result in a change of intensity, color, or fluorescence.

[0213] In some embodiments, the method further comprises, prior to step (b), contacting the one or more nucleic acid molecules with a detection agent (e.g., fluorescent label or mass amplifier), wherein binding of the detection agent to the one or more nucleic acid molecules enhances the change in the property of the nanostructures.

[0214] The test sample may be, e.g., a cell lysate (e.g., a cell lysate produced from cells grown in a bioreactor), a crude viral extract, a viral lysate (e.g., viral lysate produced from viral particles grown in a bioreactor) or a reaction mixture (e.g., of an amplification reaction) derived therefrom. A portion of the sample may be applied to any of the sensors described herein. Depending upon the circumstances, the sample may be or need not be diluted before application to the sensor. It is contemplated that the methods described herein are particularly useful in the characterization of adenoviral particles (e.g., adenoviral particles, adeno-associated virus (AAV) particles, lentiviral particles, baculovirus particles, or engineered viral particles of any one of the foregoing).

[0215] In certain embodiments, the one or more nucleic acid molecules are DNA or RNA molecules. In certain embodiments, the one or more nucleic acid molecules are DNA molecules. Depending upon the circumstances, the functionalized nanostructures can comprise a capture agent (e.g., antibody or antigen-binding fragment thereof) capable of binding to the one or more nucleic acid molecules. A capture agent can function to facilitate binding of the nucleic acid molecule by a nanostructure. Depending on the circumstances, the capture agent binds directly to a nucleic acid molecule or the capture agent binds to a capture label linked to the nucleic acid molecule. In certain embodiments, nanostructures in the first region, the nanostructures in the second region, and the nanostructures in the third region each comprise a distinct capture agent and amplicons bound by the first region and the amplicons bound by the second region comprise different capture labels. In certain embodiments, the amplicons are produced by amplification of nucleic acid molecules harvested from the viral particles, and in certain embodiments, the amplicons comprise a capture label.

[0216] In certain embodiments, the capture agent is selected from the group consisting of: an antibody that binds a capture label, an aptamer that binds a capture label, streptavidin, a nucleic acid molecule, a poly-A nucleic acid sequence, a poly-T nucleic acid sequence, a poly-C nucleic acid sequence, a poly-G nucleic acid sequence, an azide, an alkyne, a dibenzocyclooctyne, a tetrazine, and a transcyclooctene. In certain embodiments, the capture label is selected from the group consisting of: a target antigen, an aptamer-binding partner, biotin, a fluorophore (e.g., fluorescein, Tamra, Cy5, or Cy3), digoxigenin, a nucleic acid molecule, a poly-A nucleic acid sequence, a poly-T nucleic acid sequence, a poly-C nucleic acid sequence, a poly-G nucleic acid sequence, an azide, an alkyne, a dibenzocyclooctyne, a tetrazine, and a transcyclooctene.

[0217] In some embodiments, a pair of the capture agent and the capture label can be selected from the group consisting of: an antibody and a target antigen, an aptamer and an aptamer-binding partner, biotin and streptavidin, an antibody that binds a fluorophore and a fluorophore bound by the antibody (e.g., fluorescein, Tamra, Cy5, or Cy3), an anti- digoxigenin antibody and digoxigenin, a nucleic acid molecule and a complementary nucleic acid molecule, an aptamer capable of binding a nucleic acid molecule and a nucleic acid molecule bound by the aptamer, a poly-A nucleic acid sequence and a poly-T nucleic acid sequence, a poly-A nucleic acid sequence and an antibody that binds the poly-A nucleic acid sequence, a poly-T nucleic acid sequence and an antibody that binds the poly-T nucleic acid sequence, a poly-C nucleic acid sequence and a poly-G nucleic acid sequence, a poly-C nucleic acid sequence and an antibody that binds the poly-C nucleic acid sequence, a poly-G nucleic acid sequence and an antibody that binds the poly-G nucleic acid sequence, and a click chemistry pair (e.g., an azide and an alkyne, an azide and a dibenzocyclooctyne, or a tetrazine and a transcyclooctene).

[0218] Depending upon the actual assay conducted, the amplicons can comprise a detection label capable of binding to a detection agent. In certain embodiments, a detection agent binds to a nucleic acid molecule and enhances detection of the nucleic acid upon binding to a nanostructure e.g., by enhancing the change in a property of the nanostructure). In certain embodiments, the detection agent binds or is linked directly to the nucleic acid molecule. In certain embodiments, the detection agent binds to a detection label on the one or more nucleic acid.

[0219] In certain embodiments, the detection agent is selected from the group consisting of: streptavidin, an antibody that binds the detection label, an antibody conjugated to horseradish peroxidase (HRP), an oligonucleotide, an aptamer, an oligonucleotide binding partner, an azide, an alkyne, a dibenzocyclooctyne, a tetrazine, and a transcyclooctene. In certain embodiments a detection label is selected from the group consisting of biotin, fluorescein, digoxigenin, 3,3',5,5'-Tetramethylbenzidine (TMB), an oligonucleotide, an azide, an alkyne, a dibenzocyclooctyne, a tetrazine, and a transcyclooctene. In certain embodiments, a pair of the detection agent and the detection label can be selected from the group consisting of: biotin and streptavidin, fluorescein and an anti-fluorescein antibody, digoxigenin and an anti- digoxigenin antibody, 3,3',5,5'-Tetramethylbenzidine (TMB) and an antibody conjugated to horseradish peroxidase, an oligonucleotide and a complementary oligonucleotide, an oligonucleotide and an aptamer, an oligonucleotide and its binding partner, a click chemistry pair (e.g., an azide and an alkyne, an azide and a dibenzocyclooctyne, or a tetrazine and a transcyclooctene). In certain embodiments, the detection agent is streptavidin and the detection label is biotin. In certain embodiments, the detection agent is an anti-fluorescein antibody and the detection label is fluorescein. In certain embodiments, the detection agent is an anti-digoxigenin antibody and the detection label is digoxigenin. In certain embodiments, the detection agent is an antibody conjugated to HRP and the detection label is TMB. In certain embodiments, the detection agent is an oligonucleotide and the detection label is a complementary oligonucleotide. In certain embodiments, the detection agent is an oligonucleotide and the detection label is an aptamer. In certain embodiments, the detection agent is an oligonucleotide and the detection label is and a binding partner of the oligonucleotide. In certain embodiments, the detection agent and the detection label are a click chemistry pair. In certain embodiments, the click chemistry pair is an azide and an alkyne. In certain embodiments, the click chemistry pair is an azide and a dibenzocyclooctyne. In certain embodiments, the click chemistry pair is a tetrazine and a transcyclooctene.

[0220] In certain embodiments, the amplicons comprise double stranded nucleic acid. Depending upon the assay selected, a first strand of the double stranded nucleic acid can comprises a detection label and a second strand of the double stranded nucleic acid can comprise a capture label. In certain embodiments, the functionalized nanostructures each comprise an antibody or antigen binding fragment thereof that binds the capture label.

[0221] In certain embodiments, prior to step (a), the amplicons are produced using a first primer optionally coupled to a detection label and a second primer optionally coupled to a capture label and a thermostable polymerase. The first primer can hybridize to a first locus on the one or more nucleic acid molecules and the second primer can hybridize to a second locus on the one or more nucleic acid molecules. Depending upon the region to be detected, the first locus can be located at a 5' end of a first strand of the one or more nucleic acid molecules and the second locus can be located at a 5' end of a second strand of the one or more nucleic acid molecules, wherein the first strand and second strand are complementary. Alternatively, the first locus can be located at a 5' end of a first strand of the one or more nucleic acid molecules and the second locus can be located at an interior region of a second strand of the one or more nucleic acid molecules, wherein the first strand and second strand are complementary. Alternatively, the first locus can be located at an interior region of a first strand of the one or more nucleic acid molecules and the second locus is located at a 5' end of a second strand of the one or more nucleic acid molecules, wherein the first strand and second strand are complementary. Alternatively, the first locus can be located at an interior region of a first strand of the one or more nucleic acid molecules and the second locus can be located at an interior region of a second strand of the one or more nucleic acid molecules, wherein the first strand and second strand are complementary.

[0222] It is contemplated that the primers used in the amplification (e.g., the first primer and/or the second primer) are resistant to exonuclease digestion. This provides certain advantages and permits the opportunity to degrade and residual parental nucleic acid molecules in the sample before initiating the detection steps. Accordingly, in certain circumstances, it may be desirable to expose the samples to an exonuclease (e.g., Lambda exonuclease, RecJF, exonuclease VIII, or other exonuclease) so that nucleic acid molecules not containing the first primer or the second primer are digested by exposure to the exonuclease. Depending upon the circumstances, an exonuclease can be introduced to remove any unincorporated primers. Suitable exonucleases for removal of unincorporated primers include, but are not limited to exonuclease I, exonuclease T, and exonuclease VII.

[0223] In some embodiments, when the method is performed using two or more replicates, wherein each replicate comprises a distinct first primer and/or second primer, it is possible to detect the presence or absence of a plurality of loci within the one or more nucleic acid molecules.

[0224] In certain embodiments, the method comprises measuring the amount or concentration of viral particles in the biological sample. This information can then be used to determine what amount or fraction of the total viral particles (i) lack a viral genome entirety, (ii) contain a full viral genome and optionally one or more transgenes disposed therein, (iii) contain fragments of the viral genome and/or fragments of the one or more transgenes disposed therein, or (iv) contain a complete genome or a fragment thereof and/or a complete transgene or a fragment thereof, but which also contain an insertion, deletion or substitution of a given nucleotide or group of nucleotides.

[0225] Although the number of viral particles can be measured using a variety of approaches, for speed and efficiency, the number of viral particles can be measured using functionalized nanostructures functionalized to bind the viral particles. Accordingly, in certain embodiments, the method further comprises, prior to step (c): contacting a substrate comprising a plurality of functionalized nanostructures with a second test sample comprising a plurality of viral particles, wherein the functionalized nanostructures are capable of binding the viral particles; and then detecting a change in a property of the nanostructures thereby to determine the concentration of the viral particles in the biological sample. In certain embodiments, the method further comprises, prior contacting the substrate with the second test sample, contacting the viral particles with a detection antibody capable of binding the viral particles. The detection antibody can be selected from the group consisting of: an antibody that specifically binds a viral protein of a specific serotype of the viral particle (e.g., an anti-VPl antibody, an anti-VP2 antibody, an anti-VP3 antibody), a serotype non-specific antibody, an antibody that specifically binds a specific serotype of the viral particle (e.g., an anti-AAV9 antibody, an anti-AAV5 antibody, an anti-AAV2 antibody, or an anti-AAV8 antibody), an antibody that specifically binds a lentiviral particle, an antibody that specifically binds a lentiviral capsid protein, an antibody that binds to a baculovirus particle, and an antibody that binds to a baculovirus capsid protein. It is contemplated that the detection antibody can be conjugated with a second detection agent selected from the group consisting of streptavidin, HRP, HRP and TMB, HRP and DAB, a nucleic acid molecule, a bead (e.g., a polystyrene bead or a magnetic beads), a nanoparticle (e.g., a gold nanoparticle), and any other optically detectable bead or nanoparticle (e.g., a bead or nanoparticle that has a diameter of 10 nm to 2,000 nm).

[0226] Depending upon the circumstances, the first test sample and the second test sample can be derived from a single viral culture grown in a bioreactor.

Exemplary Assays

[0227] It is contemplated that a number of assays can be used to characterize the presence, absence and/or amount of the various groups or viral particles in the sample.

[0228] As a non-limiting example, FIGs. 26A and 26B show a workflow for detecting oligonucleotides of interest. Capture oligonucleotides are attached to the surface of the nanostructures, which have a sequence that completely or partially matches with a target oligonucleotide sequence FIG. 26A. The hybridization of the target oligonucleotides to the capture oligonucleotides can be detected by changes in the optical properties of the nanostructures (FIG. 26B).

[0229] With reference to FIG. 27A, capture oligonucleotides can be attached to the surface of the nanostructures. The target nucleic acid analyte is permitted to hybridize to the capture oligonucleotides (FIG. 27B). Biotinylated probe oligonucleotides are permitted to hybridize to the target nucleic acid (FIG. 27C). As an optional additional step, streptavidin-HRP can be added to and permitted to bind to the biotin of the biotinylated probe, after which a substrate for the HRP can be added (e.g., TMB), which is catalyzed by HRP to form a nonsoluble deposit on the nanostructure, creating a significant signal enhancement (FIG. 27D).

[0230] With reference to FIG. 28A, capture oligonucleotides complementary to the target nucleic acid molecules are conjugated to the nanostructures. In FIG. 28B, the target nucleic acid molecules are added and permitted to hybridize to the capture oligonucleotides. As an optional additional step in FIG. 28C, a circular DNA molecule added to and permitted to hybridized to the probe oligonucleotides. In FIG. 28D more oligonucleotides can be formed or deposited on the nanostructures by rolling circle amplification, creating a significant signal enhancement.

[0231] With reference to FIGs. 29A-29B, proteins such as bovine serum albumin with solvent accessible lysine s-amines are adsorbed to the surface of a nanostructure or a chemical modification can be implemented on the surface of the nanostructure to incorporate one or more primary amines. A nucleic acid oligonucleotide with a terminal 3’ ribonucleotide base (FIG. 29A) oxidized using sodium periodate can then be reacted with the lysine s-amines of the adsorbed protein (FIG. 29B). The resulting secondary aldimine Schiff base is reduced using sodium borohydride to generate a stable covalent linkage between the immobilized protein or the chemically modified surface and the capture DNA oligonucleotide (FIG. 29B)

[0232] With reference to FIGs. 30A and 30B, a nucleic acid oligonucleotide modified with hydrophobic groups, such as cholesterol (FIG. 30A) is adhered to the nanostructures through hydrophobic interaction (FIG. 30B). With reference to FIGs. 31A and 31B, nucleic acid modified with any chemical/biological moiety (e.g., digoxigenin) that is an antigen for a specific antibody adsorbed on nanostructures.

Methods of Detecting DNA andRNA Using Nanostructures

[0233] With reference to FIG. 32, double stranded DNA (dsDNA) from a sample of interest sample is heated to denature into single strands or remove and secondary structures in the presence of excess biotinylated detection and/or capture oligonucleotide. The capture and detection oligonucleotide pair are strand specific and can be designed to detect either strand. Thereafter, the temperature is reduced to the annealing temperature of the capture and detection oligonucleotides, and then rapidly chilled to 4° C. In cases where capture may be conducted after hybridization, i.e. when the capture is modified with a hydrophobic or chemical/biological entity that lends antibody specificity, the oligonucleotides can be annealed together with the sample. In the case where the capture oligonucleotide is covalently attached to an adsorbed protein, only the detection probe is annealed. The annealed samples are then applied and permitted to adhere to the nanostructures described hereinabove.

[0234] As a non-limiting example, neutravidin-HRP can be added permitted to bind to the biotin, after which TMB can be catalyzed by the HRP to form a non-soluble deposit on the nanostructures. The concentration of specific nucleic acids in the samples can be measured using nano structures disclosed herein.

Method of Detecting Transgenes from Viral Particles

[0235] In various embodiments, the present disclosure provides methods for detecting and/or quantifying nucleic acids encoding viral transgenes using nanostructures.

[0236] With reference to FIG. 33, viral particles, e.g., N particles are first treated with DNase I to remove any non-encapsulated DNA. Thereafter, the DNase I is inactivated. Then, proteinase K is added to digest the viral capsid and release the transgene containing nucleic acids, followed by enzyme inactivation. Single-stranded DNA (e.g., from AAV genomic DNA) is converted to double-stranded DNA, e.g., using a polymerase. The transgene is then digested with an appropriate restriction enzyme to create transgene fragments. The fragments are then 3 ’-end labeled with a biotinylated nucleotide using the enzyme - terminal deoxynucleotidyl transferase. The release of the transgene, restriction digestion and target DNA biotinylation can be performed successively without any DNA purification between steps. A DNA standard (e.g., plasmid DNA) for the transgene is digested with restriction endonuclease and 3 ’-end labeled in the same manner as the viral DNA. The digested and 3 ’-end labeled DNA is then permitted to hybridize with an excess of a modified (e.g., digoxigenin modified) oligonucleotide - the capture probe - that is complementary to the target sequence. The hybridized 3 ’-end labeled target DNA and the capture probe is then coupled to the nanostructures via an antibody that binds capture probe modification (e.g., an anti -digoxigenin antibody). As a non-limiting example, neutravidin- HRP can be added to permitted to bind to the to the biotin on the target DNA and permitted to react with TMB to form a non-soluble deposit on the surface of the nanostructures. The concentration of specific nucleic acids in the samples can then be measured using structures disclosed herein. Method of Detecting Viral Particle Titers on Nanostructures

[0237] In various embodiments, the present disclosure provides methods of detecting viral particle titers using nanostructures.

[0238] With reference to FIG. 34, nanostructures are coupled to viral serotype specific antibodies. All remaining regions of the nanostructures that are unoccupied by the antibody are coated with an unrelated protein (e.g., fish gelatin). The viral particles (from a sample or control standard) are then affinity captured on the nanostructures. As an optional additional step, a biotinylated antibody with affinity to the viral particle can be added for signal amplification. Neutravidin-HRP can be added and permitted to bind to the biotin, and the HRP can be used to convert the substrate TMB into a non-soluble deposit on the nanostructures. The concentration of specific viral particles in the samples can be measured using the nanostructures disclosed herein.

Quantification of Viral Genomes using Nanostructures

[0239] In various embodiments, the present disclosure provides methods of quantifying viral genomic DNA using nanostructures.

[0240] The viral genome quantification is a flexible assay for quantifying regions of interest in the viral transgene. The approach utilizes oligonucleotides hybridize to known viral genome sequences in a series of steps that result in a detectable DNA copy. To be detectable, the DNA is conjugated to a capture label on one end to facilitate binding to the nanostructures (or nanoneedles), and a detection label on the other end to facilitate the addition of mass to the nanostructures (or nanoneedles) to enable detection by light scattering. In addition, quantitation is based on a standard curve created from the parental plasmid DNA. Several embodiments are described below.

Method of Detecting Viral Capsids

[0241] Also described herein is a method of detecting the presence, or quantifying the amount, of a viral capsid in a sample of interest.

[0242] FIG. 35 shows an exemplary workstream for detecting viral capsids. First, a capture antibody capable of binding viral particles is bound to a nanostructure. Second, a sample of crude extract from a bioreactor comprising a plurality of viral particles is added to the substrate under conditions that allow binding of the viral particles to the capture antibodies bound to the nanostructures. Third, a detection antibody that is capable of binding to the viral particles and that also comprises a ligand for a detection agent is added to the substrate under conditions that allow binding of the detection antibodies to the viral particles. Fourth, a detection agent is added to the substrate under conditions that allow binding of the detection agent to the ligand on the detection antibody. Finally, a change in a property of the nanostructures (e.g., light scattering) caused by binding of the viral particles, detection antibody, and detection agent to the nanostructures is detected to determine the presence, absence, and/or amount of the viral particles in the sample.

Method of Detecting Viral Nucleic Acids

[0243] Also described herein is a method of detecting the presence, or quantifying the amount, of viral nucleic acids in a sample of interest.

[0244] FIG. 36 shows an exemplary workflow for detecting viral nucleic acids. First, a capture antibody that is capable of binding to a capture probe is bound to a substrate defining a plurality of nanostructures under conditions that allow binding of the capture antibody to the nanostructures. Second, a sample of crude extract from a bioreactor comprising a plurality of viral particles is obtained, and viral nucleic acids (e.g., comprising a transgene) are released from the viral particles. Third, viral nucleic acid are amplified using a first primer (Pl) comprising a capture label and a second primer (P2) comprising a detection label that is capable of binding to a detection agent to generate a double-stranded DNA (dsDNA) molecule comprising a first strand comprising a capture label at its 5' end and a second strand comprising a detection label at its 5' end. Fourth, a detection agent is added to the substrate under conditions that allow binding of the detection agent to the detection label on the dsDNA molecule. Finally, a change in a property of the nanostructures (e.g., light scattering) caused by binding of the dsDNA molecule and detection agent to the nanostructures is detected to determine the presence, absence, and/or amount of the viral nucleic acids within the viral particles in the sample.

[0245] In certain embodiments, adjacent nanostructures are functionalized with different materials to capture different labels. For example, one nanostructure may be functionalized to capture one labeled oligonucleotide and an adjacent nanostructure in the same well, or a separate well, can be functionalized to capture another labeled oligonucleotide. In this way, multiple analytes (e.g., regions of interest of viral nucleic acids) in the same sample can be distinguished simultaneously. For example, multiple nanostructures can be used to detect multiple first-labels, and multiple oligonucleotides. For example, multiple nucleic acid sequences of interest can be detected in a single well, or separate wells, using multiple first, second, and/or third oligonucleotide sets. In another example, different nucleic acid lengths can be distinguished using multiple first, second, and third oligonucleotide sets by hybridizing them along the length of the nucleic acid of interest and comparing the quantification of each of them to characterize the mixture of various nucleic acid lengths.

[0246] FIG. 37 depicts an exemplary nucleic acid labeling scheme that may be used for nanostructures capture and detection. First, an unlabeled first oligonucleotide is used as a primer to generate a copy of the nucleic acid of interest (e.g., a viral nucleic acid). Second, two labeled oligonucleotides are added to facilitate capture and detection. The second oligonucleotide (labeled A) comprises a capture label and the third oligonucleotide (labeled B) comprises a detection label. The second and third oligonucleotides are permitted to hybridize to the nucleic acid copy. The resulting sample mixture is added to a substrate defining a plurality of functionalized nanostructures, whereupon the capture label is bound by binding agents (e.g., antibodies) immobilized on the surface of nanostructures. The detection label can be bound by detection antibodies to generate a change in a property of the nanostructures (e.g., light scattering) that can be detected to determine the presence, absence, and/or amount of the nucleic acid of interest in the sample.

[0247] FIG. 38 depicts an example of nucleic acid characterization by multiple capture oligonucleotides and multiple nanostructures. First, an unlabeled first oligonucleotide is used as a primer to generate a copy of nucleic acids of interest (e.g., viral nucleic acids). Both full length and partial length targets may be copied as shown. Second, multiple labeled oligonucleotides are added to the sample mixture, each directed to a different region (labeled A, B, and C). Finally, the sample mixture can be captured on two separate nanostructures, each functionalized to capture a different capture label and provide information on the presence of different points along the region of interest. For example, labeled oligonucleotides that are close to the first oligonucleotide (e.g., the second and third oligonucleotides) can be used to measure both complete and partial viral genomes. This method can be used, e.g., for establishing a gross ratio of full-to-empty viral particles. In contrast, the distal oligonucleotide can only bind to transgene copies that have both ends (first and fourth oligonucleotides), such as the full-length copy. Nanostructures functionalized with different antibodies can be multiplexed in a single well or in separate wells.

[0248] FIG. 39 depicts an example of a nanosensor well containing functionalized nanostructures for capture and detection of multiple analytes. Analytes captured can include a viral capsid, a full-length nucleic acid (e.g., containing a transgene), a partial sequence transgene, or other analytes. In this example, two different first oligonucleotides comprising capture labels Al or A2 are used in the initial nucleic acid copy step. The copies are then permitted to hybridize to the second oligonucleotide comprising a detection label (B). A sample containing the Al/B nucleic acid copy, the A2/B nucleic acid copy, and the intact capsid is loaded onto a single substrate containing 3 nanostructures, each functionalized for a different capture antibody against Al, A2, and the viral capsid.

Short Transgene Sequence Detection and Quantification

[0249] In certain embodiments, full/empty capsid titer is measured to assess viral genome packaging efficiency.

[0250] In some embodiments, the assay is a quantification of a short sequence of the transgene to determine the proportion of capsids containing DNA to those with none. For this approach, a pair of closely adjacent oligonucleotides, one conjugated to a capture label and the other to a detection label, can be used to amplify a small region of the parental viral DNA. The amplification can be done by PCR using as few as 2 to 6 PCR thermal cycles for samples that are >1 x 10⁹ viral genomes/mL, with standard PCR reagents (polymerase, dNTPs, buffers, and the labeled oligonucleotides as primers).

[0251] In some embodiments, full/empty capsid titer is measured by thermocycling using only a single labeled primer to generate unidirectional copies of the parental genome. This approach can result in linear amplification and labeling of the transgene with one of the two labels required. The second label (capture or detection) can then be added by hybridizing a probe to the amplified single stranded DNA. This method may be preferred to the PCR amplification since it avoids exponential amplification of PCR which may be less accurate.

[0252] FIG. 40 shows an exemplary workstream to generate a double stranded detectable PCR product. Capture and detection labels can selected from commonly used oligonucleotide modifications. For example, a capture oligonucleotide may be a fluorescein labeled primer that can be captured by anti-fluorescein antibodies on the nanoneedles and a detection oligonucleotide may be a biotin labeled primer that can be conjugated to streptavidin to provide additional mass to permit detection by light scattering.

[0253] Full Length Transgene Quantification

[0254] Following a similar approach to the short region quantification, one can position the capture and detection primers on opposite ends of the transgene to achieve quantification of only the full-length transgene. FIGs. 41A and 41B illustrate exemplary workflows to generate the double stranded detectable PCR product. Briefly, a viral transgene is contacted with a first primer (which can comprise a capture label) and a second primer (which can comprise a detection label). Thereafter, two to six rounds of PCR are performed. The resulting samples are applied to a functionalized nanostructure as disclosed herein, and the detected signal is plotted as a function of concentration of viral genomes (VG) in the sample. An exemplary graph showing a standard curve of readouts (in arbitrary “Nano Units”) as a function of viral genome concentration (in VG/mL) is depicted in FIG. 41C.

Multiple Region Quantification

[0255] In certain embodiments, a nucleic acid of interest can be interrogated across multiple regions of interest. In some embodiments, transgenes that have both terminal ends, and additional internal genomic sequences the following approach can be measured.

[0256] FIG. 42 illustrates the workflow to generate the double stranded detectable PCR product. All four primer sequences must exist to generate a positive signal.

[0257] In an exemplary workflow, unlabeled, exonuclease resistant, primers at both ends of the transgene are first used to PCR-copy the full length of the transgene. Exonuclease resistance can be achieved in multiple ways, for example by incorporating phosphorothioate linkages into the primer sequence. A small number of thermocycling cycles, for example 2- 6, can be used to ensure the PCR amplification of the transgene remains relatively linear for accurate quantification. Next, the parental genetic material is destroyed, which can be achieved for example by digesting the parental material with an exonuclease, while the copied amplicons containing the exonuclease resistant 5’ ends, remain intact. Afterwards, an internal region of the amplicon can be amplified using capture and detection labeled primers. Finally, the resulting double stranded labeled PCR product are captured on the nanoneedle surface and can be detected as described above.

EXAMPLES

Example 1: Process For Detecting and Quantifying Nucleic Acids Using Nanosensors [0258] This example describes an exemplary workflow for detecting and quantifying nucleic acid molecules of a known sequence of interest in a heterogeneous mixture of nucleic acid molecules. The method uses several oligonucleotides in a series of steps to ensure that: (i) the sample contains a nucleic acid sequence of interest, (ii) the nucleic acid sequence of interest is captured on the nanostructure, and (iii) the nucleic acid sequence of interest is detected on the nanostructure. Furthermore, the method is designed to identify up to 3 regions of interest simultaneously. [0259] In one approach, the method comprises the following steps:

1. A nanostructure is functionalized with capture chemistry such as for example, an antibody, a protein, a nucleic acid, neutravidin, streptavidin, or any other capture chemistry (e.g., as described in PCT/US2020/020204).

A first oligonucleotide is added to a sample, e.g., a cell culture sample. The first oligonucleotide is designed to hybridize to an end-region of the nucleic acid molecule region of interest. The first oligonucleotide may be labeled to facilitate the capture or the detection on the nanostructure, where the label may be, for example, biotin, fluorescein, Cy5, Texas Red, a fluorophore, digoxygenin, a peptide, a modified DNA or RNA base, an aptamer, or any label that can be captured by an antibody, streptavidin, neutravidin, a nucleic acid sequence. The label may be conjugated to the first oligonucleotide on the 5’ end, the 3’ end, or internally. The first oligonucleotide may be of any length, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides or longer.

2. Next, materials are added to the sample mixture to allow the first oligonucleotide to act as a primer in a PCR reaction and to generate a unidirectional copy of the region of interest.

The reagents added to the sample mixture may include salt-buffer, dNTPs, DNA polymerase, reverse transcriptase, MgCl, or any other reagents known in the art to be required in DNA polymerization. The unidirectional copy of the region of interest template may be generated by multiple cycles of unidirectional copying. In one embodiment, once the unidirectional copies are created, the parental nucleic acids are destroyed, for example, by using a DNA or RNA nuclease specific to the parental nucleic acids. This can be accomplished by using a nuclease specific to double stranded nucleic acids such as T7 exonuclease, exonuclease III, exonuclease VIII, or lambda exonuclease, or if the parental nucleic acids are RNA they may be eliminated by heating, or digestion with RNAse H.

3. Next a labeled, second oligonucleotide designed to hybridize to a region on the copied nucleic acid of interest is added to the sample.

The first and second oligonucleotides can be positioned to interrogate the structure and sequence of the nucleic acid of interest. For example, the first oligonucleotide can be positioned at one end of the nucleic acid of interest and the second oligonucleotide can be positioned at the opposite end of the nucleic acid of interest or at any position along the nucleic acid of interest. The first and second oligonucleotides can overlap a nucleic acid sequence variant region of interest to inform on a presence of a genetic variant. The second oligonucleotide may be labeled to facilitate the capture or detection on the nanostructure. The label may be a biotin, fluorescein, Cy5, Texas Red, a fluorophore, digoxygenin, a peptide, a modified DNA or RNA base, an aptamer, or any label that can be captured by an antibody, streptavidin, neutravidin, a nucleic acid sequence. A label may be conjugated to the second oligonucleotide on its 5’ end, 3’ end, or internally. The second oligonucleotide may be of any length, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides or longer. Next, a labeled third oligonucleotide is added.

The first, second, and third oligonucleotides can be positioned to interrogate the structure and sequence of the nucleic acid of interest. For example, the first oligonucleotide can be positioned at one end of the nucleic acid of interest, the second oligonucleotide can be positioned at the opposite end of the nucleic acid of interest, and the third oligonucleotide may be positioned in between the first and the second oligonucleotides positions. The first, second, and third oligonucleotides can be positioned at any location along the nucleic acid of interest. The third oligonucleotide may be labeled to facilitate the capture or detection on the nanostructure. The label may be a biotin, fluorescein, Cy5, Texas Red, a fluorophore, digoxygenin, a peptide, a modified DNA or RNA base, an aptamer, or any label that can be captured by an antibody, streptavidin, neutravidin, a nucleic acid sequence. A label may be conjugated to the third oligonucleotide on its 5’ end, 3’ end, or internally. The third oligonucleotide may be of any length, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides or longer. Next, the mixture including the sample and the hybridized oligonucleotides is added to the functionalized nanostructure for capture.

At least one of the oligonucleotides (first, second, or third oligonucleotides must be conjugated to a label that will be captured by the nanostructure’s capturing moiety. Next, detection materials designed for at least one of the first, second, or third oligonucleotides are added to the nanostructure well. At least one of the oligonucleotides (first, second, or third oligonucleotides) must be a substrate to the nanostructure’s detection chemistry. The detection materials can include, for example, an antibody, streptavidin, neutravidin, HRP, TMB, or any other material that can be captured by the first, second, or third oligonucleotide to generate a signal, for example, as described in PCT/US2020/020204.

[0260] The sample can be prepared using a specifically designed capture primer and detection primer by the following steps:

1. Release of the viral genome from the capsid: treat the sample with DNAse I by using appropriate buffers and enzymes and incubation at 37°, followed by inactivation of DNase I at 75°C for 10 min. After DNAse I treatment, digest the capsids with proteinase K at 50°C, followed by inactivation of proteinase K at 95°C for 10 min.

2. Preparation of DNA standard: quantify the plasmid DNA that bears the relevant AAV genome using the method of choice (e.g., agarose gel quantification, UV spectrometry, etc.).

3. PCR amplification with the relevant primers Next, PCR-amplify the sample and the standard using the appropriate primers. The forward primer has the following structure: 5’ poly(A) tail, a spacer, and then the transgene specific sequence (see, FIG. 43 A). The reverse primer is 5’ biotinylated (see, FIG. 43B). Amplify the sample for the appropriate number of cycles, based on the required sensitivity. Usually, only a low number of cycles e.g., 6) is sufficient to detect 1.00e+10 molecules/mL (see, FIG. 44).

4. Detection with nanoneedles: pre-image the nanoneedle plate. Then, coat the plate with anti-DIG antibody overnight at room temperature. Wash and block the plate with 1% BSA containing 500 nM of an anti-DIG poly(T) universal capture oligonucleotide, and incubate at room temperature for 1 hour. Wash the plate and apply the PCR amplified standards and/or samples and incubate at room temperature for 1 hour. Wash the plate and apply mass amplifier that binds to biotin. Dry the plate under nitrogen gas/air, and post-image the plate (see, FIGs. 45A-45B).

[0261] An exemplary readout of a method described herein is shown in FIG. 46.

[0262] In another approach, the sample can be prepared using a unidirectional linear amplification of the viral genome by the following steps: 1. Release of viral genome from the capsid: treat the sample with DNAse I by using appropriate buffers and enzymes and incubation at 37°, followed by inactivation of DNase I at 75°C for 10 min. After DNAse I treatment, digest the capsids with proteinase K at 50°C, followed by inactivation of proteinase K at 95°C for 10 min.

2. Preparation of DNA standard: quantify the plasmid DNA that bears the relevant AAV genome using the method of choice (e.g., agarose gel quantification, UV spectrometry, etc.). Generate a standard curve from the prepared standard by serial dilution.

3. Unidirectional amplification of the sample and standards: amplify the sample and the standards using a single biotin labeled primer (the detection primer) in a standard PCR reaction (with dNTPs, buffer and DNA polymerase). Thermal-cycle 20 times to generate biotin labeled, single-stranded copies of the sample and the standards.

4. Hybridize the capture probe to the sample and the standard: add a poly(A) labeled capture probe to the sample and standards. Add fresh DNA polymerase and thermal cycle as in PCR for 1 cycle.

5. Detection with nanoneedles: pre-image the nanoneedle plate. Then, coat the plate with anti-DIG antibody overnight at room temperature. Wash and block the plate with 1% BSA containing 500 nM of an anti-DIG poly(T) universal capture oligo and incubate at room temperature for 1 hour.

6. Wash the plate and apply the amplified standards and samples and incubate at room temperature for 1 hour. Wash the plate and apply a mass amplifier that binds to biotin. Dry the plate under nitrogen gas/air, and post-image the plate.

[0263] An exemplary readout of such a method is shown in FIGs. 47A and 47B. In FIG. 47A, both the capture primer and detection probes are positioned proximal to the 3’ ITR and can therefore quantify both full length and 3’ truncated viral genomes. In FIG. 47B, the capture primer is adjacent to the 3’ ITR and the detection probe is positioned in the farthest distal region of the viral genome - adjacent to the 5’ ITR, as a result, this approach can quantify full length genomes.

[0264] In certain embodiments, the sample is prepared for the detection of empty capsids versus full capsids versus transgenes without lysing the viral particles. A quick analysis of AAV viral particles can be useful for gene therapy process development and manufacturing QC. For example, it can be important to differentiate between fully packaged AAV viral particles and empty particles. This method uses a nanostructure sensor to detect single AAV viral particles, and further characterize its empty or full character by monitoring light scattering in real time. In this approach, AAV antibodies are captured on the nanostructures. A sample that contains empty, partially full, and full viral particles are flowed onto a sensor with containing nanostructures. A light scattering signal from the nanostructure is continuously recorded by a photodiode or a low-noise camera. Due to light source intensity and detector dark noise, a fluctuation of the signal with a drift is expected. Nevertheless, the binding of the viral particle to a nanostructure will be detected as a step-wise signal in the time trace.

[0265] The typical mass of an empty AAV capsid is about 3.70 MDa, a full-length transgene is about 4.7 kb or 1.55 MDa, a fully packaged AAV with intact transgene is about 5.25 MDa, which represents > 30% change of the mass. 30% of light scattering change can be detected by identifying the “step” size in the binding signal. FIG. 48 shows a real-time trace of the light scattering on the nanostructure in three different scenarios: empty capsids (1), capsids containing partial genomes (2), and capsids containing full genomes (3).

Example 2: Measurement of Viral Particle Titer

[0266] This example describes the use of a nanostructure-based method for the measurement of viral titer in a sample.

[0267] A capture antibody was diluted to a working concentration of 5 pg/mL in buffer. 20 pL of capture antibody, serotype specific to AAV9 capsid protein was pipetted into each well of the nanostructure containing sensor chip. The sensor chip was covered and incubated for 1 hour at room temperature. Each well was aspirated and washed three times using buffer on a plate washer. The sensor chip was then blocked by adding 10 pL of buffer to each well and incubated at room temperature for a minimum of 1 hour, followed by washing three times with buffer. AAV9 control samples were prepared in lx PROGEN assay buffer according to the manufacturer’s instructions. A two-fold dilution series was prepared using buffer in low bind tubes. Then 10 pL of AAV9 control dilutions in buffer were added to each well to make a standard titration curve. The sensor chip was then covered and incubated for 2 hours at room temperature. The sensor chip was then washed 3 times using buffer, aspirating between each wash. 10 pL of the AAV9 antibody in its biotinylated form at lx concentration diluted in buffer was added to each well. The sensor chip was covered and incubated for 1 hour at room temperature, and then washed with buffer as conducted previously. For signal detection, 10 pL of reagent was added to each well, and the sensor chip was covered and incubated for 30 minutes. The chip was then washed (3x), and 10 pL of reagent was then pipetted into each well and allowed to incubate for 15 minutes. After 15 minutes, the chip was submerged in distilled water, then dried and imaged. FIG. 49 shows the viral particle titer, from 10⁸ parti cl e/mL at the lower limit, to approximately 10¹² particles/mL at the upper limit, with an average CV of 9%.

Example 3: Measurement of Viral Genome Titer

[0268] This Example describes the measurement of the concentration of viral genomic DNA in a viral sample.

[0269] An AAV containing sample was first treated with DNase I to remove any nonencapsulated DNA. Proteinase K was then added and incubated and subsequently deactivated. Restriction digestion of the transgene was performed to create transgene fragments. The capture probe was designed to 100% match the (+) strand of the transgene fragment after restriction digestion. Following restriction, 8 pg of restricted plasmid DNA and 50 pL of sample transgene fragment were biotinylated. The release of the transgene, restriction digestion and target DNA biotinylation were done successively without cleanup between the steps.

[0270] The standard plasmid was diluted to 150 pL in PBS and 1 mM EDTA to a final plasmid concentration of 20 nM. A three-fold serial dilution of the standard plasmid was prepared followed by addition of 1 pL capture probe to each standard dilution to achieve a final capture concentration of 200 nM. 1 pL of capture probe was added directly to the commercial Vector Builder sample after labeling to achieve a final capture concentration of 200 nM. Samples and standards were then heat denatured for 5 minutes and then rapidly cooled to allow hybridization of the capture probe to the target sequence.

[0271] Standards and samples were then added to the sensor chip. The capture probe was designed to be 100% specific to the fragment plasmid and transgene. For coupling to the nanostructure, a 3’ digoxigenin label is incorporated in each capture probe sequence for binding to the nanostructure, which is facilitated via an anti-digoxigenin antibody linked to each nanostructure. The AAV9 viral genome titer was performed utilizing a control plasmid for standard control and commercially available sample from Vector Builder. When designing the hybridization assay, target probe was designed to be specific to the (+) strand of the genome. It should be noted that with multiplexing capability, assays can be designed to independently target both (+) and (-) strands.

[0272] Along with the standard, an AAV9 sample sourced from Vector builder was tested to compare the nanostructure determined concentration to that by qPCR. The manufacturer specified concentration of the viral genome (vg) was 8.22 x 10¹¹ vg/mL as determined by qPCR using primers for the ITR region of the transgene. Assuming a packaging of (+) and (-) strands at 50% each, this would calculate to a (+) vg/mL of 4.11 x 10¹¹. The nanostructure measured a concentration of 3.11 x 10¹¹ (+) vg/mL, after adjusting for dilution during enzymatic steps required for viral genome release, and restriction digestion and 3’- biotinylation of the transgene. FIG. 50 shows the viral genome titer from IO¹⁰ to 10¹² viral genomes/mL with an average CV across the dynamic range of 3.4%.

[0273] This approach permits specific targeting to both positive and negative strands of the encapsulated genome independently and may lead to further understanding of packaging efficiency.

Example 4: Improved Detection of Viral Capsids

[0274] Viral capsid samples were produced in a bioreactor and titered using a standard ELISA method or using the method described in Example 2. Standard curves were prepared for each method. The linear dynamic range for the ELISA method ranged from about 2* 10⁸ to about 1 x 10¹⁰ capsids/mL (see, FIG. 51A), and the curve for the method using nanostructures ranged from about 2* 10⁸ to about 2* 10¹¹ capsids/mL (see, FIG. 51B). These results indicate that the method using functionalized nanostructures has an improved dynamic range for detection of viral capsids over that achieved with the ELISA assay.

[0275] Furthermore, quantification of capsid titers measured by the nanostructure method were compared to an existing ELISA-based method. Briefly, samples were diluted to fall within the dynamic ranges for each assay as indicated in FIGs. 51A and 51B and analyzed using the nanostructure and an ELISA-based method. Capsid quantification using the nanostructures showed a high degree of correlation with values achieved using an ELISA assay on samples obtained across different sample steps (see TABLE 4). TABLE 4

[0276] To assess effects of assays at different titers on detection of viral capsids, the method detailed in Example 2 was carried out on viral samples at six known concentrations per sample measured across nine different substrates (nine sets of nanoneedles) measured by three different users over three different instruments (see, FIG. 52). These results indicate that the method using nanostructures produced reproducible results over a range of titers over a number substrates, instruments and users.

[0277] To assess effects of different cell lysis buffer systems on the detection of viral capsids, the method essentially as described in Example 2 was carried out on HEK293T cells grown in 100% Complete BalanCD HEK293 media (BCD) supplemented with 4 mM Glutamax and lysed with one of the following lysis buffers, JT Baker lysis buffer, AAV-max lysis buffer, or NP40 lysis buffer, and capsids were spiked by serial dilution into the resulting lysates. The results are summarized in FIGs. 53 and 54. These results indicate that using nanostructures, it was possible to accurately estimate the capsid titer under crude conditions (FIG. 53), and that no matrix interference was noted using the crude samples as there was linearity between the samples estimated in crude cell background down to l.Oe⁹ capsids/mL (FIG. 54)

Example 5: Improved Detection of Viral Genomic DNA

[0278] Viral capsids were produced in a bioreactor and assessed using the method described in FIG. 55A. To assess whether the nanostructure-based method successfully measures partial versus full-length genomes, full-length viral genome segment was generated from plasmid DNA using an appropriate restriction enzyme. Released transgene DNA was probed via digital droplet PCR (ddPCR) using probes specific for the ITR, or via nanostructures using the method essentially as described in Example 1, and detecting the full length of the viral genome. A portion of the full-length viral genome was further digested by another restriction enzyme that creates an incision at one discreet location within the genomic segment to mimic a truncated genome (FIG. 55B). The full-length and truncated samples were combined with probe sets for detection of the full-length transgene sequence or a partial region of the transgene. As shown in FIG. 55C, the probes for detection of the full-length transgene produce a signal upon binding to a nucleic acid comprising the full-length transgene but not a truncated transgene, however the probes for detection of the partial region produce a signal upon binding to either the full transgene or truncated nucleic acid.

[0279] Full-length and truncated viral genomes were quantified and mixed in fixed molar ratios (100% full-length [0% partial]; 75% full-length:25% partial; 50% full-length: 50% partial; 25% full-length:75% partial; and 100% partial [0% full-length]) and then measured using probes specific for the truncated region or the full-length viral genome (see, FIG. 56A). The probes for the truncated region gave similar signals for both the truncated and the full- length viral genome in all tested mixtures (FIG. 56B). In contrast, the probe for the full- length region detected maximum signal for the sample that only had the full-length viral genome fragment and gave progressively weaker signal when mixed 3: 1, 1 : 1, or 1 :3 with the partial viral genome sample (FIG. 56C). The signal was equivalent to background when there were no full-length viral genome fragments and only partial genome segments. This indicates that the signal derived from the probes for the full-length viral genome was specific and that partial and full-length viral genomes can be specifically quantified allowing the measurement of the true value of the full-length genome as well as characterize the partial genomes in the sample.

[0280] Nanostructure-based full-length genome analysis was compared with an existing droplet digital PCR (ddPCR). As shown in FIG. 57, the nanostructure-based method can detect the presence of the full viral transgene sequence, whereas the ddPCR method utilizes a probe detecting the inverted terminal repeat (ITR) sequence. The analysis was carried out on a mixed population of nucleic acids containing full-length genomic sequences and truncated sequences. As shown in TABLE 5, the ddPCR-based method resulted in a nearly two-fold higher level of detection of viral genomes, which can be attributed to the undesirable of partial genomes by the ddPCR probe. TABLE 5

[0281] In a parallel experiment, nucleic acids from AAV9 samples were purified using an affinity column and iodixanol purification. Purified samples were analyzed both using ddPCR detection of a probe detecting a 0.06 kb region of the viral transgene sequence and the nanostructure method detecting the full viral genome. As shown in TABLE 6, the ddPCR- based method resulted in a nearly two-fold higher level of detection of viral genomes before and after purification, which can be attributed to the detection of partial genomes by the ddPCR probe.

TABLE 6

Example 6: Detection of Multiple Target Regions within a Viral Genomic Sequence [0282] This example describes an approach for detecting multiple target regions within a viral genomic sequence.

[0283] To estimate transgene integrity, three different probe sets including a capture probe that hybridized at one end of the transgene, which was the same for each, and a detection probe that hybridized a position either 0.6, 1.7 or 2 kb away from the site hybridized by the capture probe. FIG. 58A shows a schematic representation of where all three probe sets would give signal for the full-length transgene but would only show signal only from the regions that can amplify with probes contained within a partial genome. When using the plasmid, as expected, quantification was equivalent with all three probe sets since the entire sequence of the transgene was equimolar (see, FIG. 58B). However, when the sample was measured with the same probe sets gave equivalent signal for the 1.7 and 2 kb spaced probe sets, but was higher for 0.6 kb probe set (FIG. 58C). This indicates that there were no detected truncations between 1.7 kb and 2 kb, but there were genome species in the population containing truncations between the 0.6 kb and 1.7 kb region.

INCORPORATION BY REFERENCE

[0284] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc. , whether supra or infra, are hereby incorporated by reference in their entirety for all purposes. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

EQUIVALENTS

[0285] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

WHAT IS CLAIMED IS:

1. A method of determining the presence, absence, and/or amount of one or more groups of viral particles in a biological sample, the method comprising:

(a) contacting a sensor defining a plurality of functionalized nanostructures with a first test sample comprising one or more nucleic acid molecules under conditions that permit the functionalized nanostructures to bind the one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are harvested from the viral particles in the biological sample or are amplicons derived therefrom;

(b) after step (a), detecting a change in a property of the nanostructures thereby to determine the presence, absence, and/or amount of the one or more nucleic acid molecules; and

(c) after step (b), using the presence, absence, and/or amount of the one or more nucleic acid molecules and, optionally a concentration of viral particles in the biological sample, to determine the presence, absence, and/or amount of the one or more groups of viral particles within the first test sample.

2. The method of claim 1, wherein the functionalized nanostructures each comprise a capture agent.

3. The method of claim 1 or 2, wherein the amplicons are produced by amplification of nucleic acid molecules harvested from the viral particles.

4. The method of claim 3, wherein the amplicons comprise a detection label capable of binding to a detection agent.

5. The method of claim 4, wherein the detection label and the detection agent are selected from the group consisting of: biotin and streptavidin, fluorescein and an antifluorescein antibody, digoxigenin and an anti-digoxigenin antibody, 3, 3', 5,5'- Tetramethylbenzidine (TMB) and an antibody conjugated to horseradish peroxidase (HRP), an oligonucleotide and a complementary oligonucleotide, an oligonucleotide and an aptamer, an oligonucleotide and its binding partner, a click chemistry pair (e.g., an azide and an alkyne, an azide and a dibenzocyclooctyne, or a tetrazine and a transcy cl ooctene) .

6. The method of any one of claims 2-5, wherein the capture agent is an antibody or an antigen-binding fragment thereof capable of binding the one or more nucleic acid molecules.

7. The method of any one of claims 1-6, wherein the amplicons comprise a capture label.

8. The method of claim 7, wherein the capture agent and the capture label are selected from the group consisting of: a target antigen and an antibody, an aptamer-binding partner and an aptamer, biotin and streptavidin, a fluorophore (e.g., fluorescein, Tamra, Cy5, or Cy3) and an antibody that binds the fluorophore, digoxigenin and an anti-digoxigenin antibody, a nucleic acid molecule and a complementary nucleic acid molecule, a nucleic acid molecule and an aptamer capable of binding the nucleic acid molecule, a poly-A nucleic acid sequence and a poly-T nucleic acid sequence, a poly- A nucleic acid sequence and an antibody that binds the poly-A nucleic acid sequence, a poly-T nucleic acid sequence and an antibody that binds the poly-T nucleic acid sequence, a poly-C nucleic acid sequence and a poly-G nucleic acid sequence, a poly- C nucleic acid sequence and an antibody that binds the poly-C nucleic acid sequence, a poly-G nucleic acid sequence and an antibody that binds the poly-G nucleic acid sequence, and a click chemistry pair (e.g., an azide and an alkyne, an azide and a dibenzocyclooctyne, or a tetrazine and a transcyclooctene).

9. The method of claims 7 or 8, wherein the functionalized nanostructures each comprise an antibody or antigen binding fragment thereof that binds the capture label.

10. The method of any one of claims 1-9, further comprising, prior to step (c):

(i) contacting an additional substrate comprising a plurality of functionalized nanostructures with an additional test sample comprising a plurality of viral particles, wherein the functionalized nanostructures of the additional substrate are capable of binding the viral particles; and (ii) after step (i), detecting a change in a property of the nanostructures of the additional substrate thereby to determine the concentration of the viral particles in the biological sample.

11. The method of claim 10, wherein the first test sample and the additional test sample are derived from a single viral culture and/or cell culture grown in a bioreactor.

12. The method of any one of claims 10 or 11, further comprising, prior to step (ii), contacting the viral particles with a detection antibody capable of binding the viral particles.

13. The method of claim 12, wherein the detection antibody is selected from the group consisting of an antibody that binds a viral protein of a specific serotype of the viral particle (e.g., an anti- VP 1 antibody, an anti-VP2 antibody, an anti-VP3 antibody), a serotype non-specific antibody, an antibody that binds a specific serotype of the viral particle (e.g., an anti-AAV9 antibody, an anti-AAV5 antibody, an anti-AAV2 antibody, or an anti-AAV8 antibody), an antibody that binds a lentiviral particle, an antibody that binds a lentiviral capsid protein, an antibody that binds to a baculovirus particle, and an antibody that binds to baculovirus capsid protein.

14. The method of any one of claims 10-13, wherein the detection antibody is conjugated to a second detection agent selected from the group consisting of: streptavidin, HRP, HRP and TMB, HRP and 3,3 '-Diaminobenzidine (DAB)), a nucleic acid molecule, a bead (e.g., a polystyrene bead or a magnetic bead), and a nanoparticle (e.g., a gold nanoparticle).

15. A method of determining the presence, absence, and/or amount of two or more groups of viral particles in a biological sample, the method comprising:

(a) contacting a first region, a second region, and a third region of a sensor defining a plurality of functionalized nanostructures with a test sample comprising one or more nucleic acid molecules and one or more viral particles or viral capsid proteins thereof under conditions that permit the functionalized nanostructures to bind the one or more nucleic acid molecules and/or the one or more of the viral capsid proteins, wherein the first region of the sensor is configured to bind a first nucleic acid molecule, the second region of the senor is configured to bind a second, different nucleic acid molecule, and the third region of the sensor is configured to bind the viral particles or viral capsid proteins, and wherein the one or more nucleic acid molecules are harvested from the viral particles in the biological sample or are amplicons derived therefrom;

(b) after step (a), detecting a change in a property of the nanostructures thereby to determine the presence, absence and/or amount of the first nucleic acid molecule, the second nucleic acid molecule, and the one or more viral particles or viral capsid proteins; and

(c) after step (b), using the presence, absence, and/or amount of the one or more nucleic acid molecules of the first test sample, the one or more nucleic acid molecules of the second test sample, and the one or more viral particles or viral capsid proteins in the biological sample to determine the presence, absence, and/or amount of the two or more groups of viral particles within the biological sample.

16. The method of claim 15, wherein the functionalized nanostructures comprise a capture agent.

17. The method of claim 15 or 16, wherein the amplicons are produced by amplification of nucleic acid molecules harvested from the viral particles.

18. The method of claim 17, wherein the amplicons comprise a detection label capable of binding to a detection agent.

19. The method of claim 18, wherein the detection label and the detection agent are selected from the group consisting of biotin and streptavidin, fluorescein and an antifluorescein antibody, digoxigenin and an anti-digoxigenin antibody, 3, 3', 5,5'- Tetramethylbenzidine (TMB) and an antibody conjugated to horseradish peroxidase (HRP), an oligonucleotide and a complementary oligonucleotide, an oligonucleotide and an aptamer, an oligonucleotide and its binding partner, a click chemistry pair (e.g., an azide and an alkyne, an azide and a dibenzocyclooctyne, or a tetrazine and a transcy cl ooctene) .

20. The method of any one of claims 16-19, wherein the capture agent is an antibody or an antigen-binding fragment thereof capable of binding the one or more nucleic acid molecules.

21. The method of any one of claims 17-19, wherein the amplicons comprise a capture label.

22. The method of claim 21, wherein the capture agent and the capture label are selected from the group consisting of: an antibody and a target antigen, an aptamer and an aptamer-binding partner, biotin and streptavidin, a fluorophore (e.g., fluorescein, Tamra, Cy5, or Cy3) and an antibody that binds the fluorophore, digoxigenin and an anti-digoxigenin antibody, a nucleic acid molecule and a complementary nucleic acid molecule, a nucleic acid molecule and an aptamer capable of binding the nucleic acid molecule, a poly-A nucleic acid sequence and a poly-T nucleic acid sequence, a poly- A nucleic acid sequence and an antibody that binds the poly-A nucleic acid sequence, a poly-T nucleic acid sequence and an antibody that binds the poly-T nucleic acid sequence, a poly-C nucleic acid sequence and a poly-G nucleic acid sequence, a poly- C nucleic acid sequence and an antibody that binds the poly-C nucleic acid sequence, a poly-G nucleic acid sequence and an antibody that binds the poly-G nucleic acid sequence, and a click chemistry pair (e.g., an azide and an alkyne, an azide and a dibenzocyclooctyne, or a tetrazine and a transcyclooctene).

23. The method of any one of claims 21 or 22, wherein the capture agent is an antibody or antigen binding fragment thereof that binds the capture label.

24. The method of any one of claims 21-23, wherein nanostructures in the first region, the nanostructures in the second region, and the nanostructures in the third region each comprise a distinct capture agent; and amplicons bound by the first region and amplicons bound by the second region comprise different capture labels.

25. The method of any one of claims 1-24, wherein the one or more nucleic acid molecules each independently comprise (i) a full length transgene, (ii) a fragment of the transgene, or (iii) a full length transgene or fragment thereof comprising a nucleotide insertion, deletion or substitution.

26. The method of any one of claims 1-25, further comprising, prior to step (b), contacting the one or more nucleic acid molecules with a detection agent, wherein binding of the detection agent to the one or more nucleic acid molecules enhances the change in the property of the nanostructures.

27. The method of any one of claims 1-26, wherein the biological sample is a viral lysate and/or a cell lysate.

28. The method of claim 27, wherein the viral lysate is produced from viral particles grown in a bioreactor and/or the cell lysate is produced from cells grown in a bioreactor.

29. The method of any one of claims 1-28, wherein the viral particles are adenoviral particles, adeno-associated viral (AAV) particles, lentiviral particles, baculovirus particles, or engineered viral particles of any one of the foregoing.

30. The method of any one of claims 1-29, wherein the viral particles are adenoviral particles.

31. The method of any one of claims 1-30, wherein the one or more nucleic acid molecules are DNA or RNA molecules.

32. The method of any one of claims 1-31, wherein the one or more nucleic acid molecules are DNA molecules.

33. The method of any one of claims 1-32, wherein the amplicons comprise double stranded nucleic acid.

34. The method of claim 33, wherein a first strand of the double stranded nucleic acid comprises a detection label and the second strand of the double stranded nucleic acid comprises a capture label.

35. The method of any one of claims 1-34, wherein, prior to step (a), the amplicons are produced using a first primer optionally coupled to a detection label and a second primer optionally coupled to a capture label and a thermostable polymerase.

36. The method of claim 35, wherein the first primer hybridizes to a first locus on the one or more nucleic acid molecules and the second primer hybridizes to a second locus on the one or more nucleic acid molecules.

37. The method of claim 36, wherein the first locus is located at a 5' end of a first strand of the one or more nucleic acid molecules and the second locus is located at a 5' end of a second strand of the one or more nucleic acid molecules, wherein the first strand and second strand are complementary.

38. The method of claim 36, wherein the first locus is located at a 5' end of a first strand of the one or more nucleic acid molecules and the second locus is located at an interior region of a second strand of the one or more nucleic acid molecules, wherein the first strand and second strand are complementary.

39. The method of claim 36, wherein the first locus is located at an interior region of a first strand of the one or more nucleic acid molecules and the second locus is located at a 5' end of a second strand of the one or more nucleic acid molecules, wherein the first strand and second strand are complementary.

40. The method of claim 36, wherein the first locus is located at an interior region of a first strand of the one or more nucleic acid molecules and the second locus is located at an interior region of a second strand of the one or more nucleic acid molecules, wherein the first strand and second strand are complementary.

41. The method of any one of claims 35-40, wherein the first primer and the second primer are resistant to exonuclease digestion.

42. The method of claim 41, wherein nucleic acids not containing the first primer or the second primer are digested by exposure to an exonuclease.

43. The method of any one of claims 35-42, wherein the method is performed over two or more replicates, wherein each replicate comprises a distinct first primer and/or second primer, thereby detecting the presence or absence of a plurality of loci within the one or more nucleic acid molecules.

44. The method of any one of claims 1-43, wherein the property of the nanostructures is an optical property.

45. The method of any one of claims 1-44, wherein the sensor comprises a plate defining a plurality of addressable wells, each well comprising a grid disposed therein, wherein the grid comprises the plurality of nanostructures.

46. The method of any one of claims 1-45, wherein each nanostructure comprises a nanoneedle.

47. The method of any one of claims 1-46, wherein the nanostructures are integral with at least one of a planar support, a spherical surface, or a flexible substrate.

48. A method of determining the presence, absence, and/or amount of a plurality of regions within a target nucleic acid, the method comprising:

(a) preparing a plurality of reaction mixtures comprising:

(i) the target nucleic acid;

(ii) a first primer that hybridizes to a first locus on the target nucleic acid, optionally wherein the first primer is coupled to a detection label;

(iii) a second primer that hybridizes to a second locus on the target nucleic acid, optionally wherein the second primer is coupled to a capture label; and

(iv) a thermostable polymerase, wherein each reaction mixture comprises a distinct first primer and/or second primer that hybridize to distinct loci within the target nucleic acid;

(b) performing a plurality of PCR cycles on the plurality of reaction mixtures, thereby generating amplicons comprising a region of the target nucleic acid between the first and second loci; and

(c) detecting the amplicons from each reaction mixture, thereby determining the presence, absence, and/or amount of each region within the target nucleic acid.

49. A method of determining the presence, absence, and/or amount of a plurality of subregions within target complementary nucleic acid sequences, the method comprising:

(a) preparing a first reaction mixture comprising: (i) a first target nucleic acid sequence that is in a forward orientation,

(ii) a first primer that hybridizes to a first locus on the first target nucleic acid sequence,

(iii) a second target nucleic acid sequence that it is in a reverse orientation to the first nucleic acid sequence,

(iv) a second primer that hybridizes to a second locus on the second target nucleic acid sequence, and

(v) a thermostable polymerase;

(b) performing a plurality of PCR cycles on the first reaction mixture to generate a plurality of amplicons, a first amplicon comprising the first primer and a sequence complementary to the first target nucleic acid sequence and a second amplicon comprising a second primer and a sequence complementary to the second nucleic acid sequence;

(c) enzymatically digesting the first target nucleic acid sequence and the second target nucleic acid sequence to produce a second reaction mixture;

(d) adding to the second reaction mixture a third primer that hybridizes to a first locus of the first amplicon, the third primer coupled to a detection label and a fourth primer that hybridizes to a second locus of the second amplicon, the fourth primer coupled to a capture label, and performing a plurality of PCR cycles on the second reaction mixture thereby generating a plurality of double stranded amplicons, wherein a first strand of each double stranded amplicon corresponds a first subregion within the first target nucleic acid sequence and a second strand of each double stranded amplicon corresponds to a second subregion within the second target nucleic acid sequence; and

(e) detecting the plurality of double stranded amplicons, if present, thereby to determine the presence, absence, and/or amount of the first and second subregions within the first and second target nucleic acid sequences.

50. The method of claim 49, wherein, prior to step (e), a double strand amplicon is captured on the surface of a nanostructure via said capture label.

51. The method of claim 49 or 50, wherein a mass amplifier is bound by said detection label.

52. The method of any one of claims 49-51, wherein the detecting step comprises detecting an optical signal.

53. The method of claim 52, wherein the optical signal is light scattering.

54. A sensor for characterizing a nucleic acid molecule in a sample, the sensor comprising:

(i) a first region comprising a first series of nanostructures comprising capture agents attached thereto;

(ii) a nucleic acid complex bound to at least one nanostructure in the first series of nanostructures, the nucleic acid complex comprising a first nucleic acid sequence comprising a capture probe affixed thereto and a second nucleic acid sequence comprising a detection probe affixed thereto, wherein the first nucleic acid sequence is hybridized to the second nucleic acid sequence, and the nucleic acid complex is bound to the at least one nanostructure via at least one of the capture agents affixed thereto, and

(iii) a mass amplifier bound to the detection probe, whereupon binding of the nucleic acid complex and the mass amplifier to the at least one nanostructure produces a detectable signal in the first series of nanostructures.

55. The sensor of claim 54, wherein the first nucleic acid sequence and/or the second nucleic acid sequence is an amplicon derived from the nucleic acid molecule.

56. A sensor for characterizing a nucleic acid molecule in a sample, the sensor comprising:

(a) a first region comprising:

(i) a first series of nanostructures comprising a first series of capture agents attached thereto;

(ii) a first nucleic acid complex bound to at least one nanostructure in the first series of nanostructures, the nucleic acid complex comprising a first nucleic acid sequence comprising a first capture probe affixed thereto and a second nucleic acid sequence comprising a first detection probe affixed thereto, wherein the first nucleic acid sequence is hybridized to the second nucleic acid sequence, and the nucleic acid complex is bound to the at least one nanostructure in the first series of nanostructures via at least one of the capture agents affixed thereto; and

(iii) a first mass amplifier bound to the first detection probe;

(b) optionally, a second region comprising:

(i) a second series of nanostructures comprising a second series of capture agents attached thereto;

(ii) a second nucleic acid complex bound to at least one nanostructure in the second series of nanostructures, the nucleic acid complex comprising a third nucleic acid sequence comprising a second capture probe affixed thereto and a fourth nucleic acid sequence comprising a second detection probe affixed thereto, wherein the third nucleic acid sequence is hybridized to the fourth nucleic acid sequence, and the second nucleic acid complex is bound to the at least one nanostructure in the second series of nanostructures via at least one of the capture agents affixed thereto; and

(iii) a second mass amplifier bound to the second detection probe; and

(c) a third region comprising:

(i) a third series of nanostructures comprising a third series of capture agents attached thereto; and

(ii) a viral particle or viral capsid protein thereof bound to at least one nanostructure in the third series of nanostructures; whereupon binding of the first nucleic acid complex and the first mass amplifier to the at least one nanostructure in the first series of nanostructures produces a detectable signal in the first series of nanostructures, binding of the second nucleic acid complex and the second mass amplifier to the at least one nanostructure in the second series of nanostructures produces a detectable signal in the second series of nanostructures, and/or binding of the viral particle or viral capsid protein thereof to the at least one nanostructure in the third series of nanostructures produces a detectable signal in the third series of nanostructures.

57. The sensor of claim 56, wherein the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, and/or the fourth nucleic acid sequence is an amplicon derived from the nucleic acid molecule.

58. The sensor of any one of claims 54-57, wherein each capture probe is an antibody or antigen binding fragment thereof.

59. The sensor of any one of claims 54-58, wherein each detection probe is an antibody or antigen binding fragment thereof.

60. The sensor of claim 59, wherein each mass amplifier is a ligand bound the antibody or antigen binding fragment thereof.

61. The sensor of any one of claims 54-60, wherein each series of nanostructures are integral with at least one of a planar support, spherical surface, or a flexible substrate.

62. The sensor of any one of claims 54-61, wherein the detectable signal is an optically detectable signal.

63. The sensor of claim 62, wherein the optically detectable signal is light scattering produced by the at least one nanostructure.

64. The sensor of any one of claims 54-63, wherein the nucleic acid molecule is harvested from a viral particle.

65. The sensor of claim 64, wherein the viral particle is an adenoviral particle, an AAV particle, a lentiviral particle, or a baculovirus particle.

66. The sensor of claim 65, wherein the viral particle is an AAV particle.

67. A sensor for characterizing nucleic acid molecules in a sample, the sensor comprising: a first region and a second region, the first region comprising a first series of nanostructures each comprising capture agents attached thereto capable of binding at least a first nucleic acid complex and producing a detectable signal indicative of a concentration of the nucleic acid molecules within a first concentration range, wherein the series of nanostructures are integral with at least one of a planar support, a spherical surface, or a flexible substrate, and the second region comprising a second series of different nanostructures comprising capture agents attached thereto capable of binding a second nucleic acid complex and producing a detectable signal indicative of a concentration of the nucleic acid molecules within a second, different concentration range, wherein the series of nanostructures are integral with at least one of a planar support, a spherical surface, or a flexible substrate, wherein (i) the first and second nucleic acid complexes each comprise a first nucleic acid sequence comprising a capture probe attached thereto and a second nucleic acid sequence comprising a detection probe affixed thereto and the first nucleic acid sequence is hybridized to the second nucleic acid sequence, and each nucleic acid complex is bound to the nanostructure via at least one of the capture agents affixed thereto, and (ii) the sensor is capable of quantifying the amount of nucleic acid molecules in the sample across both the first concentration range and the second concentration range.

68. The sensor of claim 67, wherein the nucleic acid sequences of the first and second complexes are amplicons derived from the nucleic acid molecules.

69. The sensor of claim 67 or 68, wherein the nucleic acid complexes further comprise a mass amplifier bound via each detection probe.

70. The sensor of claim 67 or 68, wherein the concentration of nucleic acid molecules in the sample, if within the first concentration range, is determined from a number of individual nanostructures in the first series that have bound molecules of analyte, and wherein the concentration of nucleic acid molecules in the sample, if within the second concentration range, is determined by analog detection of a substantially uniform change in a detectable property of the nanostructures in the second region as a function of the concentration of nucleic acid molecules in the sample.

71. The sensor of any one of claims 67-70, wherein the first concentration range has a lower detectable value than that of the second concentration range.