SENSING PEPTIDES CROSS-REFERENCE TO RELATED APPLICATION

This is a PCT Application, which claims priority to U.S. Provisional Application No.

61/671,934, filed July 16, 2012.

BACKGROUND OF THE INVENTION

A. FIELD OF THE INVENTION

The present invention relates to compositions, devices, and methods for detecting antigens small molecules, and peptides, including bacterial quorum sensing peptides.

B. DESCRIPTION OF RELATED ART

Monitoring the course of bacterial infection - especially antibiotic resistant strains - is paramount in improving patient outcomes and decreasing communal disease persistence.

Unfortunately, means of monitoring infection progression are laboratory- based and temporally inefficient.

Bacterial infections are generally diagnosed by obtaining specimen samples from patients, extracting bacterium from the specimen samples, culturing the bacterium in the laboratory, and performing bacterial identification via performing genotypic characterization using polymerase chain reaction (PCR) strategies. This general strategy is disadvantageous for a number of reasons. First, obtaining bacteria cells via biopsy from patient specimen can become surgically-invasive. Further, the time necessary to perform the specimen purification, cell culturing and genotypic characterization could take 4-7 days (non-rapid, temporally inefficient). These procedures must also be performed in a laboratory (non-portable, not point- of-care, specialized training a requirement). Other attempts at developing point-of-care strategies for bacterial diagnostics are limited because they require bacterial cell surfaces and/or intracellular constituents for identification - again possibly requiring invasive methods for obtaining such a specimen.

Quorum sensing (QS) bacteria produce and release chemical signal molecules termed autoinducers whose external concentration increase as a function of increasing cell-population density [37]. Equivalently, bacteria detect the accumulation of a minimal threshold stimulatory concentration of these autoinducers and alter gene expression, and therefore behavior, in response to their impingement. Using such signal-response systems, bacteria synchronize particular behaviors on a population wide-scale and thus function as multicellular organisms. For example, increase in concentrations autoinducing peptide (AIP), the QS protein used in Staphylococcus aureus infections, activates a gene-regulated switch which ensures that the entire population switches from low-cell-density to the high-cell-density state. Even further, S. aureus strains are classified on the basis of the sequence of their thiolactone-containing AIPs. At present, four different AIPs and thus four different groups of S. aureus, are known.

Monoclonal antibodies are monospecific antibodies that are equivalent due to the fact that they are made by identical immune cells that are clones of a unique parent cell. Monoclonal antibodies have monovalent affinity, where they bind to the same epitope of the targeted antigen. Alternatively, polyvalent antibodies are obtained from different B cell resources. They are a combination of antibody molecules secreted against a specific antigen, yet identifying a different epitope. Polyvalent antibodies are produced by the inoculation of a suitable mammal (mouse, rabbit, goat) with the antigen. This causes B-lymphocytes to produce IgG immunoglobulins specific for the antigen. Resultant polyvalent IgG is purified from the mammal's serum.

Anti-metatype antibodies are antiimmunoglobulins that specifically recognize an antibody-liganded active site but lack specificity for either the ligand or the unliganded antibody. Polyvalent anti-metatype antibodies will be able to recognize different epitopes of an antibody-liganded complex, yet will not bind the unliganded antibody nor the free antigen/ligand. To obtain the polyvalent antibodies targeted only to the complex, the antibodies are cross-reacted with the appropriate antigen-complex for appropriate purification.

SUMMARY OF THE INVENTION

This disclosure provides compositions for detecting the presence or concentration of a target antigen, said compositions comprising a plurality of nanoparticles. Each nanoparticle comprises a core comprising iron oxide, and a shell comprising at least one plasmon active metal, where an exterior surface of the nanoparticle is functionalized with a plurality of anti- metatype polyvalent antibodies, each independently configured to bind to at least a portion of a complex comprising a monoclonal antibody bound to the target antigen ("monoclonal antibody - target antigen complex"). When a monoclonal antibody - target antigen complex is contacted with the composition, at least some of the plurality of nanoparticles bind to the monoclonal antibody - target antigen complex to form a nanoparticle-complex aggregate. The nanoparticle absorbs electromagnetic radiation at a first peak wavelength, and the nanoparticle-complex aggregate absorbs electromagnetic radiation at a second peak wavelength different from the first wavelength. At least one of the first peak wavelength and the second peak wavelength are in the near-infrared region or infrared region of the electromagnetic spectrum. This disclosure also provides methods of detecting the presence or concentration of a target antigen. The method comprises contacting a composition comprising the monoclonal antibody - target antigen complex with a composition comprising a plurality of nanoparticles. Each nanoparticle comprises a core comprising iron oxide, and a shell comprising at least one plasmon active metal. An exterior surface of the nanoparticle is functionalized with a plurality of anti-metatype polyvalent antibodies, each independently configured to bind to at least a portion of the target antigen-monoclonal antibody complex. When the composition is added to a solution comprising the monoclonal antibody - target antigen complex, at least some of the plurality of nanoparticles bind to the monoclonal antibody - target antigen complex to form a nanoparticle-complex aggregate. The nanoparticle absorbs electromagnetic radiation at a first peak wavelength, and the nanoparticle-complex aggregate absorbs electromagnetic radiation at a second peak wavelength different from the first wavelength, and wherein at least one of the first peak wavelength and the second peak wavelength are in the near-infrared region or infrared region of the electromagnetic spectrum. The method further comprises measuring a change in the absorbance of near-infrared or infrared light at the first peak wavelength, the second peak wavelength, or both.

The disclosure further provides an anti-metatype polyvalent antibody specific for a monoclonal antibody - target antigen complex, wherein the target antigen is a bacterial quorum sensing peptide.

The disclosure further provides a method of detecting a bacterial quorum sensing peptide in a sample, the method comprising contacting the sample with an anti-metatype polyvalent antibody specific for a monoclonal antibody - target antigen complex, wherein the target antigen is a bacterial quorum sensing peptide.

The disclosure further provides a method of diagnosing a bacterial infection, the method comprising detecting the presence of a bacterial quorum sensing peptide in a sample.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 demonstrates various examples of nanoparticle within the scope of this disclosure. (A) demonstrates the various components of an exemplary nanoparticle core comprising a magnetic core (1), an optional subsidiary layer (2), a dielectric layer (3), and a plasmon active metal layer (4). (B) demonstrates various arrangements of antibodies attached to an exemplary nanoparticle core.

FIG. 2 is an illustration of an exemplary use of the nanoparticles as disclosed herein. (A) illustrates addition of a monoclonal antibody is added to a sample comprising the target of interest (ex. quorum sensing peptide) to form an antibody-target complex. (B) illustrates addition of a nanoparticle composition comprising at least one anti-metatype antibody to the antibody-target complex.

FIG. 3 illustrates binding of the antibodies to the target. (A) illustrates a binding entity specific for the target (5), an anti-metatype antibody (6), and the target (7). (B) illustrates binding of the binding entity specific for the target (5) to the target (7) to form a target complex. (C) illustrates binding of the anti-metatype antibody (6) to the target complex.

FIG. 4 illustrates nanoparticles binding to bacterial quorum sensing peptide complexes to form an aggregate.

FIG. 5 illustrates a plurality of aggregates.

FIG. 6 is a plot showing a change in the peak absorbance of nanoparticles caused by aggregation.

FIG. 7 is a graph of a serum titer of a monovalent polyclonal antibody raised against AIP-1 (single epitope peptide) evidencing high-affinity epitopal antibody recognition with specificity.

FIG. 8 is a graph of a serum titer of a monoclonal antibody raised against AIP-1 (single epitope peptide evidencing high-affinity epitopal antibody recognition with specificity.

FIG. 9 is a graph demonstrating a kinetic change in absorbance upon nanoparticle aggregation in the presence of angiopoietin-2 (ang-2) at a concentration 2.2 μg/ml. The graph of the result of this phantom study evidences the nanoparticle aggregation process using an exemplary protein and nanoparticles conjugated with analogous polyclonal antibodies, anti-ang- 2 pAb.

DETAILED DESCRIPTION OF THE INVENTION

This disclosure provides compositions, devices and methods for detecting the presence or concentration of target antigens, small molecules, and peptides such as bacterial quorum sensing peptides.

The compositions, devices and methods may be used, for example, to diagnose and/or monitor the progression of a bacterial infection using easily obtainable, low- volume, patient fluid samples (blood, saliva, sputum, mucus, fecal matter, genital secretions, urine, cerebrospinal fluid, inflamed joint fluid). The concentration of the target antigen, such as a bacterial quorum sensing peptide, may be directly related to other systemic indications, such as the cell mass of a bacteria population during an infection. Using this correlation, the progression of an infection - both before and after treatment - can be monitored, including in a point-of-care, personalized fashion.

These compositions, devices and methods may provide for a rapid (<30 min), single- step, sensitive, solution-phase, antigen detection method based on intermolecular recognition nanoparticulate self-assembly. Briefly, photonically-active, magnetic nanoparticles with a constant peak absorbance are conjugated with anti-metatype polyvalent antibodies against a target antigen-analogous monoclonal antibody complex. Following incubation with a solution containing the target antigen and monoclonal antibodies, the nanoparticles bind with the target antigen thereby causing the nanoparticles to aggregate into self-assembled structures. This 'programmed- agglomeration' may cause the peak absorbance of the particles to change - thus providing a detectable property that is directly correlated to the presence and concentration of the target antigen. In the case of targeting bacterial quorum sensing peptides in solution, detecting the presence and concentration of the protein may allow for the determination of the species, substrain and approximate cell mass of a bacteria population. The nanoparticles used for this method have absorption peaks outside of the biologically- sensitive, visible wavelength range, thus allowing for easy analysis of biological samples (e.g. blood, saliva, sputum, etc.).

The description below, with reference to the figures, provide additional details of the methods, devices and compositions of this disclosure:

1. Nanoparticles

In an aspect, nanoparticles are provided which are adapted to aggregate in the presence of a target molecule, and wherein aggregation is detectable. In an aspect, the nanoparticles are photonically active. In an aspect, the nanoparticles are rendered photonically active by possessing surface materials on the nanoscale which exhibit surface plasmon resonance (SPR). Localized SPRs are collective electron charge oscillations on the surface of metallic nanoprobes which are excited by light and exhibit enhanced near-field amplitude at some resonant wavelength dictated by the nanoparticle compositions. The nanoparticles usually contain metallic outer surfaces of plasmon active materials including, but not limited to, gold, silver, copper and platinum. Further, a number of internal shells of plasmon active metals or dielectric materials may be containined under the outer metallic layer which may also modify the resonant wavelength of the nanoscale material.. Examples of photonically active nanoparticles include, but are not limited to, photonically-active (i.e. gold, silver, platinum, copper) colloid, photonically inactive core and photonically-active shell particles, and multilayered, metallodielectric particles such as nanospheres, magnetonanoshells, matryoshka and multistrata nanoparticles. Preferably, the nanoparticles have a different peak absorbance in a dissociated state versus an agglomerated state. Even more preferably, the peak absorbance of the nanoparticles is a function of the degree of aggregation, such that the peak absorbance mathematically correlates with the degree of aggregation.

In an aspect as illustrated at FIG. 1A, the nanoparticle may comprise four layers: a magnetic core (1); an optional subsidiary layer (2); a dielectric layer (3); and a plasmon active metal layer (4). The magnetic core (1) enables the nanoparticle with super-paramagnetic characteristics for analysis of changes in bulk magnetic properties upon aggregation as well as enabling the separation of nanoparticles, and their bound proteins, from the sample fluid using strong magnets. By way of example and not limitation, the magnetic core may comprise iron oxide e.g., Fe ₂ 0 ₃ or Fe ₃ C"4. The optional subsidiary layer (2) acts as an intermediary and spacer layer assisting the deposition of dielectic material. By way of example and not limitation, the subsidiary layer may be an aminosilane (e.g., APTES, APTMS, APDEMS, APEMS, etc.). The dielectric layer (3) augments the surface plasmon resonance of the outer metallic shell of the nanoparticles thus red shifting the resonance frequency into the near-infrared. By way of example and not limitation, the dielectric layer may be a silicate or silicon oxide (such as Si0 ₂ ). The plasmon active metal layer (4) enables surface plasmon resonance and thus the resultant absorptive nature of the nanoparticles. By way of example and not limitation, the plasmon active metal layer comprises one or more plasmon active metals (e.g., gold, silver, copper, platinum).

In an aspect, nanoparticle aggregation is mediated by binding of the nanoparticle to a target composition. Preferably, binding is mediated by an entity capable of specifically binding to the target composition ("binding entity") provided on the surface of the nanoparticle. In an aspect, the target composition comprises a peptide. As used herein, the term "peptide" embraces any compound comprising a peptide bond linking a pair of amino acids, including for example, oligopeptides, polypeptides, and full-length proteins. In an aspect, the binding entity specifically binds the peptide. In another aspect, the antibody specifically binds a complex comprising the peptide, such as a ligand-receptor complex or an antibody-antigen complex, but does not substantially bind the peptide alone. In an aspect, the surface of the nanoparticles may be functionalized with a polyvalent antibody. Polyvalent antibodies may be utilized (as opposed to monoclonal antibodies) due to their ability to bind to different structures or motifs on the surface of an antigen. This allows a single antigen to simultaneously be agglutinated with more than one polyvalent antibody (and thus more than one nanoparticle). When linked to the nanoparticles, this ability encourages the formation of nanoparticle-complex aggregates, which also may be referred to as "immuno- agglomerates". These nanoparticle-complex aggregates may be configured to absorb electromagnetic radiation at a second peak wavelength that is different from the first peak wavelength, such as a wavelength in the UV, visible, infrared (IR) and near- infrared (NIR) regions of the electromagnetic spectrum.

The polyvalent antibodies may be attached or otherwise conjugated to the surface of the nanoparticles using any of a number of different methods. For example, a heterobifunctional polyethylene glycol (PEG) linker may be used, such as OPSS-PEG-NHS ((ortho- pyridyl)disulfide-PEG-Succinimidyl Ester), where the OPSS sulfur groups may bind strongly to the gold surface, and the NHS groups may form an amide linkage the PEG to amine containing residues on the antibodies, thereby forming an amide linkage. Alternatively or additionally, a streptavidin group may be conjugated to the exterior surface of the nanoparticle (e.g., using a glycin-saline buffer solution of commercially procured streptavidin molecules) thus allowing for functionalization of the surface with biotinylated-polyvalent antibodies. A plethora of other functionalization strategies are readily available to those skilled in the art.

In an aspect, the polyvalent antibody is an anti-metatype polyvalent antibody. Anti- metatype antibodies are anti-immunoglobulins that specifically recognize an antibody-liganded active site but lack specificity for either the ligand or the unliganded antibody. Polyvalent anti- metatype antibodies thus are able to recognize different epitopes of an antibody-liganded complex, yet will not bind to either the unliganded antibody or the free antigen/ligand. Thus, in a further aspect, the anti-metatype polyvalent antibody is capable of binding to a complex comprising the target antigen and a monoclonal antibody bound thereto (referred to hereafter as a "monoclonal antibody - target antigen complex"), but does not bind to either the monoclonal antibody or to the antigen in isolation.

In an aspect, the anti-metatype polyvalent antibody is a polyclonal antibody. To obtain polyclonal antibodies targeted only to the monoclonal antibody - target antigen complex, a suitable mammal (e.g. mouse, rabbit, goat) may be inoculated with a monoclonal antibody - target antigen complex comprising the target antigen and a monoclonal antibody. This causes B-lymphocytes to produce IgG immunoglobulins specific for the antibody-antigen complex, which may then be purified from the mammal's serum. These purified antibodies are then affinity-purified, utilizing common methods such as high performance liquid chromatography, reverse-phase chromotography or bioaffmity chromatography using the monoclonal antibody - target antigen complex, such that only antibodies recognizing the antigen and monoclonal antibody bound together are harvested. The harvested antibodies may be further purified using affinity chromatography to remove antibodies specific for the monoclonal antibody in isolation and the target antigen in isolation.

Other polyvalent molecules such as single-chain fragment variable recombinant antibodies as well as a wealth of other polyvalent materials can be utilized for this methodology as well.

Other affinity molecules include, but are not limited to, peptide-binding peptides, recombinant proteins, ligand receptors and their analogous fragments or recombinant forms which bind their corresponding analytes.

In an aspect as illustrated at FIG. IB, the nanoparticle comprises a first binding entity specific for the target (5) and a second binding entity (6) specific for a complex comprising the target but does not bind the target. In an aspect as illustrated at FIG. 1C, the nanoparticle comprises the first binding entity (5), but not the second binding entity (6). In an aspect as illustrated at FIG. ID, the nanoparticle comprises the second binding entity (6), but not the first binding entity. Preferably, the first binding entity (1) is an antibody specific for the target, or a fragment of such an antibody that retains the ability to bind to the target; and the second binding entity (6) is an anti-metatype antibody specific for a complex comprising the first binding entity bound to the target, or a fragment of such an anti-metatype antibody that retains the ability to bind to the complex.

In another aspect, which also may be illustrated by FIG. IB, the first binding entity (5) is specific for a first portion of a complex comprising the target, while the second binding entity (6) is specific for a second portion of a complex comprising the target but does not bind the target.

In an aspect, the nanoparticle is an FeOx-Si02-Au-antibody nanoparticle. An FeOx- Si02-Au-antibody nanoparticles of this disclosure may include a superparamagnetic iron-oxide core (e.g., Fe ₂ 0 ₃ or Fe ₃ 0 ₄ ), a first shell formed of a dielectric material (e.g., Si0 ₂ , among others) surrounding the core, and a second shell formed of one or more plasmon active metals (PAMs; e.g., gold, silver, copper, platinum) surrounding the first core. Depending on the overall conformation of these nanoparticles, they may be configured to absorb electromagnetic radiation at a first peak wavelength, such as a wavelength in the UV, visible, infrared (IR) and near- infrared (NIR) regions of the electromagnetic spectrum.

In order to provide nanoparticles which have so many functional layers, each shell must be carefully added through controlled fabrication methods. These methods permit the fabrication of extremely thin shells (as small as 1-2 nm to maintain an overall particle diameter less than about lOOnm, such as less than about 90nm, less than about 80nm, less than about 70nm and preferably, less than about 60 nm) while still ensuring magnetic material retention throughout the fabrication process. Generally, the methods include coating a superparamagnetic iron oxide particle (e.g., Fe ₂ 0 ₃ or Fe ₃ C"4) with a first [optional] aminosilane (e.g., APTES, APTMS, APDEMS, APEMS, etc.) to form an aminated core, coating the aminated core with a first shell formed of a dielectric material (e.g., Si0 ₂ , among others) using sonication, coating the first shell with a second aminosilane (e.g., APTES, APTMS, APDEMS, or APEMS, a cylic aminosilane such as N-n-butyl- aza-2,2-dimethoxysilacyclopentane, etc.) to form an aminated first shell, and coating the aminated first shell with a second shell formed of one or more plasmon active metals (e.g., gold, silver, copper, platinum).

2. Methods of using the nanoparticles

In an aspect, the nanoparticles as described herein may be used to detect the presence of an target in a sample. Methods of using the nanoparticles include contacting a composition containing a target antigen with the nanoparticles discussed above. The composition may include, but is not limited to, ex vivo samples from a subject, in vitro culture extractions, etc. Compositions therefore may include non-target biological molecules (e.g., proteins, DNA, lipids, etc.) that are capable of absorbing light in the UV and visible regions of the electromagnetic spectrum.

In an aspect, the method comprises: (a) contacting a nanoparticle as disclosed herein with a sample suspected of comprising the target under conditions sufficient for the binding entity to bind to the target; and (b) detecting aggregation of the nanoparticles, wherein aggregation of the nanoparticles indicates the presence of the target in the sample.

In an aspect, a composition is provided comprising a monoclonal antibody - target antigen complex, and said composition is contacted with nanoparticles comprising an anti- metatype polyvalent antibody as discussed above. The composition may be generated by contacting a sample comprising the target antigen with a monoclonal antibody capable of binding to the target antigen to form the monoclonal antibody - target antigen complex. The monoclonal antibody and the nanoparticles may be added to the sample in any order. For example, the sample may be contacted with the monoclonal antibody either before or after the sample is contacted with the nanoparticles. Alternatively, the sample may be contacted with a composition comprising both the monoclonal antibody and the nanoparticles. In an aspect, the monoclonal antibody is provided as a free antibody. In another aspect, the monoclonal antibody is attached to a nanoparticle. In another aspect, the monoclonal antibody may be bound to or adapted to be bound to a solid phase, such as a bead. By using a solid phase, the monoclonal antibody - target antigen complex may be purified from the sample before detection.

In an aspect, a nanoparticle as illustrated at FIG. ID is provided comprising a binding entity (6), wherein the binding entity is an anti-metatype antibody specific for a complex comprising a monoclonal antibody bound to the target, or a fragment of such an anti-metatype antibody that retains the ability to bind to the complex. In such an aspect, aggregation is mediated by two binding events as illustrated at FIG. 3. First, the target binds to a monoclonal antibody (5) (in free form or optionally bound to a solid phase) to form a complex comprising the antibody and the target (FIG. 3B). Second, the binding entity (6) binds to the complex, thereby causing aggregation (FIG. 3C).

In an aspect, a plurality of first nanoparticles as illustrated at FIG. IB is provided comprising a first binding entity (5), and a second binding entity (6), wherein the first binding entity (5) is specific for the target and a second binding entity (6) is specific for a complex comprising the target but does not bind the target. In such an aspect, aggregation is mediated by two binding events as illustrated at FIG. 3. First, the target (7) binds to the first binding entity (5) to form a complex comprising the target immobilized to a first nanoparticle (FIG. 3B). Second, the second binding entity (6) disposed on a second nanoparticle of the plurality binds to the complex, thereby causing aggregation (FIG. 3C). Preferably, the first binding entity (5) is an antibody specific for the target, or a fragment of such an antibody that retains the ability to bind to the target; and the second binding entity (6) is an anti-metatype antibody specific for a complex comprising the first binding entity bound to the target, or a fragment of such an anti-metatype antibody that retains the ability to bind to the complex.

In an aspect, a first nanoparticle as illustrated at FIG. IB is provided comprising a first binding entity (5), and a second binding entity (6), wherein the first binding entity (5) and the second binding entity (6) are specific for a complex comprising the target but does not bind the target itself. In such an aspect, aggregation is mediated by three binding events. First, a free binding entity binds to the target to form a target-containing complex (element 7 of FIG. 3A). Second, the first binding entity binds to a first portion of the complex (FIG. 3B). Third, the second binding entity binds to a second, different, portion of the complex (FIG. 3C). Preferably, the free binding entity is a monoclonal antibody specific for the target, or a fragment of such an antibody that retains the ability to specifically bind the target; the first binding entity is an anti-metatype antibody specific for a complex comprising the free binding entity bound to the target, or a fragment of such an antibody that retains the ability to bind to the complex; and the second binding entity is an anti-metatype antibody specific for a complex comprising the free binding entity bound to the target, or a fragment of such an anti-metatype antibody that retains the ability to bind to the complex.

In another aspect, a first nanoparticle as illustrated at FIG. 1C is provided comprising the first binding entity (5), and a second nanoparticle as illustrated at FIG. ID is provided comprising the second binding entity (6), wherein the first binding entity specific for the target; and the second binding entity is specific for a complex comprising the first binding entity bound to the target. In such an aspect, aggregation is mediated by two binding events as illustrated at FIG. 3. First, the target binds to the first binding entity to form a complex comprising the target immobilized to a first nanoparticle (FIG. 3B). Second, the second binding entity binds to the complex, thereby causing aggregation (FIG. 3C). Preferably, the first binding entity (5) is an antibody specific for the target, or a fragment of such an antibody that retains the ability to bind to the target; and the second binding entity (6) is an anti-metatype antibody specific for a complex comprising the first binding entity bound to the target, or a fragment of such an anti-metatype antibody that retains the ability to bind to the complex.

In another aspect, a first nanoparticle as illustrated at FIG. 1C is provided comprising the first binding entity (5), and a second nanoparticle as illustrated at FIG. ID is provided comprising the second binding entity (6), wherein the first binding entity (5) and the second binding entity (6) are specific for a complex comprising the target but do not bind the target itself. In such an aspect, aggregation is mediated by three binding events. First, a free binding entity binds to the target to form a target-containing complex (element 7 of FIG. 3A). Second, the first binding entity binds to a first portion of the complex (FIG. 3B). Third, the second binding entity binds to a second, different, portion of the complex (FIG. 3C). Preferably, the free binding entity is a monoclonal antibody specific for the target, or a fragment of such an antibody that retains the ability to specifically bind the target; the first binding entity is an anti- metatype antibody specific for a complex comprising the free binding entity bound to the target, or a fragment of such an antibody that retains the ability to bind to the complex; and the second binding entity is an anti-metatype antibody specific for a complex comprising the free binding entity bound to the target, or a fragment of such an anti-metatype antibody that retains the ability to bind to the complex.

In an aspect, a method is provided wherein: (a) the nanoparticle comprises four layers:

(al) a magnetic core, (a2) an optional subsidiary layer, (a3) a dielectric layer, and (a4) a plasmon active metal layer; and (b) detection of aggregation comprises measuring the change in absorbance over time of the nanoparticles in solution.

As illustrated in FIG. 4 and FIG. 5, binding of the target to the binding entities as described above links the nanoparticles to one another. Preferably, the size of the aggregates is a function of the concentration of the target in the sample. In such an embodiment, the size of the aggregate is a measure of the concentration of the target.

FIG. 8 shows the results of a scanning kinetic experiment showing the aggregation of magnetonanoshells labeled with antibodies against a target analyte in solution over time. Magnetonanoshells were aggregated using the following protocol. The exemplary analyte and antibody pair are recombinant human angiopoietin-2 (R&D Systems, 623-AN) and human angiopoietin-2 antibody (R&D Systems, AF623). 1.7 ml of 10X PBS was added to the -3.7 ml solution of MNS-PEG-Ab. Analyte was diluted to 2.2 μ^πιΐ in 10X PBS. In a spectrophotometer, the absorbance protocol was zeroed by using a blank composed of 10X PBS (1.7 ml), sodium bicarbonate (1.9 ml), and DI H20 (1.8 ml). 864 ml of MNS-PEG-Ab was added to a low volume cuvette and measured for a T=0 absorbance. Absorbance scans across a spectral range of 300-1 lOOnm was measured for 30 minutes every 30 seconds. At T=60 seconds, 36 of analyte was added to the solution. Progressive kinetic decrease was observed in the absorbance measurements over time; such are shown in the figure. Such decrease in absorptivity of the nanoparticles solution is not seen in experiments using expired analyte solutions as a control. This decrease in absorbance represents the aggregation of MNSs and further show the detection of the presence of the analyte. Further, the aforementioned experiments can be repeated and performed using exactly the same prescriptions and methods while using quorum sensing proteins and antibodies raised against these quorum sensing antigen as the corresponding antibodies and analytes, Further, to show that this technique can be used as a correlative method for analyte concentration determination, a serial dilution creating a range of analyte concentrations (across a range of physiologically relevant analyte concentrations) can performed which, upon repeated aggregation reactions, can generate a standard curve in accordance with Beer's law. The formula which represents this curve may be used to determine unknown analyte concentrations in biological media.

3. Anti-Quorum Sensing Antibodies and Anti-Metatype Antibodies

In an aspect, anti-quorum sensing antibodies, and anti-metatype antibodies specific for quorum sensing-antibody complexes are described herein. As shown in the ELISA depicted in FIG. 7, polyclonal antibodies against the AIP-1 quorum sensing peptide have been generated with high epitopal affinities. Polyclonal antibodies were generated by inoculating rabbits with Keyhole limpet hemocyanin (KLH) bound AIP-1 peptide. Harvested antibodies were measured using ELISA. Equivocally, as shown in the ELISA depicted in FIG. 8, monoclonal antibodies against the AIP-1 quorum sensing peptide have been generated with high epitopal affinities. Monoclonal antibodies were generated through mouse immunization, cell fusion and parental clone screening, and monoclonal harvest from cell culture.

4. An exemplary method of detecting a quorum sensing peptide using the nanoparticles

Step l:Fe( -SiC)?-Au-antibodv nanoparticles are formed by surface functionalizing

Fe( -Si02-Au nanoparticles with anti-metatype polyvalent antibodies specific for a target antigen (e.g., a bacterial quorum sensing peptide) complexed with an analogous monoclonal antibody

As shown in FIG. 1, an FeO _x -Si0 ₂ -Au-antibody nanoparticles of this disclosure may include a superparamagnetic iron-oxide core (e.g., Fe ₂ 0 ₃ or Fe ₃ 0 ₄ ), a first shell formed of a dielectric material (e.g., Si0 ₂ , among others) surrounding the core, and a second shell formed of one or more plasmon active metals (PAMs; e.g., gold, silver, copper, platinum) surrounding the first core. Depending on the overall conformation of these nanoparticles, they may be configured to absorb electromagnetic radiation at a first peak wavelength, such as a wavelength in the UV, visible, infrared (IR) and near- infrared (NIR) regions of the electromagnetic spectrum.

In order to provide nanoparticles which have so many functional layers, each shell must be carefully added through controlled fabrication methods. These methods permit the fabrication of extremely thin shells (as small as 1-2 nm to maintain an overall particle diameter less than about lOOnm, such as less than about 90nm, less than about 80nm, less than about 70nm and preferably, less than about 60 nm) while still ensuring magnetic material retention throughout the fabrication process. Generally, the methods include coating a

superparamagnetic iron oxide particle (e.g., Fe ₂ 0 ₃ or Fe ₃ 0 ₄ ) with a first [optional] aminosilane (e.g., APTES, APTMS, APDEMS, APEMS, etc.) to form an aminated core, coating the aminated core with a first shell formed of a dielectric material (e.g., Si0 ₂ , among others) using sonication, coating the first shell with a second aminosilane (e.g., APTES, APTMS, APDEMS, or APEMS, a cylic aminosilane such as N-n-butyl- aza-2,2-dimethoxysilacyclopentane, etc.) to form an aminated first shell, and coating the aminated first shell with a second shell formed of one or more plasmon active metals (e.g., gold, silver, copper, platinum).

In an aspect, the surface of the nanoparticles may be functionalized with polyvalent antibodies. Polyvalent antibodies may be utilized (as opposed to monoclonal antibodies) due to their ability to bind to different structures or motifs on the surface of an antigen. This allows a single antigen to simultaneously be agglutinated with more than one polyvalent antibody (and thus more than one nanoparticle). When linked to the nanoparticles, this ability encourages the formation of nanoparticle-complex aggregates, which also may be referred to as "immuno- agglomerates". These nanoparticle-complex aggregates may be configured to absorb electromagnetic radiation at a second peak wavelength that is different from the first peak wavelength, such as a wavelength in the UV, visible, infrared (IR) and near- infrared (NIR) regions of the electromagnetic spectrum.

The polyvalent antibodies may be attached or otherwise conjugated to the surface of the nanoparticles using any of a number of different methods. For example, a heterobifunctional polyethylene glycol (PEG) linker may be used, such as OPSS-PEG-NHS ((ortho- pyridyl)disulfide-PEG-Succinimidyl Ester), where the OPSS sulfur groups may bind strongly to the gold surface, and the NHS groups may form an amide linkage the PEG to amine containing residues on the antibodies, thereby forming an amide linkage. Alternatively or additionally, a streptavidin group may be conjugated to the exterior surface of the gold shell (e.g., using a glycin-saline buffer solution of commercially procured streptavidin molecules) thus allowing for functionalization of the surface with biotinylated-polyvalent antibodies. A plethora of other functionalization strategies are readily available to those skilled in the art.

In an aspect, the polyvalent antibody is an anti-metatype polyvalent antibody. Anti- metatype antibodies are anti-immunoglobulins that specifically recognize an antibody-liganded active site but lack specificity for either the ligand or the unliganded antibody. Polyvalent anti- metatype antibodies thus are able to recognize different epitopes of an antibody-liganded complex, yet will not bind to either the unliganded antibody or the free antigen/ligand. Thus, in a further aspect, the anti-metatype polyvalent antibody is capable of binding to a complex comprising the target antigen and a monoclonal antibody bound thereto (referred to hereafter as a "monoclonal antibody - target antigen complex"), but does not bind to either the monoclonal antibody or to the antigen in isolation.

In an aspect, the anti-metatype polyvalent antibody is a polyclonal antibody. To obtain polyclonal antibodies targeted only to the monoclonal antibody - target antigen complex, a suitable mammal (e.g. mouse, rabbit, goat) may be inoculated with a monoclonal antibody - target antigen complex comprising the target antigen and a monoclonal antibody. This causes B-lymphocytes to produce IgG immunoglobulins specific for the antibody-antigen complex, which may then be purified from the mammal's serum. These purified antibodies are then affinity-purified, utilizing common methods such as high performance liquid chromatography, reverse-phase chromatography or bioaffmity chromatography using the monoclonal antibody - target antigen complex, such that only antibodies recognizing the antigen and monoclonal antibody bound together are harvested. The harvested antibodies may be further purified using affinity chromatography to remove antibodies specific for the monoclonal antibody in isolation and the target antigen in isolation.

Other polyvalent molecules such as single-chain fragment variable recombinant antibodies as well as a wealth of other polyvalent materials can be utilized for this methodology as well.

By way of example and not limitation, the anti-metatype polyvalent antibody is a polyclonal antibody raised against a monoclonal antibody - target antigen complex, wherein the target antigen is a quorum sensing peptide. Exemplary quorum sensing peptides are described at, for example, Reference [37], which is herein incorporated by reference in its entirety.

Step 2:Fe( -SiC)?-Au-antibody nanoparticles are added to solutions containing target antigens (e.g., quorum sensing peptide).

Methods of using the nanoparticles include contacting a composition containing a target antigen with the nanoparticles discussed above. The composition may include, but is not limited to, ex vivo samples from a subject, in vitro culture extractions, etc. Compositions therefore may include non-target biological molecules (e.g., proteins, DNA, lipids, etc.) that are capable of absorbing light in the UV and visible regions of the electromagnetic spectrum.

In an aspect, a composition is provided comprising a monoclonal antibody - target antigen complex, and said composition is contacted with nanoparticles comprising an anti- metatype polyvalent antibody as discussed above. The composition may be generated by contacting a sample comprising the target antigen with a monoclonal antibody capable of binding to the target antigen to form the monoclonal antibody - target antigen complex. The monoclonal antibody and the nanoparticles may be added to the sample in any order. For example, the sample may be contacted with the monoclonal antibody either before or after the sample is contacted with the nanoparticles. Alternatively, the sample may be contacted with a composition comprising both the monoclonal antibody and the nanoparticles.

In an aspect, the monoclonal antibody is provided as a free antibody.

In another aspect, the monoclonal antibody is attached to a nanoparticle.

In another aspect, the monoclonal antibody may be bound to or adapted to be bound to a solid phase, such as a bead. By using a solid phase, the monoclonal antibody - target antigen complex may be purified from the sample before detection.

Step 3: Antigen complexes are bound to one or more antibodies on Fe( -SiC)?-Au- antibody nanoparticles, thereby causing aggregation of the nanoparticles.

The anti-metatype polyvalent antibodies functionalized on the surfaces of the nanoparticles bind to the monoclonal antibody - target antigen complexes. This allows a single antigen, with only a single binding site, to possess multiple recognition sites, due to the binding of the analogous monoclonal antibody, allowing them to be agglutinated with more than one anti-metatype polyvalent antibody (or more than one nanoparticle), thus causing the nanoparticles to aggregate into colloidal nanoparticle-complex agglomerates though the self- assembly driven multi- bridging of the constructs across the desired peptide complexes.

Step 4: Further agglomeration of the nanoparticles yields microparticles which have a significant difference in extinction characteristics.

After a number of self-assembly driven aggregation events, the nanoparticle-complex aggregates may form colloidal particles growing to diameters larger than 1 μιη. These colloidal particles formed from the nanoparticle-complex aggregates may have significantly different magnetic characteristics (difference between a single superparamagnetic nanoparticle and a cohort of closely associated superparamagnetic nanoparticles) as well as different extinction properties with respect to the absorbance of electromagnetic radiation.

Step 5: The change in extinction characteristics can be directly related to the concordant bacterial quorum protein concentration and thus the bacterial cell load during an infection.

In the presence of a monoclonal antibody - target antigen complex, the nanoparticles agglomerate, thereby causing a change in the extinction characteristics of the nanoparticles. Specifically, non-aggregated nanoparticles may absorb electromagnetic radiation at a first peak wavelength, and nanoparticle-complex aggregates may absorb electromagnetic radiation at a second peak wavelength different from the first wavelength. In some cases, a slight red-shifting of the extinction peak may occur upon aggregation. Monitoring the change in absorption at either the first or second wavelengths may allow for the detection of the presence or concentration of the target antigen (e.g., via the use of a standard curve).

Moreover, a change in the magnetic properties of the solution also may indicate the formation of immuno-agglomerates and thus permit the detection of target antigens as well. Superparamagnetic nanoparticles, such as magnetonanoshells, possess differing magneto- relaxometric properties when they are in dispersed and aggregated states.

In order to make sure that non-target biological molecules do not interfere with the detection of changes in either the first or second wavelengths, the peak absorbance of either the non-aggregated nanoparticles, the nanoparticle-complex aggregates, or both, may be in the IR or NIR regions rather than the UV-visible regions of the electromagnetic spectrum. Any absorption-based assays utilizing UV and visible regions necessarily would be inhibited by the background absorbance of non-target biological components.

A device may be used in conjunction with the compositions disclosed herein. For example, the device may include reader and a cartridge or cassette. The reader may include a spectrophotometer and a receiver for receiving the cartridge or cassette. The cartridge or cassette may include a sample reservoir loaded with the compositions disclosed herein, and having a volume for receiving a sample that potentially comprises the target antigen. In some embodiments, the device may function as a cassette reader for One-use' cartridges. Different cartridges may be loaded with different compositions configured to detect the presence or concentration of different target antigens, such as different bacterial quorum sensing peptides associated with different bacteria strains. The devices and methods disclosed herein may be used to analyze samples from a patient several times a day, so as to monitor the course or duration of an infection.

Advantages

Overall, this disclosure provides compositions, devices and methods that simultaneously offer five major technical advantages other technologies:

1. When used to detect bacterial quorum sensing peptides, the compositions, devices and methods do not require the presence of actual bacterium for diagnosis;

2. The devices and compositions are portable, thus allowing for use at the point-of-care;

3. The methods may be performed very quickly and efficiently without the need for culturing samples in a lab, or conducting time-consuming PCR experiments; 4. The devices, compositions and methods permit for the use of crude, unpurified biological samples taken from a patient or subject;

5. The compositions, devices and methods are easy to use, thus obviating the need for extensive training;

The methods and apparatus disclosure herein are not limited in their applications to the details of construction and the arrangement of components described herein. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also it is to be understood that the phraseology and terminology used herein is for the purpose of description only, and should not be regarded as limiting. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures, are not meant to be construed to indicate any specific structures, or any particular order or configuration to such structures. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the invention.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10%> to 30%>, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

Further, no admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinency of any of the documents cited herein. Reference

s

The following references are herein incorporated by reference in their entireties for all purposes:

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