WO2017138969A1 - Compositions and related methods for quantitative detection of antigens using glucometer readout - Google Patents

Abstract

This disclosure provides compositions and related kits, devices, systems, and methods that incorporate antigen detection compositions comprising an antigen-binding molecule tethered to an artificial vesicle. The antigen-binding molecule selectively binds to an antigen of interest and the artificial vesicle encapsulates glucose.

Description

COMPOSITIONS AND RELATED METHODS FOR QUANTITATIVE DETECTION OF ANTIGENS USING GLUCOMETER READOUT

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Provisional Application

No. 62/294,903, filed February 12, 2016, and U.S. Provisional Application No. 62/310,558, filed March 18, 2016, each of which is incorporated by reference herein in its entirety.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under R21 OHO 10768 awarded by Centers for Disease Control and Prevention/National Institute for Occupational Safety and Health (CDC/NIOSH). The Government has certain rights in the invention.

BACKGROUND

Despite the high sensitivity of well-developed methods, such as enzyme-linked immunosorbent assay (ELISA), spectroscopy, electrochemistry, and chromatography, these approaches have serious limitations, including high cost and requiring complicated instrumentation and highly trained personnel in laboratories. On-site, or point of care (POC) detection systems, can be highly beneficial alternatives for use at home or in the field for personal healthcare, disease diagnostics, and commercial applications because of potential simplicity, accuracy, and low cost. Although different types of immunosensors have been developed to detect antigens of interest, including disease-related proteins and biomarkers, only a limited number of products, such as personal glucose meters (GM) test strips, are commercially available to the public. To expand the application of existing GM technologies, several efforts have been made to modify the GM approach to enable measurement of targets beyond glucose. For example, glucose meters and functional DNA sensors have been modified for a variety of non-glucose analytical targets, including cocaine, adenosine, interferon-γ and uranium. This approach typically incorporates an invertase enzyme by releasing it from a functional DNA conjugate to generate the detectable signal. This same concept has been further applied to detect prostate-specific antigen (PSA) and DNA. Other efforts have focused on replacing the enzyme inside test strips of GM with other enzymes to quantify cholesterol, lactate, alcohol and ornithine instead of glucose. However, as with most handheld devices, the sensitivity of the assays was lacking, and the signal amplification had to proceed without the use of any laboratory-based devices.

To address sensitivity problems, several technologies use nanomaterials in combination with electrochemical devices as signal amplifiers. These nanomaterials include gold nanoparticles, quantum dots, magnetic nanoparticles, silica nanoparticles and carbon nanomaterials, which are used as carriers or tags for signal enhancement. However, such additional nanomaterial reagents introduce significant reagent cost and operational complication in the preparation of the sensory platform. For example, the releasing step from the nano-tags tends to involve strong acid, which may have a detrimental effect on functional biological components, such as enzymes.

Accordingly, despite the advances in the art of diagnostic and antigen detection technologies, a need remains for a simple, inexpensive platform that can accurately detect and quantify any antigen of interest. The present disclosure addresses this and related needs.

SUMMARY

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In one aspect, the disclosure provides a composition comprising an antigen binding molecule tethered to an artificial vesicle. The antigen binding molecule selectively binds to an antigen of interest, and the artificial vesicle encapsulates glucose.

In some embodiments, the antigen binding molecule is an antibody, antibody-like molecule, lectin, receptor, aptamer, or a functional antigen binding domain thereof. In some embodiments, the antigen-binding molecule comprises an antigen-binding fragment of an antibody or antibody like molecule. In some embodiments, the antibody-like molecule is a single chain antibody, a bispecific antibody, an Fab fragment, or an F(ab)2 fragment. In some embodiments, the single-chain antibody is a single chain variable fragment (scFv), single-chain Fab fragment (scFab), VHH fragment, VNAR, or nanobody. In some embodiments, the artificial vesicle is a liposome. In some embodiments, the artificial vesicle is a micelle. In some embodiments, the artificial vesicle is covalently tethered to the antigen binding molecule. In some embodiments, the artificial vesicle is non-covalently tethered to the antigen binding molecule. In some embodiments, the artificial vesicle encapsulates glucose at a concentration of at least about 0. 1 mg/mL. In some embodiments, the liposome is at least about 100 nm in diameter. In some embodiments, the interior volume of the liposome is at least about 4.00 x lO^"13 nL.

In another aspect, the disclosure provides a method for detecting an antigen of interest. The method comprises contacting a sample to the composition described herein under conditions sufficient to permit the selective binding of the antigen-binding molecule in the composition to the antigen of interest; lysing the artificial vesicle to release the glucose; and detecting the presence of the glucose, thereby indicating the presence of the antigen of interest.

In some embodiments, the method also comprises immobilizing the antigen of interest. In some embodiments, the method further comprises removing unbound composition prior to lysing the artificial vesicle. In some embodiments, the step of detecting the presence of the glucose comprises using a glucose meter. In some embodiments, detecting the presence of the glucose comprises quantifying the amount or concentration of the glucose. In some embodiments, the amount or concentration of glucose detected indicates a quantity of the antigen of interest. In some embodiments, the sample is contacted to the composition in solution. In some embodiments, the sample is contacted to the composition on a lateral flow strip. In some embodiments, the composition, i.e., the described antigen binding molecule, binds to the antigen of interest indirectly by binding to a primary antigen binding molecule that selectively binds the antigen of interest.

In another aspect, the disclosure provides a device for detecting and/or quantifying an antigen of interest in a sample, comprising the composition described herein. In some embodiments, the device further comprises a capture reagent that specifically binds to the antigen of interest in a manner that is non-competitive with antigen binding molecule. In some embodiments, the capture reagent is immobilized or configured to be immobilized on a surface. In some embodiments, the capture reagent is tethered to a magnetic particle. In some embodiments, the device comprises a lateral flow strip. In some embodiments, the device further comprises a primary antigen binding reagent that specifically binds directly to the antigen of interest and wherein the described composition, i.e., the antigen binding reagent including the glucose encapsulating vesicle, binds the antigen of interest indirectly as a secondary detection reagent that specifically binds the primary antigen binding reagent.

In another aspect, the disclosure provides a kit for detecting and/or quantifying an antigen of interest in a sample, comprising the composition described herein. In some embodiments, the kit further comprises a capture reagent that specifically binds to the antigen of interest in a manner that is non-competitive with antigen binding molecule. In some embodiments, the capture reagent is immobilized or configured to be immobilized on a surface. In some embodiments, the capture reagent is tethered to a magnetic particle. In some embodiments, the kit further comprises a magnet. In some embodiments, the kit further comprises a glucose meter. In some embodiments, the kit further comprises a primary antigen binding reagent that specifically binds the antigen of interest and wherein the described composition, i.e., the antigen binding reagent including the glucose encapsulating vesicle, binds the antigen of interest indirectly as a secondary detection reagent that specifically binds the primary antigen binding reagent.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIGURE 1 is a schematic representation of an exemplary solution-based embodiment of the present invention in which the measurement principle is based on immuno-recognition together with a portable mini glucose meter (GM).

FIGURE 2A is a transmission electron miscroscopy (TEM) image of a representative glucose encapsulating liposome.

FIGURE 2B graphically illustrates the diameter distribution of glucose encapsulating liposome determined by dynamic light scattering measurements.

FIGURES 3A-3D graphically illustrate the XPS full spectra of Ab2-GEL (3 A) and high-resolution spectra of Nls (3B), P2p (3C) and S2p (3D).

FIGURE 4A graphically illustrates the QCM measurements during the immuno- recognition. The secondary recognition was Ab₂-GEL (a) and Ab₂ (b), respectively.

FIGURE 4B graphically illustrates the optimization of incubation time for the first (a) and secondary (b) immunoreaction, respectively. FIGURE 4C graphically illustrates the effect of the ratio of GEL/phospho- p53¹⁵Ab₂ on the detected glucose response.

FIGURE 5A graphically illustrates the calibration curve for the detection of phospho-p53¹⁵ using a personal glucose meter.

FIGURE 5B graphically illustrates glucose responses under different interfering agents.

FIGURE 6 schematically illustrates an exemplary strip-based embodiment of the present disclosure where the detection process is implemented on test trip device linked with a personal GM.

FIGURE 7A is a scanning electron miscroscopy (SEM) image of glucose encapsulating liposomes nanoparticles.

FIGURE 7B graphically illustrates an UPLC measurement of hollow liposomes (a), glucose encapsulating liposomes (b) and 0.25g/mL glucose (c).

FIGURE 8 A graphically illustrates the effect of GEL/p53¹⁵ Ab₂ ratio on GM readout.

FIGURE 8B graphically illustrates the effect of flow time of the sample along with the test strip on GM readout.

FIGURE 8C graphically illustrates the effect of the BSA concentration on GM responses of phospho-p53¹⁵ samples (dark bars) and control (light bars). 1.0 ng/mL phospho-p53¹⁵ was used.

FIGURE 9 graphically illustrates the relationship between GM responses and the concentration of phospho-p53¹⁵.

DETAILED DESCRIPTION

Compositions, devices, kits, systems, and related methods are provided to enable the detection and/or quantification of any antigen of interest that leverages existing, off-the-shelf glucose meters to detect glucose as an indirect antigen signal amplifier. The disclosure is based on the inventors' development and characterization of reagents that combine an antigen binding molecule with encapsulated glucose. Generally, upon binding to the antigen of interest, the encapsulated glucose can be released into the environment and detected and/or quantified with existing glucose detection technology. The inventors established that the amount of antigen present is proportional to the resulting glucose level, thus permitting use of the glucose level as a proxy for antigen detection and/or quantification. Because relatively high levels of glucose can be incorporated into the antigen detection reagents, the glucose is useful to amplify the detectable signal. The described reagents can be incorporated into a variety of detection formats, assays, and devices and readily customized for any specific antigen of interest.

Accordingly, in one aspect, a composition is provided. The composition comprises an antigen-binding molecule tethered to an artificial vesicle. The antigen-binding molecule selectively binds to an antigen of interest. Additionally, the artificial vesicle encapsulates glucose. The composition is useful as a detection reagent that can amplify the signal indicating presence and quantity of the antigen of interest by providing a quantum of detectable glucose only after binding to the antigen of interest. Thus, in the discussion below, the composition is also referred to as "detection reagent". Antigen-binding molecule

As used herein, the term "antigen" refers to any particular molecule or moiety of interest for which detection and/or quantification is desired without limitation except that the antigen must be capable of being specifically bound by a binding molecule, as described below. The antigen can be a biomarker for a condition, disease, or presence of an infectious agent. As a non-limiting example, the antigen can be a cell-surface receptor or excreted protein that is characteristic of a transformed cell or pathogen. In another example, the antigen can be protein that is an allelic variant, in phosphorylated form, or in a misfolded state as compared to a reference protein. In other non-limiting embodiments, the antigen can be an environmental factor, such as a contaminant or allergen.

The antigen-binding molecule of the present disclosure can bind the antigen of interest (i.e., target analyte) directly, as illustrated in FIGURES 1 and 6. However, the present disclosure also encompasses embodiments where the antigen-binding molecule that comprises the encapsulated glucose binds to the antigen of interest indirectly, for example, as a "secondary" detection reagent. In context of a secondary detection reagent, the antigen-binding molecule can selectively bind to any component of a primary antigen detection reagent, such as the Fc domain of a primary antibody, where the primary antibody selectively binds to the target analyte. In this case, the secondary detection reagent can be used in, for example, sandwich immune-assays according to known techniques and assay formats. Thus, for such a secondary detection reagent the antigen- binding aspect refers to the binding of a primary detection reagent as the specific antigen, whereas the primary detection reagent specifically binds to the antigen of interest (i.e., target analyte). As used herein, the term "antigen-binding molecule" refers to any molecule having an ability to bind to a specific molecule (e.g., antigen of interest or an aspect of a primary antigen detection reagent) with a specific affinity (i.e., detectable over background). Exemplary categories of antigen-binding molecules that can be used in the context of the present invention include antibodies, antibody derivatives (also referred to as "antibody-like molecules"), functional antigen-binding portions of antibodies or antibody-like molecules, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, functional antigen-binding portions of proteins that comprise a ligand-binding portion of a receptor that specifically binds a particular antigen, ligands themselves (or moieties that incorporate a ligand), antigen-binding scaffolds (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, and other scaffolds based on naturally occurring repeat proteins, etc., [see, e.g., Boersma and Pluckthun, Curr. Opin. Biotechnol. 22:849-857, 2011, and references cited therein, incorporated herein by reference]), aptamers or antigen binding portions thereof, and nucleic acid molecules with sequences sufficiently complementary to a target "antigen" nucleic acid sequence so as to promote hybridization.

In some embodiments, the affinity reagent is an antibody. As used herein, the term "antibody" encompasses antibodies and antibody fragments thereof, derived from any antibody -producing mammal (e.g., mouse, rat, rabbit, and primate including human), that specifically bind to an antigen of interest. Exemplary antibodies include polyclonal, monoclonal and recombinant antibodies; multispecific antibodies (e.g., bispecific antibodies); humanized antibodies; murine antibodies; chimeric, mouse-human, mouse- primate, primate-human monoclonal antibodies; and anti-idiotype antibodies. The antigen-binding molecule can be any intact antibody molecule or fragment thereof (e.g., with a functional antigen-binding domain).

An antibody fragment is a portion derived from or related to a full-length antibody, preferably including the complementarity-determining regions (CDRs), antigen binding regions, or variable regions thereof. Illustrative examples of antibody fragments useful in the present disclosure include Fab, Fab', F(ab)2, F(ab')2 and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments, and the like. A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide can further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. Antibody fragments can be produced recombinantly, or through enzymatic digestion.

Antibodies can be further modified to suit various uses. For example, a "chimeric antibody" is a recombinant protein that contains the variable domains and complementarity-determining regions (CDRs) derived from a non-human species (e.g., rodent) antibody, while the remainder of the antibody molecule is derived from a human antibody. A "humanized antibody" is a chimeric antibody that comprises a minimal sequence that conforms to specific complementarity-determining regions derived from non-human immunoglobulin that is transplanted into a human antibody framework. Humanized antibodies are typically recombinant proteins in which only the antibody complementarity-determining regions (CDRs) are of non-human origin.

Production of antibodies can be accomplished using any technique commonly known in the art. For example, the production of a polyclonal antibody can be accomplished by administering an immunogen containing the antigen of interest to an antibody-producing animal. For example, the antigen of interest (also referred to as "target antigen") can be administered to a mammal (e.g., a rat, a mouse, a rabbit, a chicken, cattle, a monkey, a pig, a horse, a sheep, a goat, a dog, a cat, a guinea pig, a hamster) or a bird (e.g., a chicken) so as to induce production of a serum containing an antigen-specific polyclonal antibody. The target antigen can be administered in combination with other components known to facilitate induction of a B-cell response, such as any appropriate adjuvant known in the art. Furthermore, the polyclonal antibody reagent can be further processed to remove or subtract any antibody members that have unacceptable affinity for antigens that are not the antigen of interest. The resulting polyclonal antibody reagent will exhibit enhanced specificity for the antigen of interest and are useful for detection and quantification purposes. Many approaches for adsorption of polyclonal antibody reagents to reduce cross-reactivity exist, are familiar to persons of ordinary skill in the art, and are encompassed by the present disclosure.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,

2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas

563-681 (Elsevier, N.Y., 1981), incorporated herein by reference in their entireties. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art.

Antibody fragments that recognize specific epitopes can be generated by any technique known to those of skill in the art. For example, Fab and F(ab')₂ fragments of the invention can be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')₂ fragments). F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain. Further, the antibodies of the present invention can also be generated using various phage display methods known in the art.

As used herein, the term "aptamer" refers to oligonucleic or peptide molecules that can bind to specific antigens of interest. Nucleic acid aptamers usually are short strands of oligonucleotides that exhibit specific binding properties. They are typically produced through several rounds of in vitro selection or systematic evolution by exponential enrichment protocols to select for the best binding properties, including avidity and selectivity. One type of useful nucleic acid aptamers are thioaptamers, in which some or all of the non-bridging oxygen atoms of phophodiester bonds have been replaced with sulfur atoms, which increases binding energies with proteins and slows degradation caused by nuclease enzymes. In some embodiments, nucleic acid aptamers contain modified bases that possess altered side-chains that can facilitate the aptamer/target binding.

Peptide aptamers are protein molecules that often contain a peptide loop attached at both ends to a protamersein scaffold. The loop typically has between 10 and 20 amino acids long, and the scaffold is typically any protein that is soluble and compact. One example of the protein scaffold is Thioredoxin-A, wherein the loop structure can be inserted within the reducing active site. Peptide aptamers can be generated/selected from various types of libraries, such as phage display, mRNA display, ribosome display, bacterial display and yeast display libraries. In some embodiments, the antigen-binding molecule is a receptor molecule or comprises a binding domain of a receptor molecule. The receptor molecule can be any receptor known that can specifically bind the antigen of interest as the ligand. As a non-limiting example, the antigen-binding domain can be a lectin molecule. In another embodiment, the antigen-binding molecule is or contains a protein binding domain that enables the detection of the target protein as the antigen of interest. In yet another embodiment, the antigen-binding molecule can be or comprise a ligand or portion of a ligand that is specific for a receptor or a binding domain of a protein, which receptor or a binding domain of a protein would then serve as the antigen of interest.

As used herein, the term "selectively binds" refers to the ability of the antigen- binding molecule to bind to the antigen of interest, without significant binding to other molecules, under standard conditions known in the art. The antigen-binding molecule can bind to other peptides, polypeptides, or proteins, but with lower affinity as determined by, e.g., immunoassays, BIAcore, or other assays known in the art. However, antigen-binding molecule preferably does not cross-react with other antigens.

Artificial vesicle

The composition provided herein comprises an artificial vesicle. The term "vesicle" generally refers to a closed structure formed by amphiphilic molecules and contains a solvent, which is often water or an aqueous solution. The amphiphilic molecules comprise a hydrophilic region and at least one hydrophobic region, such as a steroid group or at least one hydrophobic tail. The hydrophobic tails are typically hydrocarbon chains of variable length (e.g., about 14-22 carbon) and can have varying degrees of saturation. Illustrative, non-limiting amphiphilic molecules are phospholipids. Exemplary phospholipids include phosphatidylcholine, distearoyl phosphatidylcholine (DSPC), hydrogenated soybean phospholipids (HSPC), and polyethyleneglycol-distearoyl phosphoethanolamine (PEG-DSPE). Other phospholipids can also be used in preparing the vesicles of the invention, including dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC) and dioleylphosphatidyl ethanolamine (DOPE), sphingoglycolipid and glyceroglycolipid. These phospholipids are used for making the vesicle, alone or in combination of two or more or in combination with a lipid derivative where a non-polar substance such as cholesterol or a water soluble polymer such as polyethylene glycol has been bound to the lipid. A wide variety of amphiphilic molecules are routinely used to generate artificial vesicles to predictably obtain desired vesicle characteristics.

Vesicles of the present disclosure encompass liposomes, which have one or more bilayers of amphiphilic molecules. Liposomes with a single lipid bilayer are often called unilamellar liposomes. These range from small (often termed "small unilamellar vesicles" or SUV) to large (often termed "small unilamellar vesicles" or LUV) to giant (often termed "giant unilamellar vesicles" or GUV). However, liposomes can also be multilamellar, which are often termed "large multilamellar vesicles" or MLV. The MLV have several bilayers of amphiphilic molecules, with each layer being comprised of the same or different combinations of amphiphilic molecules. Additionally, the liposomes can be multivesicular vesicles (MVV), in which several non-concentric vesicles are encapsulated within a single outer layer. The layers are typically separated by the aqueous solvent in which they were prepared. Methods for preparing liposomes are well-known in the art and can be tailored to a variety of preferred parameters, including preferred size; the design of uni- or multi-lamellar structure; the physicochemical characteristics of material (e.g., glucose) to be encompassed, i.e. trapped, within the interior space of the liposome; the concentration of the material (e.g., glucose) to be encompassed within the interior space; the nature of the medium for the assay in which the final liposome will be employed; the intended stability and shelf-life of the liposomes; and the like. Generally described, the amphiphilic molecules can be dissolved in a suitable solvent to form a thin film. The initial solvent is replaced by with an aqueous solvent and disrupting force is applied to disperse the amphiphilic molecules. The disrupting force can be sonification, homogenization, freeze and thaw cycling, dialyzing away a detergent solution from lipids, or other known methods. As indicated above, characteristics such as size of the liposomes, lamellar structure, and the like, can be controlled using a variety of known techniques including the duration of sonication. See Kirby, C.J. and Gregoriadis, G., "A simple procedure for preparing liposomes capable of high encapsulation efficiency under mild conditions," Liposome Technology, CRC Press, Boca Raton, FL, 1984, 1, pp 19-27, incorporated herein by reference in its entirety. A specific example of a reversed-phase evaporation method for forming glucose encapsulating liposomes is described in more detail below and in Ho, J. A., Hsu, H.W., Anal. Chem. 75, 4330-4334, 2003, and Szoka Jr, F., Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA 75, 4194-4198, 1978, each incorporated herein by reference in its entirety.

Vesicles of the present disclosure also encompass micelle particles. Micelles are particles with the amphiphilic molecules arranged in a unilayer (as opposed to bilayer) structure. Micelles can comprise surfactant molecules and/or amphiphilic block copolymers and, like liposomes, can be readily constructed to encapsulate materials (e.g., glucose. See, e.g., U.S. Patent Pub. No. 2007/0122440, incorporated herein by reference in its entirety.

The vesicles of the present disclosure can range from as small as about 2 nm to about 3 μΜ in diameter. For example, micelles can range from about 2 nm to about 20 nm or more in diameter. Liposomes are typically larger than micelles and can range from about 20 nm to about 100 nm (a range typical for SUV), about 100 nm to about 400 nm (a range typical for LUV), about 1 μιη and larger (a range typical for GUV), or about 200 nm to about 3 μιη (a range typical for MLV and MVV). Thus, in some embodiments, the artificial vesicles (e.g., liposomes) have a diameter of about 2 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 50 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1 μπι, 1.5 μπι, 2 μπι, 2.5 μπι, or 3 μπι, or any range therein. As used herein, the term about indicates a variance of 10% of the reference value. Additionally, it is noted that when considering a plurality of the described composition, such as incorporated in an assay reagent within a kit or assay system/device, the multiple iterations of the described composition need not consistently have a specific an common diameter, but instead can encompass a range of diameters. In any event, the desired diameter of liposome can be obtained by optimization of the assembly process or size extrusion through filters with preset pore size.

The interior volume of the vesicles (e.g., liposomes) is related to the particular diameter of the vesicle structure and the nature of the amphiphilic molecules creating the membrane. For example, the liposomes described in more detail below exhibited a rather narrow diameter range around 176 nm and a lipid bilayer thickness of about 4 nm, which produced an average volume of 2.54 x 10^"12 μυ It will be appreciated that the internal volume of the vesicles can be readily optimized to a preferred value based on the obtained size of the vesicles and the thickness of the obtained bilayer, which are parameters that are readily controlled. Accordingly, the volume of the internal space of the vesicles (e.g., liposomes) can range from about 4.07χ10^"13μΙ- to about 3.15X10^_1 L, which generally correspond with liposomes having diameters of from about 100 nm to about 400 nm, respectively.

Components of vesicles (e.g., liposomes) can include cholesterol, surfactants, HSPC, DSPE, and the like, as are well-known in the art. As is well understood, vesicle components include those that are typically are amphiphilic (such as phospholipids) and have components such as cholesterol, which can contribute the permeability, stability and/or performance of the vesicle. These are often useful to optimize the stability and/or susceptibility of the vesicles to lysis, as is well-understood in the art.

The disclosed artificial vesicles encapsulate glucose. This is generally accomplished by including glucose of a desired concentration in the solution as the amphiphilic molecules are dispersed and aggregated into the vesicular structure. Typically, the solution of formed vesicles is then filtered, washed, or otherwise separated from all un-encapsulated glucose from the liposome collection. The concentration of glucose in the solution and the volume of the interior space of the vesicle will determine the amount of glucose present in the formed vesicles. For example, as described in more detail below, a 5 mg/ml glucose solution was used to generate liposomes with an average volume of 2.54 x lO^-12 \L, resulting in an estimated ~4 x l0⁵ encapsulated glucose molecules. It will be appreciated that the amount of encapsulated can be readily optimized by altering the initial concentration of glucose in the formation step and/or assembling vesicles with larger or smaller interior spaces. For applications where an antigen of interest is expected to be rare, higher concentrations of glucose may be desired so as to provide for a significant amplification of detectable signal. In contrast, if the antigen of interest is expected to be rather abundant, the amount of glucose does not need to be as high. Accordingly, the disclosed composition encompasses embodiments wherein a vesicle encapsulates at least about 0.1 mg/mL glucose up to about 800 mg/mL. In terms of absolute quantity of glucose, the disclosed composition encompasses embodiments wherein a vesicle encapsulates from about 1 x 10⁴ to about 1 x 10⁸ glucose molecules.

In some embodiments, the vesicle also encapsulates glucose oxidase.

Tether

As described above, the artificial vesicle is tethered to the antigen-binding molecule. The antigen-binding molecule can be tethered to the vesicle using any acceptable method or structure known in the art. For example, the tether can comprise a covalent bond between the antigen-binding molecule and any component of the vesicle. Alternatively, the tether can comprise a non-covalent bond between the antigen-binding molecule and any component of the vesicle.

A covalent tether can be accomplished by providing the antigen-binding molecule with a domain that can integrate into the membrane structure of the vesicle. The vesicular domain can be incorporated during the vesicle assembly process, which integrates with the hydrophobic moieties of the amphiphilic molecules within the vesicle membrane, thus expressing the antigen-binding domain of the antigen-binding molecule on the exterior of the formed vesicle. Alternatively, the vesicular domain of the antigen binding molecule can be inserted into a preformed vesicle. See, e.g., Allen T.M., et al. Cell. Mol. Biol. Lett. 7(2):218-219, 2002, incorporated herein by reference in its entirety. By virtue of the vesicular domain and the antigen-binding domain of the composition being covalently linked, and the vesicular domain being integrated into the vesicle membrane so as to be considered part of the vesicle, the antigen-binding molecule is considered to be covalently tethered to the vesicle.

Alternatively, the vesicle can be preformed with a surface moiety that can be functionalized for subsequent covalent binding of the antigen binding molecule. For example, techniques such as amine modification, carbohydrate modification, and disulfide modification can be utilized to functionalize the surface of the vesicle for covalent attachment of the antigen-binding molecule. For example, one of the most widely used approaches to attach antibodies to liposomes is the reaction of sulfhydryl groups with maleimide functional groups on the liposome surface.

In other embodiments, the antigen-binding molecule is non-covalently tethered to the vesicle. For example, as is well-known in the art, biotin forms non-covalent bonds with streptavidin and avidin, with high specificity and affinity (Kd of 10-14 mol/1 to 10-15 mol/1). Thus, as in the specific example described in more detail below, amphiphilic molecules incorporating biotin, such as DSPE-biotin, can be included in the formed vesicles, and avidin or streptavidin-containing antigen binding molecules can be contacted thereafter to form a noncovalent tether between the vesicle and the antigen- binding molecule. However, it will be appreciated that the arrangement of the biotin and avidin/streptavidin moieties can be switched between the vesicle and antigen-binding molecule to achieve the same noncovalent tethering. Methods

In another aspect, methods of using the described composition are provided. The methods use the described composition as a detection reagent to enable the detection of any antigen of interest in a sample or environment. As will be appreciated, the methods can be performed in a variety of formats optimized for suitable application. In one embodiment, the method comprises contacting a sample to the composition described herein under conditions sufficient to permit the selective binding of the antigen-binding molecule of the composition to the antigen of interest present in the sample. In some embodiments, the unbound composition is removed. The artificial vesicle in the bound composition is lysed to release the encapsulated glucose. The presence of glucose detected in the method is indicative of the presence of antigen of interest. In some embodiment, the released glucose is quantified as an amount or concentration, which value is indicative of the level of antigen of interest.

In some embodiments, the antigen of interest is immobilized. The immobilization can occur before or after the contacting step with the detection reagent described above. In some embodiments, components of the sample are immobilized directly to a suitable solid substrate. In some embodiments, a "capture" reagent can be used that binds to the antigen of interest in a manner that is not competitive with the antigen-binding molecule of the above-described composition, which serves as the "detection reagent". The capture reagent can be any appropriate reagent, such as an antibody, antibody-like molecule, aptamer, receptor, or antigen binding fragments thereof, as described above. The capture reagent can be attached to a surface, such as to a solid bead or nanoparticle, a solid substrate, and/or a surface of a well. Once bound to the antigen of interest, the antigen of interest can be effectively immobilized. In an exemplary embodiment, as described in more detail below, the capture reagent is attached to a magnetic particle. Once bound to the antigen of interest, a magnetic field can be applied to immobilize the "captured" antigen to a location in a tube or on a lateral flow strip.

Once the capture reagent-antigen-detection reagent complex is formed, the immobilization permits a rinsing or washing step to remove all unbound detection reagent, i.e., composition comprising glucose encapsulating vesicles, prior to the lysing step so as to avoid a false signal. Accordingly, any detectable glucose will be attributed to the presence of antigen rather than unbound detection reagent. The lysing step can be performed after the detection reagent is bound to the antigen of interest, and preferably after unbound detection reagent has been removed. The lysing step can be accomplished by any acceptable technique known to reliably rupture vesicle membranes. Methods include exposing the antigen-complexed vesicles to a detergent, which have surfactant qualities that lower the interfacial tension between the vesicle and the surrounding environment, thus causing the disassembly and dispersion of the amphiphilic molecules from the vesicle structure. Non-limiting examples of useful detergents include SDS, Triton X-100, X-114, CHAPS, DOC, NP-40, octyl thioglucoside, octyl glucoside, dodecyl maltoside, or other organic solvents with strong polarity. Other techniques for lysing the vesicles include sonication and electroporation, which can be applied to interrupt the integrity of the vesicle membrane.

The presence and/or quantification of free glucose can be readily determined using existing glucose meter (GM) technologies. Numerous GMs are commercially available from manufacturers such as Abbott Diabetes, Bayer, Advocate, Diagnostic Devices, Entra Health Systems, Hypoguard, HealthPia, Nipro Diagnostics, Lifescan, Nova Biomedical, Relion, Roche, Nova, U.S. Diagnostics, and WaveSense, and can be readily applied to the present method to determine the resulting presence and amount of glucose. The differences among the above illustrative GMs generally address the detection stability, sensitivity and the detection limits for glucose. Choices among these or other available GMs can be readily informed and balanced considering the particular needs and parameters of the assay.

As indicated above, the glucose can be quantified to indicate the amount or level of antigen in the sample. This can be determined by referring to the determined level of glucose and referring to a look up chart to determine the associated level of antigen that is indicated by the detected glucose level. Because the relationship between glucose level and antigen level will depend on the characteristics of the detection reagent (e.g., the amount of vesicle encapsulated glucose associated with each antigen-binding molecule) a control panel of known antigen amounts can be assayed using the same detection reagent or reagent batch as used for the experimental sample in the method.

Assay formats

The disclosed detection reagent composition and related methods described above can be readily incorporated or configured into a wide variety of assay formats known in the art for detection and quantification. Such assays for the detection and/or quantification of an antigen of interest typically involve incubation of the sample that potentially contains the antigen of interest with the detection reagent described above, and detection/quantification via the provision of glucose only after binding between the detection reagent and antigen of interest. In some embodiments, the components of the sample are immobilized, such as through the use of a capture reagent.

Immunoassays encompassed by the present disclosure can be organized in any of a number of different formats recognized in the art. For example, the sample can be brought in contact with, and components therein can be immobilized onto a solid phase support or a carrier, such as nitrocellulose, a plastic well, beads, magnetic particles, and the like. Such solid phase supports or carriers are capable of immobilizing cells, cell particles or soluble proteins. The solid phase support or carrier can then be washed with suitable buffers followed by treatment with the detection reagent. The solid phase support or carrier can then be washed with the buffer a second time to remove unbound detection reagent. The amount of bound antigen of interest on the solid phase support or carrier can then be detected by lysing the vesicles of the bound detection reagent, where the resulting amount of glucose is directly proportional to the amount of the antigen of interest.

Alternatively, the immunoassay format can be a "sandwich" assay, where a capture reagent can be immobilized on a solid support or carrier. The sample is captured by the captured reagent, which is either previously or thereafter immobilized on a solid phase support or carrier. A second reagent, i.e., the detection reagent that is detectably labeled is also added. Often, the capture reagent is different from the antigen-binding molecule of the detection reagent so that they do not competitively bind to the antigen.

The term "solid phase support or carrier" is intended to mean any support or carrier capable of binding an antigen of interest, or a capture reagent that can bind the antigen of interest. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. A substrate that acts as a carrier can be either soluble to some extent or insoluble for the purposes of the present disclosure. The support or carrier material can have virtually any possible structural configuration to conform to any assay format so long as the coupled target antigen is capable of binding to the corresponding detection reagent. Thus, the support or carrier configuration can be substantially spherical, as in a bead or magnetic particle, or cylindrical, as in the inside surface of a test tube, or well in a multi-well plate. Alternatively, the surface can be flat such as a sheet, test strip, etc., that would be appropriate in a lateral flow assay format.

Those skilled in the art will recognize that many other suitable carriers are available for the target antigen or a capture reagent, or will be able to ascertain the same by use of routine experimentation.

In specific embodiments, a capture reagent is conjugated onto a particle, such as a bead or magnetic particle, to facilitate collection or immobilization for further analysis.

As described below and illustrated in FIGURES 1 and 6, a capture reagent specific for a target antigen was bound to magnetic particles (Fe₃0₄ particles). Whether in solution (e.g., FIGURE 1) or on a lateral flow strip (e.g., FIGURE 6), this allowed for the immobilization of the capture reagent complexed with the target antigen and the detection reagent. This immobilization allowed for the removal of any unbound detection reagent. Thus, once the bound glucose encapsulating vesicles are lysed, an accurate quantification of glucose will serve as an amplified signal to reflect the quantity of the target antigen.

As described below and illustrated in FIGURE 6, the disclosed methods and compositions are particularly well-suited for point of care formats and devices. For example, the lateral flow test strips, such as immunochromatographic test strips (ITS) have been applied to a wide array of applications, such as environmental monitoring (see, e.g., Shim, W.B., et al., J. Agric. Food Chem. 54, 9728-9734, 2006; Bogdanovic, L, et al., J Allergy Clin. Immunol. 118, 1157-1163, 2006, each incorporated herein by reference in its entirety), food analysis (see, e.g., Wang, X. L., et al., J. Agric. Food Chem. 55, 2072-2078, 2007; Wang, S., et al., J. Agric. Food Chem. 54, 2491-2495, 2006, each incorporated herein by reference in its entirety), and disease diagnostics (see, e.g., Liu, B. W., et al., Electroanalysis 26, 1214-1223, 2014; Ge, X.X., et al., Biosens. Bioelectron. 50, 486-491, 2013; Du, D., et al., Anal. Chem. 84, 1380-1385, 2012; Ge, X.X., et al., Anal. Chem. 85, 9686-9691, 2013, each incorporated herein by reference in its entirety). Furthermore, the sensitivity of the ITS assay could be greatly improved by nanomaterials (see, e.g., Liu, G.D., et al., Anal. Chem. 79, 7644-7653 , 2007; Zou, Z.X.; Du, D., et al., Anal. Chem. 82, 5125-5133, 2010, each incorporated herein by reference in its entirety). Once the capture reagent-target antigen-detection reagent complex has been immobilized on the strip, the vesicles on the immobilized detection reagent can be lysed and quantified with a GM (see FIGURE 6). Sample

Suitable samples encompassed by the present disclosure include any relevant biological sample from an organism that might contain an antigen of interest, for example, blood, serum, plasma, sputum, pleural fluid, spinal fluid, urine, saliva, stool/feces, mucus, tissue biopsies, tissue homogenates, or a sample obtained by swabbing an area of interest on a subject. Furthermore, the target antigen could be any environmental antigen of interest and, thus, the sample can be any environmental sample, such as water, wastewater, soil, organic matter, and the like.

Device

The disclosure also provides a device for detecting and/or quantifying an antigen of interest in a sample, comprising the composition (i.e., detection reagent) described herein. The device can be configured to facilitate or run the method described above in any appropriate format. The device can further comprise a capture reagent that specifically binds to the antigen of interest in a manner that is non-competitive with antigen-binding molecule. In some embodiments, the device comprises the capture reagent immobilized on a phase support or carrier. In other embodiments, the capture reagent is configured to be immobilized within the device, such as being conjugated to a magnetic bead, whereupon application of a magnetic field immobilizes the capture reagent in the device. In some embodiments, the device comprises a lateral flow strip. Such configuration can also comprise domains configured to accept an aqueous sample, which then flows over distinct domains that include the detection reagent and the capture reagent. The flow strip can be any suitable material, such as nitrocellulose. In some embodiments, the device can integrate a GM device.

Kit

In another aspect, the present disclosure provides a kit for detecting and/or quantifying an antigen of interest in a sample. The kit comprises the composition (i.e., the detection reagent) as described herein. In some embodiments, the kit also comprises a capture reagent. In some embodiments the capture reagent binds the antigen of interest in a manner that does not compete with the binding of the detection reagent to the antigen of interest. The detection and/or capture reagent can be specifically designed to bind and detect/quantify a particular antigen of interest, as described above. In some embodiments, the assay kit also includes buffers, such as a lysis buffer, or an electromechanical component to facilitate lysis and rupture of the glucose encapsulating vesicles. In some embodiments, the kit includes magnetic particles. The particles can have the capture reagent bound thereto. Alternatively, the capture reagent can be configured to be immobilized on the particles. In some embodiments, the kit includes a device that provides a solid support. In some embodiments, the kit can comprise a lateral flow device. In some embodiments, the kit can comprise an ELISA format plate. In some embodiments, the kit comprises a magnet to facilitate reversible immobilization of the reagents in the performance of a method. In some embodiments, the kit can comprise predetermined quantities of target antigen to facilitate the generation of a standard reference for the correlation of glucose and antigen amounts. In some embodiments, the kit further comprises a glucose meter. The kit can also contain instructions to direct the performance of the method described above utilizing the components of the kit.

System

In another aspect, the disclosure provides a system for detecting and/or quantifying an antigen of interest in a sample. The system comprises the composition (i.e., detection reagent) described herein. In addition, the system can further comprise any additional reagents (e.g., capture reagents), buffers (e.g., lysis buffer), devices (e.g., lateral flow strip, GM), a magnet, and other components, as described above, to enable practice of the described methods.

Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook J., et al. (eds.) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Plainsview, New York (2001); Ausubel, F.M., et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, New York (2010); and Coligan, J.E., et al. (eds.), Current Protocols in Immunology, John Wiley & Sons, New York (2010) for definitions and terms of art.

The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

Following long-standing patent law, the words "a" and "an," when used in conjunction with the word "comprising" in the claims or specification, denotes one or more, unless specifically noted.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise," "comprising," and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to indicate, in the sense of "including, but not limited to." Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words "herein," "above," and "below," and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. It is understood that, when combinations, subsets, interactions, groups, etc., of these materials are disclosed, each of various individual and collective combinations is specifically contemplated, even though specific reference to each and every single combination and permutation of these compounds may not be explicitly disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in the described methods. Thus, specific elements of any foregoing embodiments can be combined or substituted for elements in other embodiments. For example, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed. Additionally, it is understood that the embodiments described herein can be implemented using any suitable material such as those described elsewhere herein or as known in the art.

Publications cited herein and the subject matter for which they are cited are hereby specifically incorporated by reference in their entireties.

Illustrative solution-based embodiment

The following is a description of the development and application of liposome encapsulated glucose reagents to provide signal amplification for the detection and quantification of an antigen of interest in solution. This technique facilitated the sensitive, selective, and accurate quantification of the target antigen across a wide range of concentrations.

Abstract

A new technology was developed to quantitatively detect a broad range of disease biomarkers and antigens, and was demonstrated to be portable, economical, and conveniently accessible. The reagents were generated, and subsequent measurements were performed as described in Example 1. The concept is based on releasing encapsulated glucose from antibody-tagged liposomes and subsequently detecting the released glucose using a commercial personal glucose meter (GM). The innovative aspect of this approach lies in the quantification of target biomarkers through the detection of glucose, thus expanding the applicability of the GM by broadening the range of target biomarkers instead of detecting only one analyte, glucose. Because of the bilayer membrane of liposomes, which can accommodate tens of thousands of glucose molecules, the sensitivity was greatly enhanced by using glucose encapsulating liposomes as a signal output and amplifier. To demonstrate proof of concept, the model analyte, protein 53 phosphorylated on Serine 15 (phospho-p53¹⁵), was captured by primary antibodies bound on magnetic Fe₃0₄ nanoparticles and then recognized by reporting antibodies conjugated to glucose encapsulating liposomes. Finally, the target phospho- p53¹⁵ was detected by lysing the bound liposomes to release the encapsulated glucose (4 x 10⁵ glucose molecules per liposome), which is detected with the GM. This approach was demonstrated to be a platform technology that can be readily produced to quantify a wide variety of biomarkers and/or antigens in medical diagnostics, food safety, public health, and environmental monitoring. Given the demonstrated success of this effort, sensors based on liposome encapsulated glucose, in combination with a portable GM, can be readily applied many fields such as physicians' laboratories, hospitals and the common household.

Introduction

The inventors considered liposomes to be promising candidates for the development of signal amplification. Considering the capacity in the liposomal aqueous core together with the phospholipid polar head groups, the liposomes can be readily formed to encapsulate molecules within their lumen and used as functional vesicles.

Accordingly, as described herein, the inventors generated glucose encapsulating liposomes (GEL) and used the detection of released glucose with a personal GM to quantify target biomarkers unrelated to glucose. Specifically, protein 53 phosphorylated on Serine 15 (phospho-p53¹⁵), was used as a model analyte for proof of concept. The primary antibody, Ab_{l 3} was immobilized on Fe₃0₄ magnetic nanoparticles (Fe₃04-Ab₁) to capture phospho-p53¹⁵, which was then recognized by a distinct detection antibody (Ab₂) conjugated to glucose encapsulating liposomes (GEL), i.e., Ab₂-GEL conjugate.

The phospho-p53¹⁵ was quantified after lysing the bound liposomes to release the encapsulated glucose, using a GM. Surprisingly, this method exhibited high sensitivity and a greatly enhanced signal for phospho-p53¹⁵ measurement. It is theorized that the enhanced sensitivity is the result of the high capacity of the liposomes to encapsulate a large amount of glucose (e.g., approximately 4 x 10⁵ glucose molecules per liposome). Combining the advantages of the low cost and portability of personal GMs, the approach presented here, with the use of suitable antibodies, can be readily developed into a universal technology for the detection of many other disease biomarkers.

Results and discussion

Principle of biomarker detection:

The general concept of GEL-based assay lies in the quantification of protein biomarkers by converting the signal representing the present of the biomarker into glucose concentration, which is detected by GM. Thus, this technology expands the application of GM to detect a wide range of protein biomarkers. As shown in FIGURE 1, the model analyte, phospho-p53¹⁵, was first captured by Fe3C"4-Abi magnetic beads and then recognized by Ab₂-GEL conjugates to form a sandwich immunocomplex. A magnetic force was then used to separate the immunocomplex from the solution, including remaining unbound reagents. After separation, the liposomes bound on the Fe3C"4-Abi magnetic beads were dissolved with Triton X-100 to release the encapsulated glucose, which was detectable by a GM. The content of phospho-p53¹⁵ in the samples was converted into the amount of glucose and was easily detected by a personal GM. It is theorized that the bilayer membrane of liposomes can accommodate tens of thousands of glucose molecules, the sensitivity is greatly enhanced by using glucose encapsulating liposomes (GEL) as a signal output and means of amplification. A GM is one of the few widely available devices that have been commercialized to quantitatively detect glucose in blood. With the advantages of low cost, speed, portability and simplicity, this approach expands the use of GMs for the simple, sensitive and inexpensive quantification of different kinds of non-glucose targets.

Characterization of glucose encapsulating liposomes: The glucose encapsulated liposomes were synthesized and characterized as described in Example 1, below. The synthesized liposome suspension appeared translucent with a pale blue opalescence, which is the characteristic color of liposomes. Further characterizations using TEM and a Malvern mastersizer were performed on the glucose encapsulating liposomes (observed to be evenly shaped nanoparticles), which maintained their spherical shape (see FIGURE 2A). The thin shell became well-demarcated, and no rupture of the capsule wall was observed, which strongly suggested that a typical vesicle structure had been formed. The resulting liposomes exhibited a narrow size distribution, with a diameter of approximately 176 nm (see FIGURE 2B). Considering the thickness of a lipid bilayer is 4 nm, the average volume of a single liposome is 2.53 x 10^-12 μΐ.. Glucose encapsulation efficiency was determined further by UPLC and the results showed that approximately 80% of the glucose was encapsulated in the liposomes. Ultimately, it was estimated that each liposome contained ~4 x 10⁵ glucose molecules.

The stability of the GELs was monitored by measuring the glucose content before and after lysis from the liposome. The leakages were determined to be 2.9% and 4.2% after storage for 60 and 90 days, respectively, which indicated acceptable stability of the synthesized GEL. In addition, glucose leakage during the antibody conjugation step was also explored, and negligible leakage occurred during conjugation.

In addition, XPS was employed to characterize the detection antibody (Ab2)-GEL conjugate. As shown in FIGURES 3A-3D, the full spectra of Ab2~GEL displayed the characteristic spectra of Ni _s, P2_p and S2_p. The Nls peak is mainly due to the antibodies, and the P2_p peak results from phospholipids, while the S2_p peak is due to the binding agent biotin, which connects the antibodies and liposomes. All these typical binding energies indicate successful coupling of Ab2 to GEL to form a stable conjugate.

Characterization and optimization of the immunoreaction:

To demonstrate immuno-recognition between Fe3C>4-Abi , phospho-p53¹⁵ and

Ab2~GEL, quartz crystal microbalance (QCM) measurements were performed in realtime to monitor mass changes that occurred at the quartz surface (FIGURE 4A). Before immunoreaction, a gold quartz crystal electrode assembled with Fe3C>4-Abi (Fe3C>4-Abi/Au) was first equilibrated with PBS for 1 h until the baseline was stable. Upon attachment of phosphor-p53¹⁵ to the Fe3C>4-Abi/Au particles, a rapid decrease in frequency was observed, which then attained a steady-state. As expected, the recognition between Fe3C>4-Abi and phospho-p53¹⁵ occurred on the gold surface and was completed within 35 min. Then, two different types of Ab2 were added, Ab2~GEL, and Ab2 (without the coupling liposome), for comparison. Obvious frequency drops occurred with both secondary immunoreactions and the curves tended to stabilize after 40 min. The larger decrease of Ab2~GEL (curve "a") compared to Ab2 (curve "b") was due to the additional mass introduced by the liposomes on the surface. These results confirmed that liposomes were successfully coupled to Ab2 and that immuno-recognition between Fe3C"4-Abi , phospho-p53¹⁵ and Ab2~GEL was accomplished on the surface.

Incubation time is one of the most important parameters for immunoassays. The amount of glucose released from bound liposomes increased when increasing incubation time. The GM readout tended to a steady value after immunoreaction time of 35 min (curve "a") and 40 min (curve "b"), respectively. These times were used for Fe₃0₄-Ab₁ and Ab2~GEL for recognizing phospho-p53¹⁵ in the sandwich immunoassay

(FIGURE 4B). Longer incubation times did not contribute to the signal, which might result from non-specific response. These results were consistent with the QCM responses.

The ratio of GEL to phospho-p53¹⁵Ab₂ is another key factor in signal amplification. The signal recorded with a GM is enhanced by increasing the ratio of GEL/phospho-p53¹⁵Ab2, and the maximum response is achieved at a ratio of 1 : 1

(FIGURE 4C). As expected, additional GEL can increase the total amount of glucose to increase detection response. However, reducing the content of phospho-p53¹⁵Ab2 decreases the efficiency of coupling to the captured phospho-p53¹⁵ target at the Fe₃0₄- Abi magnetic beads, which eventually results in a decreased response. Therefore, a GEL/phospho-p53¹⁵Ab₂ ratio of 1 : 1 is preferred.

Quantitative detection of phospho-p53¹⁵:

The proposed immunoassay using the Ab2-GEL conjugate as amplification label, together with GM readout, was tested in different concentrations of phospho-p53¹⁵. The targeting analytes were detected by lysing the bound liposomes to release the encapsulated glucose and detected with a personal GM. Significantly, it can be seen from FIGURE 5 A that the amount of glucose detected increased with increasing p53¹⁵ concentrations from 0.1 to 100 ng/mL with a detection limit of 50 pg/mL. In contrast, the human phospho-p53 (SI 5) ELISA kit reported a linear range of 0.3-20 ng/mL for phospho-p53¹⁵, with a detection limit of 50 pg/mL. Accordingly, the present approach is comparable to the commercial kit, but has a broader detection range. Moreover, the use of a GM as a detector in this work is easier and less expensive compared to the expensive instruments used in the ELISA. The detection limit for the targets depends primarily on the sensitivity of the glucose meter. However, the advantages of the low cost and portability of the personal GM, in conjunction with the use of suitable antibodies, provides a universal technology platform for the detection of many other antigens, such as disease biomarkers or environmental contaminants.

The selectivity of the immunosensor was investigated recommended using human plasma spiked with 1.0 ng/mL phospho-p53¹⁵ and possible interfering compounds including phospho-p⁵³³⁹² and phospho-p53⁴⁶ (FIGURE 5B). No significant signal change was observed for the spiked samples compared to the results obtained with phospho-p53¹⁵ alone, indicating good selectivity for the analytes.

To evaluate the accuracy, the immunosensor was incubated in human plasma samples spiked with standard phospho-p53¹⁵ with known concentrations. The results were compared with ELISA. The relative deviation was observed to be in the range of - 4.27% to 2.88%, indicating that the proposed method is reliable.

The intra- and inter-assay coefficients of variation were determined to demonstrate the reproducibility of the immunosensor. The intra-assay precision was evaluated with one immunosensor by assaying six replicate determinations of 1.0 ng/mL phospho-p53¹⁵. Similarly, the inter-assay precision was evaluated using six different immunosensors. The relative standard deviation of intra-assay and inter-assay was 4.3% and 1.7%, respectively. These results demonstrate that this assay platform has acceptable precision and fabrication reproducibility.

Conclusions

In summary, this work describes a novel, low-cost, and universal technology platform for sensitive detection of various kinds of biomarkers and antigens using a commercial personal glucose meter. The principle was based on a sandwich immunoassay, and the measurement was performed by lysing the bound antibody-tagged liposomes to release the encapsulated glucose, which was detectable by a glucose meter. The advantages of this approach is based on (1) new technology (e.g., GEL-based detection reagents) that convert the quantification of target biomarkers to the detection of the glucose amount; (2) being a universal method, this approach is applicable to the quantification of a wide variety of disease biomarkers through the use of suitable antibodies or other antigen-binding reagents; (3) portability and low cost, due to the use of a mini glucose meter as a detector; (4) diverse applications for commercial glucose meters, which can be expanded for the quantification of various analytes other than glucose; and (5) high sensitivity, using glucose encapsulating liposomes for signal output and for amplification.

This new GEL-based technology is a major breakthrough for the real-time, rapid and sensitive detection of disease biomarkers in medical diagnostics, food safety, and environment monitoring. It is also important for global health for diagnosis of health conditions.

Illustrative strip-based embodiment

The following is a description of the development and application of liposome encapsulated glucose reagents integrated in an immunochromatographic strip format to provide signal amplification for the detection and quantification of an antigen of interest. This technique facilitated the sensitive, selective, and accurate quantification of the target antigen across a wide range of concentrations in a point of care format, and illustrates the adaptability of the above-described system to different testing formats.

Abstract

In this project, the new glucose encapsulating liposome (GEL)-based technology described above was incorporated into an immunochromatographic test strip (ITS) platform. Specifically, primary antibody (Abi)-modified Fe₃0₄ magnetic nanoparticles (Fe₃0₄-Abi) were settled on the test zone to capture both the target phospho-p53¹⁵ and the detection antibody (Ab₂)-GEL conjugate. The measurement was based on the release and subsequent detection of encapsulated glucose from Ab₂-GEL using a commercial glucose meter (GM). The GM readout showed a linear response over the phospho-p53¹⁵ concentration range from 0.1 to 50 ng/mL, with a detection limit of 50 pg/mL. The high sensitivity was a result of the large amount of encapsulated glucose (1.8>< 10⁵ glucose molecules per liposome). The advantages of this novel approach includes: the quantification of target biomarkers by converting the signal to the detectable levels of glucose by using glucose encapsulating liposomes (GEL) for signal output and amplification, and combining the use of a test strip and glucose meter. Taking the advantage of low cost, widespread availability and portability of the test trip, together with the personal GM, the described approach can be easily developed as a universal platform technology for detection of many other disease biomarkers and antigens in medical diagnostics and environmental monitoring. Introduction

As described above, liposomes are shown to be useful tools for molecular signal enhancement by coupling their capacity to encapsulate molecules in the aquatic core or within the lipid membrane with the straightforward biofunctionalization of their phospholipid polar head groups with, for example, antibodies, facilitating the realization of multifunctional vesicles

The immunochromatographic test strip (ITS) format provides a one-step, straightforward assay for simple, rapid, inexpensive, user-friendly, and adaptable detection in various environments, and has shown promise for point-of-care (POC) assays. As a result, ITS has found broad applications, such as environmental monitoring, food analysis, and disease diagnostics. Furthermore, the sensitivity of the ITS assay could be greatly improved by nanomaterials.

In this section, the development of the ITS platform linked to a personal GM is described for detection of a target biomarker other than glucose. Here, the potential biomarker for gamma-radiation exposure, phosphorylated protein 53 on Serine 15 (phospho-p53¹⁵) was used as the model analyte for this purpose (FIGURE 6). As illustrated, detection antibodies (Ab₂) conjugated to glucose encapsulating liposomes (GEL), i.e., Ab₂-GEL, were loaded onto the conjugate pad. The primary antibody (Abi)- modified Fe₃C>4 magnetic nanoparticles (Fe₃04-Abi) were adsorbed on the test zone with the help of a small magnet inserted in the device. As the liquid sample migrates along the strip, the resulting immunocomplex is trapped on the test zone. The trapped immunocomplex was then cut off from the test trip, followed by lysing the bound liposomes to release the encapsulated glucose, which was detectable by a personal GM device. The advantages of this novel approach lies in the quantification of target biomarkers by converting the signal to the detection of the amount of glucose, thus expanding the application of commercial glucose meters to the quantification of a broad range of biomarkers other than glucose. The results show that the proposed platform exhibits higher sensitivity and a greatly enhanced signal through using liposome as a signal amplification tool as compared to a common immunchromatographic strip using colloidal gold as probes (see, e.g., Xinga, C.R., et al., Biosens. Bioelectron. 66, 445-453, 2015; Xu, H.; et al., Anal. Chem. 81, 669-675, 2009). Combining the advantages of the low-cost and portability of the test strip together with a personal GM, the described approach can be readily developed as a universal technology for the detection of many other disease biomarkers and/or environmental antigens of interest. The developed method can be deployed in physicians' offices, laboratories, nursing homes, hospitals, homes, and the field.

Results and Discussion

Characterization of liposomes:

The glucose encapsulating liposome (GEL) constructs were synthesized and characterized as describe in Example 2. The glucose encapsulating liposomes were characterized by SEM and Malvern mastersizer. As shown in FIGURE 7A, the synthesized liposome nanoparticles maintained their spherical shape and dispersed evenly in solution. The thin shell became well demarcated, and no rupture of the capsule wall was observed, which strongly suggested that a typical vesicle structure had been formed. The mean diameter of the resulting liposomes is approximately 326 nm.

The glucose encapsulation efficiency was further determined by FIPLC analysis. As shown in FIGURE 7B, the glucose peak appeared at 11 min. According to the peak area of glucose before (curve "c") and after (curve "b") embedding in the liposome, approximately 80% of the glucose was calculated to be encapsulated in the liposomes. Other parameters of the liposomes are listed in TABLE 1. From the data, each liposome was estimated to contain 1.8^χ 10⁵ glucose molecules, which was large enough to magnify the signal of the target. The concentrations of liposomes and the number of biotin molecules expressed on the liposome surface (-5150 molecules/liposome) were comparable to those previously reported.

Table 1 Characteristics of the glucose encapsulating liposome

Parameter Value

Mean diameter (nm) -326

Volume of liposome (μί) 1.68 x l0^"u

Glucose concentration (mg/mL) 5

Number of encapsulated glucose per liposome ~1.8 x l0⁵

Liposome concentration (number/mL) -l . l x lO¹³

Number of biotin molecules on the liposome surface -5150

The stability of the GEL constructs was monitored by measuring the glucose content before and after lysis from the liposome. The leakages, expressed as a ratio of the glucose quantity found in the external solution divided by the glucose amount after lysis, were determined to be 2.9% and 4.2% after storage for 60 and 90 days, respectively, which indicated acceptable stability of the synthesized GEL. In addition, glucose leakage of antibody-linked liposomes introduced on the nitrocellulose membrane was also explored, and no obvious leakage occurred during conjugation.

Optimization of Test Strip Immunoassay:

Because avidin was used to connect the GEL and Ab₂ to provide a detection reagent, it was determined that the ratio of GEL and Ab₂ is one of the most important factors affecting the response. As shown in FIGURE 8A, the signal detected by the GM increased with the increasing ratio of GEL/Ab₂, with the maximum response achieved at 2:5. As expected, the increased ratio of GEL/Ab₂ raised the total amount of glucose and, thus, increased the signal response. However, the reduced amount of Ab₂ may decrease the efficiency of the immunoreaction to capture the target phospho-p53¹⁵, and eventually decrease the response. Therefore, the 2/5 volume ratio of GEL/Ab₂ was chosen as the ideal conjugate.

The response was also found to depend on the flow time of the sample along the test strip. As shown in FIGURE 8B, the signal increased with increasing flow time and tended to stabilize after 15 min, indicating a rapid immunoreaction.

Nonspecific adsorption had another important influence on the immunoassay response. Therefore, control experiments (0 ng/mL phospho-p53¹⁵) using different concentrations of BSA in the Ab₂-GEL conjugate were performed to study the blocking effect. As shown in FIGURE 8C, both the signal from the sample and the control decreased upon increasing the BSA content in the Ab₂-GEL conjugate. This phenomenon was a result of the shield effect of the BSA agent. Minimization of the nonspecific adsorption (control signals) was achieved at 3% BSA in PBS, at which point it tended to stabilize. Although the target sample displayed much higher responses at a very low BSA concentration (0.1%), the nonspecific signal was very high. Considering the sensitivity of the response and limiting the nonspecific adsorption, 3% BSA in PBS was selected as the blocking agent.

Magnetic Fe₃0₄ nanoparticles provide an easy way to absorb Abi on the nitrocellulose membrane by simply putting a small magnet under the strip. To verify the importance of Fe₃0₄, the control experiment was carried out by immobilizing same content Abi instead of Fe₃0₄-Abi on the test zone of nitrocellulose membrane. The results showed that for the same content of phospho-p53 , the integrated immunochromatographic strip with Fe₃0₄ always had stronger response than the strip without Fe₃0₄ (not shown). The reason is that the binding force is much stronger than simply adsorption of Abi on the membrane which can reduce the loss of Abi and enhance the stability and accuracy of detection. If sensitivity is not an issue, simpler design without magnetic particle can also be used.

Triton X-100 is one of the most commonly used detergents for breaking the membrane and promoting the release of the encapsulated molecules from liposomes. However, some sources reported that Triton X-100 might denature enzymes and reduce their activity. Accordingly, the effect of Triton X-100 on glucose oxidase activity was addressed. As controls, PBS solution and OGP (a nonionic detergent) were chosen as detergents instead of Triton X-100. There was no observed response when only PBS solution was added. Meanwhile, the variation when adding Triton X-100 and OGP was the same where the personal GM signal was initially increased and then remained nearly constant with the increasing concentration of surfactant. The observed signal increase was due to the detergent-mediated rupture of the liposomal membranes, which promoted the leakage of glucose molecules. However, excess surfactants did not reduce the personal GM signal, indicated that there was no pronounced effect for Triton X-100 on glucose signal. It is hypothesized that there was little Triton X-100 exposed to glucose oxidase in the personal GM as compared to the encapsulated enzymes inside liposomes.

Analytical Performance for Electrochemical Detection of Phospho-p53¹⁵ Antigen: Under the optimal conditions, the test strip platform linked to a personal GM was challenged with different concentrations of phospho-p53¹⁵. As shown in FIGURE 9, the readout from the GM increased with increasing phospho-p53¹⁵ concentration. A linear response was obtained over the concentration range from 0.1 to 50 ng/mL with a detection limit of 50 pg/mL. These results are comparable to those of the phospho-p53¹⁵ ELISA kit, which exhibited a linear range from 0.3 to 20 ng/mL with a detection limit of 60 pg/mL for phospho-p53¹⁵. The major advantage of the presented approach over ELISA is the low cost, portable and rapid detection of the test strip platform integrated with a personal GM.

A series of phospho-p53¹⁵ human plasma samples were used to test the accuracy of the quantification approach. Phospho-p53¹⁵ human plasma samples were prepared by spiking different amounts of phospho-p53¹⁵ with known concentrations into human plasma. The results are summarized in TABLE 2, which demonstrate recoveries in the range of 94-100%, indicating that the portable device is reliable.

Table 2 Recovery of the Integrated Device in Human Plasma

Sample 1 2 3 4 5

Spiked (ng/ml) 0.5 1.0 5.0 10 20

Found (ng/ml) 0.484±0.024 1.037±0.028 4.745±0.031 9.651±0.04 20.18±0.035

Recovery (%) 96.8 103.7 94.9 96.5 100.9 To further investigate the selectivity and validate the approach for detection of the target phospho-p53¹⁵, the proposed lateral flow device was tested using human plasma as the analyte sample. Human plasma samples spiked with 1.0 ng/mL phospho-p53¹⁵ and different possible interfering agents, such as p53, phospho-p53³⁹², and phospho-p53⁴⁶, were loaded onto the test strip. No remarkable response change was observed for the mixed sample in comparison to the result obtained in the presence of phospho-p53¹⁵ alone (not shown), indicating good selectivity for the targeted phosphorylated protein.

Reproducibility and Stability of the Integrated Device:

The reproducibility of the proposed platform was evaluated by analyzing one sample on five test strips. The coefficient of variation (CV) was 5.3% at 1.0 ng/mL phospho-p53¹⁵, demonstrating the acceptable reproducibility of the proposed integrated device. The stability study was carried out by analyzing one sample (contain 1.0 ng/mL phospho-p53¹⁵) on prepared strips at 5 day intervals for one month. When the prepared strips were not in use, they were stored at 4°C. The results showed that there was no detectable loss of the initial response observed every 5 days. Furthermore, over 90% of the initial response remained after 15 days and 80% remained after one month, indicating acceptable stability. Data not shown. The proposed method thus exhibits great potential for point-of-care applications.

Conclusions

In summary, an ITS device linked to a personal GM for rapid and sensitive detection of target protein biomarkers was successfully constructed and demonstrated. The advantages of this approach include (1) the quantification of target biomarkers by converting the signal to the detection of the amount of glucose, thus expanding the application of commercial glucose meters to the quantification of a broad range of biomarkers other than glucose; (2) using glucose encapsulating liposomes for signal output and amplification, which provides high sensitivity; (3) the combination of the test strip and glucose meter to provide portability and low cost and promises great potential for field-deployable and on-site detection in homes, physicians' offices, nursing homes and hospitals. The proposed platform can be readily developed into a universal platform applicable for the quantification of broad disease biomarkers and environmental antigens through the use of suitable antibodies or other suitable binding reagents. Additionally, this technique can greatly impact global health by enabling the diagnosis of health conditions in developing countries.

EXAMPLES

Example 1

This example describes illustrative reagents, materials, and methodologies used to construct and apply an assay incorporating liposome encapsulated glucose to quantify an antigen of interest in solution.

Reagents and materials

Human phospho-p53¹⁵ ELISA kits (DuoSetsIC) containing phospho-p53¹⁵ capture antibody (Abi), phospho-p53¹⁵ antigen and biotin-detection antibody (Ab2) were purchased from R&D Systems, Inc. (Minneapolis, USA). Bovine serum albumin (BSA), human serum albumin (HSA), phosphate buffer saline (PBS), chloroform, Triton X-100, Tween-20, avidin, l-Phenyl-3-methyl-5-pyrazolone (PMP), 2-(N- morpholino)ethanesulfonic acid (MES), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma- Aldrich. Hydrogenated Soybean Phospholipids (HSPC), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[biotinyl (polyethylene glycol)-2000] (ammonium salt) (DSPE- biotin), and cholesterol were purchased from Avanti Polar Lipids Inc. (Alabama, USA). Carboxylated Fe3C"4 was purchased from Tianjin Baseline ChromTech Research Centre

(Tianjin, China).

Apparatus

Transmission electron microscopy (TEM) was performed using a JEOL 2000 transmission electron microscope. Antigen-antibody specific recognition was recorded on a quartz crystal microbalance QCM 200 (Stanford Research Systems, USA). High performance liquid chromatography (HPLC) measurements were performed on an Agilent 1100 system (Agilent, USA). X-ray photoelectron spectroscopy (XPS) measurements were conducted using a Thermo Electron MultiLab 2000 instrument (Thermo Fisher, USA). Diameter measurements were obtained using a Malvern mastersizer 2000 (Malvern, UK). Glucose concentrations were determined with a glucose meter (accu-chek, Germany).

Preparation of glucose encapsulating liposomes (GEL)-biotin

Liposomes were synthesized by a reversed-phase evaporation method (Ho, J. A.,

Hsu, H.W., Anal. Chem. 75, 4330-4334, 2003; Szoka Jr, F., Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA 75, 4194-4198, 1978). Briefly, HSPC (124 mg), cholesterol (25 mg) and DSPE-biotin (6 mg) (i.e., 159, 64 and 2 mM, respectively), were first dissolved in 15 mL mixed solvent in which the volume ratio of chloroform, isopropyl ether and methanol were 6:6: 1, and then sonicated at 45°C under nitrogen to ensure homogeneous mixing. Then, 5 mL of a warm glucose solution (5 mg/mL) was added to the lipid mixture followed by sonication for 5 min. The organic solvents were removed at 45°C, resulting in a gel-like suspension (total lipid concentration was 5 mg/mL). The suspension was then incubated in a 45°C water bath for half an hour before being passed through a 0.4 μπι polycarbonate filter over 10 times to produce a homogeneous suspension. The residual un-encapsulated glucose was removed by gel filtration on a 1.5 x 25 cm² Sephadex G-50 column at room temperature, followed by dialysis (MWCO, 12-14 kDa) at 4 °C in the dark. The morphology and average size of the GEL were measured by TEM and the Malvern mastersizer. HPLC measurements were then performed to determine the entrapment efficiency of glucose in each liposome.

Coupling of detection antibody (Ab2) to GEL

The phospho-p53¹⁵ detection antibody (Ab₂)-GEL conjugate was prepared as described in our previously reported method (Du et al., Biosens. Bioelectron. 26, 3857-3863, 2011, incorporated herein by reference in its entirety). The above prepared GEL-biotin and commercial biotin-Ab2 were bound together with native unlabeled avidin. Briefly, 100 μΕ of 0.06 mg/mL avidin was mixed with 100 μΕ of biotin-GEL and incubated for 2 h to form an avidin-biotin-GEL conjugate. Then, 100 μΕ of 7.5 mg/mL biotin-Ab₂ was added at an equimolar amount and incubated for another 2 h. Excess agents were removed by ultrafiltration. The resulting Ab2-GEL conjugate was dispersed in PBS containing 3% BSA and stored at 4 °C. Conjugation of primary antibody (Abi) with Fe3C"4 nanoparticles

A total of 1.0 mL of purified carboxylated Fe3C"4 particles (1.0 mg/mL) was blended with 400 mM EDC and 100 mM NHS in 1.0 mL of MES, pH 5.2 for activation of 30 min. The activated Fe3C>4 was separated with a magnet and washed with buffer three times so that the superfluous of EDC and NHS were separated. It was then re-dispersed in 1.0 mL of 2 mg/mL Abi , stored overnight, magnetically separated and washed with PBS. The resulting Fe₃0₄-Ab₁ preparation was dispersed in 1.0 mL of PBS with 1.0% BSA (pH 7.0) and stored at 4 °C.

Detection of target proteins

Aliquots (50 mL) of phospho-p53¹⁵ antigen (with different concentrations of desired samples) were introduced into each tube of Fe3C>4-Abi and incubated for 35 min with gentle mixing. After magnetic separation, the supernatant was removed. Then, 50 mL of Ab2~GEL was added and incubated for another 40 min. The mixture was washed by PBS containing 0.05% Tween-20. After magnetic separation, the liposomes bound to Fe304 nanoparticles were dissolved by adding 100 mL of 10 mg/mL Triton

X-100 to release the encapsulated glucose, which was then detected using a mini commercial GM. The established relationship between the target concentration and the glucose amount will be applied to the detection of desired samples.

Example 2

This example describes illustrative reagents, materials, and methodologies used to construct and apply an assay incorporating liposome encapsulated glucose to quantify an antigen of interest in a test strip format.

Reagents and Materials

The human phospho-p53(S15) ELISA kit including phospho-p53¹⁵ capture antibody (Abi), phosphor-p53¹⁵ antigen, and biotin-phospho-p53¹⁵ detection antibody (Ab₂) was purchased from R&D Systems Inc. (Minneapolis, MN, USA). Fe₃0₄-COOH was obtained from Tianjin Baseline Chrom Tech Research Centre (Tianjin, China). Tween-20, PEG 6000, bovine serum albumin (BSA), Triton X-100, n-octyl-β-ϋ- glucopyranoside(OGP), casein, trehalose, sucrose, carbodiimide hydrochloride (EDC), N- hydroxy succinimide (NHS), phosphate buffer saline (PBS) and 2-(N-morpholino) ethanesulfonic acid (MES) were acquired from Sigma-Aldrich. Hydrogenated Soybean Phospholipids (HSPC), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl (polyethylene glycol)-2000] (ammonium salt) (DSPE-biotin), and cholesterol were purchased from Avanti Polar Lipids Inc. (Alabama, USA).

Apparatus

Scanning electron microscopy (SEM) micrographs were performed on a JEOL- JSM-6700F. High performance liquid chromatography (HPLC) measurements were performed on an Agilent 1100 system (Agilent, USA). The glucose concentration was detected by a glucose meter (CONTEC Medical Systems Co., Ltd, China).

Preparation of Glucose-Encapsulating Liposomes (GEL)

Liposomes were prepared by a reversed-phase evaporation method (Ho, J. A.; Hsu, H.W., Anal. Chem. 75, 4330-4334, 2003; Szoka, F., Jr.; Papahadjopoulos, D. Proc. Natl. Acad. Sci. U.S.A. 75, 4194-4198, 1978). Briefly, HSPC (124 mg), cholesterol (25 mg) and DSPE-biotin (6 mg) were first dissolved in 15 mL of a solvent mixture consisting of 6:6: 1 volume ratios of chloroform, isopropyl ether, and methanol, and then sonicated at 45°C under nitrogen to ensure homogeneous mixing. Then, 5 mL of warm glucose solution (5 mg/mL) was added to the lipid mixture, followed by sonicating for 5 min. The organic solvents were removed at 45°C using a rotary vacuum evaporator, resulting in a gel-like suspension (total lipid concentration was 5 mg/mL). The mixture was then incubated in a 45°C water bath for half an hour before passing through the 0.4 μπι polycarbonate filter more than 10 times to produce a homogeneous suspension of uniform size. Any residual un-encapsulated glucose or trace organic solvent was removed by gel filtering on a 1.5x25 cm Sephadex G-50 column at room temperature, followed by dialysis (MWCO, 12 -14 kDa) at 4°C in the dark. The morphology and average size of the GEL was measured by SEM and a Malvern mastersizer. Then, HPLC measurements were performed to obtain the entrapment efficiency of the glucose in each liposome.

Preparation of Antibody-Glucose Encapsulating Liposome (Ab₂-GEL) Conjugate

The above prepared GEL-biotin and commercial biotin-Ab₂ were conjugated by native unlabeled avidin. Briefly, 60 μΕ of 1.0 mg/mL avidin and 400 μΕ of biotin-GEL were mixed and stirred at room temperature for 2 hours. Then, 1.0 mL of 0.86 μg/mL biotin-Ab₂ was added and the mixture was stirred for an additional 2 hours. Excess agents were removed by ultrafiltration. The resulting Ab₂-GEL conjugate was dispersed in PBS containing 3% BSA and maintained at 4°C for the following experiments. Preparation of Antibody (Abi)-Modified Fe₃0₄ Nanoparticles

First, 1.0 mL of purified carboxylated Fe₃0₄ nanoparticles (1.0 mg/mL) was mixed with 400 mM EDC and 100 mM NHS in 1.0 mL of MES (pH 5.2) for activation for 30 min and then separated by a magnet and washed with buffer three times to remove excess EDC and NHS. The particles were re-dispersed in 1.0 mL of 3.6 μg/mL Abi and kept overnight. The mixture was magnetically separated and washed three times with PBS. Finally, the resulting Fe₃0₄-Abi was re-dispersed in 1.0 mL of pH 7.0 PBS containing 1% BSA and stored at 4°C.

Fabrication of ITS based Platform and Detection of Target Proteins

Pretreatment of the test strip was applied before experiments. Briefly, the sample pad was saturated with PBS (0.01 M, PH 7.4) containing 2% PEG 6000, 2% tween-20, 1% BSA, 2% Triton X-100 and 0.5% casein. The conjugation pad was treated with PBS (0.01 M, PH 7.4) containing 5% trehalose, 0.5% casein, 15% sucrose and 1% BSA. While drying, 5 μΕ of Ab₂-GEL was introduced onto the conjugate pad. The middle portion of the nitrocellulose membrane (HFB 18004) was used as a test zone by immobilization of Fe₃0₄-Abi with a small magnet under it during immunoreaction. The strips were cut to 4 mm in width. Finally, the pretreated sample pad, conjugate pad, nitrocellulose membrane and absorption pad were assembled onto the backing card. A sample solution containing the desired concentration of phosphor-p53¹⁵ was added to the loading pad and migrated through the entire strip by capillary action, specifically binding to the Ab₂-GEL and finally resting on the test zone. The immunocomplex formed on the test zone was cut off and dissolved by adding 100 μΕ of 10 mg/mL Triton X-100 to release the encapsulated glucose, which was then detected by a commercial GM. While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMS The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A composition comprising an antigen-binding molecule tethered to an artificial vesicle, wherein the antigen-binding molecule selectively binds to an antigen of interest, and wherein the artificial vesicle encapsulates glucose.

2. The composition of Claim 1, wherein the antigen-binding molecule is an antibody, antibody-like molecule, lectin, receptor, aptamer, or a functional antigen-binding domain thereof.

3. The composition of Claim 2, wherein the antigen-binding molecule comprises an antigen-binding fragment of an antibody or antibody-like molecule.

4. The composition of Claim 2, wherein the antibody-like molecule is a single-chain antibody, a bispecific antibody, an Fab fragment, or an F(ab)2 fragment.

5. The composition of Claim 4, wherein the single-chain antibody is a single chain variable fragment (scFv), single-chain Fab fragment (scFab), \¾H fragment,

VNAR' or nanobody.

6. The composition of Claim 1, wherein the artificial vesicle is a liposome.

7. The composition of Claim 1, wherein the artificial vesicle is a micelle.

8. The composition of Claim 1, wherein the artificial vesicle is covalently tethered to the antigen binding molecule.

9. The composition of Claim 1, wherein the artificial vesicle is non-covalently tethered to the antigen binding molecule.

10. The composition of Claim 1, wherein the artificial vesicle encapsulates glucose at a concentration of at least about 0. 1 mg/mL.

11. The composition of Claim 6, wherein the liposome is at least about 100 nm in diameter.

12. The composition of Claim 6, wherein the interior volume of the liposome is at least about 4.00 x 10^-13 μΐ_^.

13. A method for detecting an antigen of interest, comprising:

contacting a sample to the composition of any one of Claims 1-12 under conditions sufficient to permit the selective binding of the antigen-binding molecule in the composition to the antigen of interest;

lysing the artificial vesicle to release the glucose; and

detecting the presence of the glucose, thereby indicating the presence of the antigen of interest.

14. The method of Claim 13, further comprising immobilizing the antigen of interest.

15. The method of Claim 13, further comprising removing unbound composition prior to lysing the artificial vesicle.

16. The method of Claim 15, wherein the step of detecting the presence of the glucose comprises using a glucose meter.

17. The method of Claim 16, wherein detecting the presence of the glucose comprises quantifying the amount or concentration of the glucose.

18. The method of Claim 17, wherein the amount or concentration of glucose detected indicates a quantity of the antigen of interest.

19. The method of Claim 13, wherein the sample is contacted to the composition in solution.

20. The method of Claim 13, wherein the sample is contacted to the composition on a lateral flow strip.

21. A device for detecting and/or quantifying an antigen of interest in a sample, comprising the composition of any one of Claims 1-12.

22. The device of Claim 21, further comprising a capture reagent that specifically binds to the antigen of interest in a manner that is non-competitive with antigen-binding molecule.

23. The device of Claim 22, wherein the capture reagent is immobilized or configured to be immobilized on a surface.

24. The device of Claim 23, wherein the capture reagent is tethered to a magnetic particle.

25. The device of Claim 21, wherein the device comprises a lateral flow strip

26. A kit for detecting and/or quantifying an antigen of interest in a sample, comprising the composition of any one of Claims 1-12.

27. The kit of Claim 26, further comprising a capture reagent that specifically binds to the antigen of interest in a manner that is non-competitive with antigen-binding molecule.

28. The kit of Claim 26, wherein the capture reagent is immobilized or configured to be immobilized on a surface.

29. The kit of Claim 28, wherein the capture reagent is tethered to a magnetic particle.

30. The kit of Claim 26, further comprising a magnet.

31. The kit of Claim 26, further comprising a glucose meter.