Metal nanoparticles in biochemical assays based on interferometry

Abstract

The present invention relates to a biochemical assay based on bio-layer interferometry (BLI) using metal nanoparticles such as gold nanoparticles (AuNPs) and the growth of the metal nanoparticles to enhance the signals. In the present methods, the target analyte molecule is first immobilized onto BLI biosensors. Biofunctionalized AuNPs then bind to the immobilized target molecules. In the present method, the detection signal is improved by growing the immobilized AuNPs on the BLI sensor. The immobilized AuNPs provide seeds to grow their size. The present method includes a step of reducing a new reagent Au (3+) to Au (0) on the existing AuNP, thus growing the size of AuNPs, which results in improved sensitivity for the detecting target biomolecules on BLI.

Description

METAL NANOPARTICLES IN BIOCHEMICAL ASSAYS BASED ON INTERFEROMETRYFIELD OF THE INVENTIONThe present invention relates to a biochemical assay based on bio-layer interferometry (BLI). The present invention uses metal nanoparticles such as gold nanoparticles and the growth of the metal nanoparticles to enhance the signals of BLI.SEQUENCE LISTINGThis application contains a ST.26 compliant Sequence Listing, which is submitted herewith in XML format via Patent Center, and is hereby incorporated by reference in its entirety. The XML copy, created on June 23, 2025, is named sequence_hsting_0766518040W001.xml and is 3,953 bytes in size.BACKGROUND OF THE INVENTIONLabel free detection methods such as bio-layer interferometry (BLI) have become standard methods in the study of receptor / ligand binding in biomedical research and in the development of therapeutics. BLI detects biomolecule binding in real time by monitoring the change in interference between light beams reflected from a proximal and a distal reflecting surface.Nanoscale materials have gained significant attention in medicine and diagnostics due to their unique physicochemical properties. Among these materials, gold nanoparticles (AuNPs) stand out as a promising candidate for various biological applications. AuNPs exhibit desirable features such as easy synthesis, surface modification, and biocompatibility. In particular, the optical properties of AuNPs make them suitable as a signal reporting agent for biosensing and bioimaging applications.BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1 illustrates one assay format schematic of the present method.FIG. 2A depicts a biosensor interferometer that includes a light source, a detector, a waveguide, and an optical assembly (also referred to as a ‘‘BLI probe'’ of a “probe’').FIG. 2B depicts an example of a BLI probe.FIG. 3 depicts another configuration of a BLI probe.FIGs. 4A-4B illustrate the principles of detection in a thin-film interferometer.FIG. 5 plots of the DNA assay results of green fluorescent protein (GFP).FIG. 6 plots of the assay results of IL6.FIG. 7 plots of the assay results of AAV5.FIG. 8 shows the signal vs. concentration in 3 different assays (GFP DNA, IL6, AAV5) using 15 nm and 50 nm as starting gold nanoparticle size for grow th.DETAILED DESCRIPTION OF THE INVENTION DefinitionsTerms used in the claims and specification are to be construed in accordance with their usual meaning as understood by one skilled in the art except and as defined as set forth below.“About,” as used herein, refers to within ± 10%, preferably ± 5% of the recited value.An "analyte-binding" molecule, as used herein, refers to any molecule capable of participating in a specific binding reaction with an analyte molecule. Examples include but are not limited to, (i) antigen molecules, for use in detecting the presence of antibodies specific against that antigen; (ii) antibody molecules, for use in detecting the presence of antigens; (iii) protein molecules, for use in detecting the presence of a binding partner for that protein; (iv) ligands, for use in detecting the presence of a binding partner; or (v) single stranded nucleic acid molecules, for detecting the presence of its complementary nucleic acid molecules.An “aspect ratio” of a shape refers to the ratio of its longer dimension to its shorter dimension.A “binding molecule,” refers to a molecule that is capable to bind another molecule of interest.“A binding pair,” as used herein, refers to two molecules that are attracted to each other and specifically bind to each other. Examples of binding pairs include, but not limited to, an antigen and an antibody against the antigen, a ligand and its receptor, complementary strands of nucleic acids, biotin and avidin, biotin and streptavidin, lectin and carbohydrates. Preferred binding pairs are biotin and streptavidin, biotin and avidin, fluorescein and antifluorescein, digioxigenin / anti-digi oxigenin.“Immobilized,” as used herein, refers to reagents being fixed to a solid surface. When a reagent is immobilized to a solid surface, it is either be non-covalently bound or covalentlybound to the surface.“A monolithic substrate,’' as used herein, refers to a single piece of a solid material such as glass, quartz, or plastic that has one refractive index.A '‘probe,” as used herein, refers to a monolithic substrate having as aspect ratio (length-to-width) of at least 2 to 1 with a thin-film layer coated on the sensing side. A probe has a distal end and a proximal end. The proximal end (also refers to probe tip in the application) has a sensing surface coated with a thin layer of analyte-binding molecules.The present invention relates to a biochemical assay based on bio-layer interferometry (BLI) using the growth of metal nanoparticles (NPs) to enhance the signals. BLI detects biomolecule binding in real time by monitoring the change in interference between light beams reflected from a proximal and a distal reflecting surface. In the present methods, the target analyte molecule is first immobilized onto the BLI biosensors. Biofunctionalized metal nanoparticles (NPs) then bind to the immobilized target molecules, which may provide a BLI interference signal. In the present method, the detection signal is improved by growing the immobilized metal NPs (such as AuNPs) on the BLI sensor. The immobilized metal NPs (about 5-100 nm) provide seeds to grow their size. The present method includes a step of growing the immobilized metal NPs by reducing a new reagent metal ions (e.g., Au (+3)) to metal (0) (for example. Au (0)) and depositing the new metal (0) on the existing metal NPs, thus growing the size of metal NPs to 4-10 pm. This results in improved sensitivity for detecting target biomolecules on the biolayer interferometry system.Metal NPs suitable for the present invention include, but are not limited to, NPs made of gold (Au), silver (Ag). platinum (Pt), palladium (Pd). and copper (Cu). Gold, silver, platinum, palladium, and copper can be synthesized as nanoparticles and then the nanoparticles can grow in size through the reduction of the metal ions to metal and depositing on the existing nanoparticles.One assay format schematic is illustrated in FIG. 1. Depending on the analyte target, the BLI biosensor is first coated with biomolecules, for example, an antibody against the analyte target. This layer allows the BLI biosensors to bind with target analyte biomolecules. Both the thickness and density of bound target biomolecules are concentration-dependent and are proportional to the optical signal. A biotinylated antibody against the analyte target is then bound to the target to introduce a biotin group onto the target.Metal NPs (such as AuNPs or AgNPs) functionalized with streptavidin are incubatedwith the biosensor to bind metal NPs with the target biomolecules through the biotinstreptavidin interaction. The core size of the metal NPs such as AuNPs is typically between 5 nm and 100 nm. The amount of bound metal NPs such as AuNPs is proportional to the amount of bound target biomolecules.After the target biomolecule and AuNPs are formed on the biosensor, the biosensor is exposed to a solution comprising one or more reducing agents and metal ions. The reducing agent can be hydroxylamine, hydrogen peroxide, or hydroquinone. The metal ion can be Au3+, Ag+, Pt2+, Pd2+, or Cu2+. The reducing agent and metal ion are pre-mixed together as the working solution at the appropriate concentration. A typical mixture is hydroxylamine mixed with 11 AuCI-i. AgNOs. PtCh, PdCh, or CuCb in water.As the reducing agent and the metal ion react with the metal NPs on the biosensors, the metal NPs grow larger in size. This size growth is captured by the biolayer interferometry. Due to the refractive index of metal nanoparticles, the increase in metal NPs size results in a negative signal shift on the interference pattern. The signal can be inverted before analysis. The final signal increases are then plotted against the known concentration of the target biomolecules to generate a standard curve. The signal generated from the unknown sample can then be calculated to obtain the concentration.Biosensor Interferometer SystemsThe present invention is suitable for several biosensor interferometer systems. FIGs. 2A-2B illustrate one example of such a system in which a solid support is a probe. FIG. 2A depicts a biosensor interferometer 100 (or simply “interferometer”) that includes a light source 102, a detector 104, a waveguide 106, and an optical assembly 108 (also referred to as a “probe”). The probe 108 may be connected to the waveguide 106 via a coupling medium.The light source 102 may emit white light that is guided toward the probe 108 by the waveguide 106. For example, the light source 102 may be a light-emitting diode (LED) that is configured to produce light over a range of at least 50 nanometers (nm), 100 nm, or 150 nm within a given spectrum (e.g., 400 nm or less to 700 nm or greater). Alternatively, the interferometer 100 may employ a plurality of light sources having different characteristic wavelengths, such as LEDs designed to emit light at different wavelengths in the visible range. The same function could be achieved by a single light source with suitable filters for directing light with different wavelengths onto the probe 108.The detector 104 is preferably a spectrometer, such as an Ocean Optics USB4000,that is capable of recording the spectrum of interfering light received from probe 108. Alternatively, if the light source 102 operates to direct different wavelengths onto the probe 108, then the detector 104 can be a simple photodetector capable of recording intensity at each wavelength. In another embodiment, the detector 104 can include multiple filters that permit detection of intensity at each of multiple wavelengths.The waveguide 106 can be configured to transport light emitted by the light source 102 to the probe 108. and then transport light reflected by surfaces within the probe 108 to the detector 104. In some embodiments the waveguide 106 is a bundle of optical fibers (e.g., single-mode fiber optic cables), while in other embodiments the waveguide 106 is a multimode fiber optic cable.As shown in FIG. 2B, the probe 108 includes a monolithic substrate 114, a thin-film layer (also referred to as an “interference layer”), and a biomolecular layer (also referred to as a “biolayer”) comprised of analyte molecules 122 that have bound to analyte-binding molecules 120. The monolithic substrate 114 is comprised of a transparent material through which light can travel. The interference layer is also comprised of a transparent material. When light is shone on probe 108, the proximal surface of the interference layer may act as a first reflecting surface and the biolayer may act as a second reflecting surface. As further described below, light reflected by the first and second reflecting surfaces may form an interference pattern that can be monitored by the interferometer 100.The interference layer normally includes multiple layers that are combined in such a manner to improve the detectability7of the interference pattern. Here, for example, the interference layer is comprised of a tantalum pentoxide (Ta2Os) layer 116 and a silicon dioxide (S1O2) layer 118. The tantalum pentoxide layer 116 may be thin (e.g., on the order of 10-40 nm) since its main purpose is to improve reflectivity at the proximal surface of the interference layer. Meanwhile, the silicon dioxide layer 118 may be comparatively thick (e.g., on the order of 650-900 nm) since its main purpose is to increase the distance between the first and second reflecting surfaces.To perform a test, the probe 108 can be suspended in a microwell 110 (or simply “well”) that includes a sample 112. Analyte molecules 122 will bind to the analyte-binding molecules 120 along the distal end of probe 108 over the course of the diagnostic test, and these binding events will result in an interference pattern that can be observed by the detector 104. The interferometer 100 can monitor the thickness of the biolayer formed along the distal end of the probe 108 by detecting shifts in a phase characteristic of the interference pattern.FIG. 3 illustrates another biosensor interferometer probe. The probe includes a monolithic substrate that has a first surface and a second surface arranged substantially parallel to one another at opposite ends of the monolithic substrate, an interference layer coated on the second surface of the monolithic substrate, and a layer of analyte-binding molecules coated on the interference layer. The interference layer will generally be comprised of magnesium fluoride (MgF2). A first interface between the monolithic substrate and the interference layer acts as a first reflecting surface when light is shone on the interferometric sensor, while a second interface between a biolayer formed by analyte molecules in a sample binding to the analyte-binding molecules and a solution containing the sample acts as a second reflecting surface when the light is shone on the probe. As described above, the thickness of the biolayer can be estimated based on the interference pattern of light reflected by the first and second reflecting surfaces.Probe 200 includes an interference layer 204 that is secured along the distal end of a monolithic substrate 202. Analyte-binding molecules 206 can be deposited along the distal surface of the interference layer 204. Over the course of a biochemical test, a biolayer will form as analyte molecules 208 in a sample bind to the analyte-binding molecules 206.

[0035] As shown in Figure 2, the monolithic substrate 202 has a proximal surface (also referred to as a “coupling side”) that can be coupled to, for example, a waveguide of an interferometer and a distal surface (also referred to as a “sensing side”) on which additional layers are deposited. Generally, the monolithic substrate 202 has a length of at least 3 millimeters (mm), 5 mm, 10 mm, or 15 mm. In a preferred embodiment, the aspect ratio (length-to-width) of the monolithic substrate 202 is at least 5 to 1. In such embodiments, the monolithic substrate 202 may be said to have a columnar form. The cross section of the monolithic substrate 202 may be a circle, oval, square, rectangle, triangle, pentagon, etc. The monolithic substrate 202 preferably has a refractive index that is substantially higher than the refractive index of the interference layer 204, such that the proximal surface of the interference layer 204 effectively reflects light directed onto the probe 200. The preferred refractive index of the monolithic substrate may be higher than 1.5, 1.8, or 2.0. Accordingly, the monolithic substrate 202 may be comprised of a high-refractive-index material such as glass (refractive index of 2.0) rather than a low-refractive-index material such as quartz (refractive index of 1.46) or plastic (refractive index of 1.32-1.49).The interference layer 204 is comprised of at least one transparent material that is coated on the distal surface of the monolithic substrate 202. These transparent material(s) aredeposited on the distal surface of the monolithic substrate 202 in the form of thin films ranging in thickness from fractions of a nanometer (e.g., a monolayer) to several micrometers. The interference layer 204 may have a thickness of at least 500 nm, 700 nm, or 900 nm. An exemplary thickness is between 500-5,000 nm (and preferably 800-1,200 nm). Here, for example, the interference layer 204 has a thickness of approximately 900-1,000 nm, or 940 nm.In contrast to conventional probes, the interference layer 204 has a substantially similar refractive index as the biolayer. This ensures that the reflection from the distal end of the probe 200 is predominantly due to the analyte molecules 208 rather than the interface between the interference layer 204 and the analyte-binding molecules 206. In some embodiments the interference layer 204 is comprised of magnesium fluoride (MgF2). while in other embodiments the interference layer 204 is comprised of potassium fluoride (KF), lithium fluoride (LiF), sodium fluoride (NaF), lithium calcium aluminum fluoride (LiCaAlFe), sodium aluminum fluoride (NasAIFe). strontium fluoride (SrF2), aluminum fluoride (AlFs). sulfur hexafluoride (SFe), etc. Magnesium fluoride has a refractive index of 1 .38, which is substantially identical to the refractive index of the biolayer formed along the distal end of the probe 200. For comparison, the interference layer of conventional probes is normally comprised of silicon dioxide, and the refractive index of silicon dioxide is approximately 1.4-1.5 in the visible range. Because the interference layer 204 and biolayer have similar refractive indexes, light will experience minimal scattering as it travels from the interference layer 204 into the biolayer and then returns from the biolayer into the interference layer 204.In one embodiment, probe 200 includes an adhesion layer that is deposited along the distal surface of the interference layer 204 affixed to the monolithic substrate 202. The adhesion layer may be comprised of a material that promotes adhesion of the analyte-binding molecules 206. One example of such a material is silicon dioxide. The adhesion layer is generally very thin in comparison to the interference layer 204, so its impact on light traveling tow ard, or returning from, the biolayer will be minimal. For example, the adhesion layer 310 may have a thickness of approximately 3-10 nm, while the interference layer 304 may have a thickness of approximately 800-1,000 nm. The biolayer formed by the analytebinding molecules 306 and analyte molecules 308 will normally have a thickness of several nm.When light is shone on probe 200, the proximal surface of the interference layer 204may act as a first reflecting surface and the distal surface of the biolayer may act as a second reflecting surface. The presence, concentration, or binding rate of analyte molecules 208 to the probe 200 can be estimated based on the interference of beams of light reflected by these two reflecting surfaces. As analyte molecules 208 attach to (or detach from) the analytebinding molecules 206, the distance between the first and second reflecting surfaces will change. Because the dimensions of all other components in the probe 200 remain the same, the interference pattern formed by the light reflected by the first and second reflecting surfaces is phase shifted in accordance with changes in biolayer thickness due to binding events.In operation, an incident light signal 210 emitted by a light source is transported through the monolithic substrate 202 toward the biolayer. Within probe 200, light will be reflected at the first reflecting surface resulting in a first reflected light signal 212. Light will also be reflected at the second reflecting surface resulting in a second reflected light signal 214. The second reflecting surface initially corresponds to the interface between the analytebinding molecules 206 and the sample in which the probe 200 is immersed. As binding occurs during the biochemical test, the second reflecting surface becomes the interface between the analyte molecules 208 and the sample.The first and second reflected light signals 212, 214 form a spectral interference pattern, as shown in FIG. 4A. When analyte molecules 208 bind to the analyte-binding molecules 206 on the distal surface of the interference layer 204, the optical path of the second reflected light signal 214 will lengthen. As a result, the spectral interference pattern shifts from TO to T1 as shown in FIG. 4B. By measuring the phase shift continuously in real time, a kinetic binding curve can be plotted as the amount of shift versus the time. The association rate of an analyte molecule to an analyte-binding molecule immobilized on the distal surface of the interference layer 204 can be used to calculate analyte concentration in the sample. Hence, the measure of the phase shift is the detection principle of a thin-film interferometer.BLI Assay-Protein AnalyteIn one aspect, the present method detects a protein analyte in a liquid sample.The method comprises the steps of: (a) obtaining a solid support having a first antibody against an analyte immobilized on the surface of the solid support; (b) contacting the surface with a liquid sample comprising the analyte for a first period of time to bind theanalyte to the surface; (c) washing the surface to remove unbound materials; (d) contacting the surface with a second antibody against the analyte conjugated with a first member of a binding pair for a second period of time; (e) washing the surface to remove unbound materials; (f) contacting the surface with metal NPs (e.g., AuNPs) coated with a second member of the binding pair for a third period of time; (g) washing the surface to remove unbound materials; (h) determining a baseline interferometry pattern; (i) contacting the surface with a solution comprising hydroxylamine and metal ions (e.g., HAuChjfor a fourth period of time, and determining a second interferometry pattern; and (j) determining the analyte concentration in the sample by measuring the interferometry phase shift between the second interferometry' pattern and the baseline interferometry pattern, and quantitating the phase shift against a calibration curve.In one embodiment, the AuNPs of step (I) have a size of 7-50 nm.In one embodiment, the binding pair is biotin and streptavidin, biotin and avidin, fluorescein and anti-fl uorescein, or digi oxigenin or anti-digioxigenin. For example, the first member of the binding pair is biotin and the second member of the binding pair is streptavidin.In step (a) of the present method, one embodiment of a solid support is a probe that has a small tip for binding an analyte is obtained. The tip has a smaller surface area with a diameter < 5 mm, preferably < 2 mm or < 1 mm. The small surface of the probe tip provides several advantages. In solid phase immunoassays, having a small surface area is advantageous because it has less non-specific binding and thus produces a lower background signal. Further, the reagent or sample carry over on the probe tip is extremely small due to the small surface area of the tip. This feature makes the probe tip easy to wash and results in negligible contamination in the wash solution since the wash solution has a larger volume. Another aspect of the small surface area of the probe tip is that it has small binding capacity. Consequently, when the probe tip is immersed in a reagent solution, the binding of the reagent does not consume a significant amount of the reagent. The reagent concentration is effectively unchanged. Negligible contamination of the wash solution and small consumption of the reagents enable the reagents and the wash solution to be re-used many times, for example, 3-10 times, 3-15 times, or 3-20 times.Methods to immobilize an antibody to the solid phase (the sensing surface of the probe tip) are common in immunochemistry and involve formation of covalent, hydrophobic or electrostatic bonds between the solid phase and a hapten. For example, an antibody can beadsorbed directly to the solid surface or by covalently binding to aminopropylsilane coated on the solid surface. Alternatively, an antibody can bind to its antigen which is immobilized on the solid surface.In step (b) of the method, the surface of the solid support is contacted with a sample solution to bind the analyte to the solid support.In steps (c), (e), and (g), the solid support is washed by a wash solution having pH of 6.0-8.5 for a short time (e.g., 5 seconds to 5 minutes, 10 seconds to 2 minutes, or 30 seconds to 1 minute), to remove unbound materials.In step (d), the solid support surface is contacted with a second antibody against the analyte conjugated with a first member of a binding pair. In one embodiment, the first and the second antibody are different.In step (I), the solid support surface is contacted with AuNPs coated with a second member of the binding pair. The AuNPs typically have a size 5-100 nm, preferably 5-50 or 7- 50 nm to provide good signals and sensitivity' for the assay. AuNPs of 7 nm can be purchased commercially, e.g.. from Luna Nanotech. AuNPs of a larger size can be prepared by reduction of Au (3+) to Au (0) with one or more reducing agents such as citrate and hydroquinone.In one embodiment, the second member of the binding pair is coated on the gold nanoparticle by adsorption. In another embodiment, the second member of the binding pair is coated on the gold nanoparticle through covalently binding to a functionalized linker that is adsorbed on the AuNPs. One example of the linker is polyethylene glycol (PEG). In one embodiment, the PEG linker saturates the AuNPs and cover the AuNPs such that cannot bind more PEGs. PEG increases the stability of AuNPs in a solution without affecting the growth of the AuNPs in a later step of the assay, and does not affect the BLI assay performance.In step (h), the baseline interferometry pattern is determined.In step (i), the solid support surface is contacted with a solution comprising hydroxylamine and HAuCU for a period of time to reduce gold ion (Au+3) to gold (Au°) and to grow the size of the AuNPs. In one embodiment, the solution comprises 0.5-20 mg / mL or 1-10 mg / mL hydroxylamine, and 1-20 mg / mL or 5-10 mg / mL HAuCI-i. The newly produced gold deposits on the existing AuNPs and grows the size of nanoparticles. Based on the microscope image, AuNPs starting from their sizes such as 15, 50, 80 nm grow to about 4-8 pm in diameter. After the AuNPs grow their size to pm level, the wavelength shifts of a second interferometry' pattern is determined.In step (j), the analyte concentration in the sample is quantitated by determining the interferometry phase shift between the second interferometry patern and the baseline interferometry patern, and quantitating the wavelength phase shift against a calibration curve to determine the analyte concentration.By growing the gold nanoparticle size from nanometer to micrometer in diameter, the BLI signal of the assay increases significantly. For example, without growing the size of 15 nm AuNPs. there is no detectable BLI signal. Without growing the size of 50 nm or 80 nm AuNPs, there is a very small signal but not sufficient for detecting an analyte.The wavelength phase shift can be monitored either kinetically or determined by the difference between starting time point (TO) and end time point (Tl) (see FIG 4B).In one embodiment, when the solid support is a probe, the reaction can be accelerated by agitating or mixing the solution in the vessel that the probe is dipped in. For example, a flow such as a lateral flow or an orbital flow of the solution across the probe tip can be induced in one or more reaction vessels, including sample vessel, reagent vessel, wash vessels, and regeneration vessel, to accelerate the binding reactions, disassociation. For example, the reaction vessels can be mounted on an orbital shaker and the orbital shaker is rotated at a speed at least 50 rpm, preferably at least 200 rpm or at least 500 rpm, such as 50- 200 or 500-1,500 rpm. Additionally, the probe tip can be moved up and down and perpendicular to the plane of the orbital flow, at a speed of 0.01 to 10 mm / second, in order to induce additional mixing of the solution above and below the probe tip.In one embodiment, during gold grow th step (i), the assay performance is improved by reaction can be accelerated by agitating or mixing the gold growth solution in the vessel that the probe is dipped in. With agitation, the signal increase (as a result of gold growth) is smooth and fits into a curve. Without agitation, the reaction may not proceed consistently, and the signal increase may be erratic.Assay Protocol-DNA AnalyteIn one aspect, the present method detects the concentration of a nucleic acid of interest in a liquid sample. The method uses one capture oligonucleotide to hybridize to one region of the nucleic acid (e.g.. DNA) of interest and to immobilize the hybridized complex to a BLI probe, and one signal oligonucleotide to hybridize to another region of the nucleic acid of interest and to generate the signals for BLI detection.The method comprises the steps of: (a) obtaining a first DNA probe (a captureoligonucleotide) that comprises a first member of a first binding pair; (b) obtaining a second DNA probe (a signal oligonucleotide) that comprise a first member of a second binding pair; wherein the first and the second DNA probes further comprise ssDNAs complementary to different regions of the ssDNA analyte, and the first member of the first binding pair and the first member of the second binding pair are different haptens; (c) hybridizing the ssDNA analyte with the first and second DNA probes to form double-stranded (ds) DNA, (d) contacting the dsDNA of (c) with a solid support comprising a second member of the first binding pair immobilized on the surface of the solid support, to bind the dsDNA on the surface of the solid support through the first binding pair; (e) washing the surface to remove unbound materials; (f) contacting the surface with AuNPs coated with a second member of the second binding pair for a first period of time; (g) washing the surface to remove unbound materials; (h) determining a baseline interferometry pattern; (i) contacting the surface with a solution comprising hydroxylamine and HAuC14 for a second period of time to grow the gold particles, and determining a second interferometry pattern; and (j) determining the analyte concentration in the sample by measuring the interferometry’ phase shift between the second interferometry pattern and the baseline interferometry pattern, and quantitating the phase shift against a calibration curve.In one embodiment, the AuNPs of step (f) have a size of 7-50 nm.In one embodiment, the first and the second binding pairs are biotin and streptavidin, biotin and avidin, fluorescein and anti-fluorescein, or digioxigenin or anti-digioxigenin.In one embodiment, wherein the first member of the second binding pair is biotin and the second member of the second binding pair is streptavidin.The details of each step are similar to those of a corresponding similar step, if any, described above in the assay protocol for protein analyte.Assay Protocol-Antibody AnalyteIn one aspect, the present method detects an antibody analyte in a liquid sample.The method comprises the steps of: (a) obtaining a solid support having an antigen of the antibody analyte immobilized on the surface of the solid support; (b) contacting the surface with a liquid sample comprising the antibody analyte for a first period of time to bind the antibody analyte to the surface; (c) washing the surface to remove unbound materials; (d) contacting the surface with an antibody that against the target antibody, e.g. an anti-human IgG antibody conjugated with a first member of a binding pair for a second period of time;(e) washing the surface to remove unbound materials; (f) contacting the surface with AuNPs coated with a second member of the binding pair for a third period of time; (g) washing the surface to remove unbound materials; (h) determining a baseline interferometry pattern; (i) contacting the surface with a solution comprising hydroxylamine and H AuCh for a fourth period of time, and determining a second interferometry pattern; and (j) determining the analyte concentration in the sample by measuring the interferometry phase shift between the second interferometry pattern and the baseline interferometry pattern, and quantitating the phase shift against a calibration curve.The details of each step are similar to those of a corresponding similar step, if any, described above in the assay protocol for protein analyte.Competitive Assay Protocol-Protein AnalyteIn one aspect, the present method detects a protein analyte in a liquid sample by a competitive assay format.The method comprises the steps of: (a) obtaining a solid support having an antibody against an analyte immobilized on the surface of the solid support; (b) contacting the surface with (i) a liquid sample comprising the analyte, and (ii) a conjugate comprising the analyte covalently linked to a first member of a binding pair for a first period of time: (c) washing the surface to remove unbound materials; (d) contacting the surface with AuNPs coated with a second member of the binding pair for a second period of time; (e) washing the surface to remove unbound materials; (f) determining a baseline interferometry pattern; (g) contacting the surface with a solution comprising hydroxylamine and HAuCk for a third period of time, and determining a second interferometry pattern; and (h) determining the analyte concentration in the sample by measuring the interferometry phase shift between the second interferometry pattern and the baseline interferometry pattern, and quantitating the phase shift against a calibration curve.In step (b), the presence of the analyte competes with the conjugate for binding to the antibody against the analyte on the solid surface. The higher concentration of the analyte, the lower signal will generate in the competitive assay.The details of each step are similar to those of a corresponding similar step, if any, described above in the assay protocol for protein analyte.The invention is illustrated further by the following examples that are not to be construed as limiting the invention in scope to the specific procedures described in them.EXAMPLESExample 1. Preparing 15 nm gold nanoparticles15 nm gold nanoparticles were prepared through the citrate reduction reaction of Au (3+) to Au (0). No strong reducing agent such as hydroquinone was used to control the size.A 250 mL Erlenmeyer flask was washed with aqua regia (three parts of hydrochloric acid and one part of nitric acid). After being rinsed thoroughly with water, 100 mL of deionized water was added to the flask along with a magnetic stirring bar. Then, 1 mL of 3% w / v sodium citrate in DI water was added to the flask. The mixture was heated until boiling. When boiling vigorously, the stirring speed was increased until even mixing, and 100 pL of 10% w / v HAuCh in DI water was added. The reaction was allowed to proceed for 10 minutes. The reaction was quenched by placing it on ice for 20 mins. This preparation yields gold nanoparticles with a core size around 15 nm. This was determined by dynamic light scattering.Example 2. Preparing 50 nm and SO nm gold nanoparticlesTo prepare 50 nm sized gold nanoparticles, 15 nm gold nanoparticles were used as the seed for the particles to grow. This is achieved by reducing HAuC14 onto the surface of gold nanoparticles. First, 100 mL of water is added to an Erlenmeyer flask along with a magnetic stirring bar to ensure mixing. Then, 3.35mL of 15 nm gold nanoparticles at 2.4 nM was added, followed by the additional of 1 mL of 10% w / v HAuCh and 1 mL of Sodium Citrate at 4.4 mg / mL. The solution was mixed by using a magnetic stirring bar. Next, 100 pL of hydroquinone (SigmaAldrich Cat# 240125) at 2.75 mg / mL was added to the solution. A strong reducing agent hydroquinone was used to grow the size to 50 nm. Citrate acted here as a protecting agent to ensure colloidal stability. Citrate not only had weak reducing power but also was adsorbed to the surface of gold nanoparticles, which provided negative charge and prevented the gold nanoparticle from aggregating by charge repulsion.The reaction was allowed to proceed for overnight at room temperature. To purify out the excessive reagent, the 50 nm gold nanoparticles were washed by centrifugating at 1000 g for 2 hours. The supernatant was discarded, and the nanoparticle pellet was resuspended in 0.02% sodium citrate in water. The nanoparticles were centrifuged again, and this process was repeated for 2 more times. The gold particle size was determined by dynamic light scattering to be 50 nm.To prepare 80 nm sized gold nanoparticles, the procedure was similar to 50 nm gold nanoparticle preparation except the amount of 15 nm gold nanoparticles and the concentrations of HAuCh and hydroquinone were adjusted. First, 100 mL of water was added to an Erlenmeyer flask along with a magnetic stirring bar to ensure mixing. Then, 0.595 mL of 15 nm gold nanoparticles at 2.4 nM in water were added into a flask, followed by the additional of 100 pL of 10% w / v HAuC14 and 100 pL of sodium citrate at 4.4 mg / mL. The solution was mixed by using a magnetic stirring bar. Next, 100 pL of hydroquinone (SigmaAldrich) at 2.75 mg / mL was added to the solution. The reaction was allowed to proceed for overnight at room temperature. To purify out the excessive reagent, the 50 nm gold nanoparticles were washed by centrifugating at 600 g for 2 hours. The supernatant was discarded, and the nanoparticle pellet was resuspended in 0.02% sodium citrate in water. The nanoparticles were centrifuged again, and this process was repeated 2 more times. The gold particle size was determined by dynamic light scattering as 50 nm.Example 3. Preparing streptavidin-coated gold nanoparticleGold nanoparticles of 15 nm, 50 nm, and 80 nm were prepared according to Examples 1 or 2.The gold nanoparticles were then functionalized by Thiol PEG 3000 Da (mPEG3k- SH; Nanocs inc.) and Azido PEG Thiol 5000 Da (N3-PEG5k-SH; Nanocs inc.). First, Ns- PEG5k-SH dissolved in water was added to the gold nanoparticles solution at a range between 0. 1 to 1 PEG molecule per nm2of the surface area of gold nanoparticles. The reaction was carried out at room temperature for 1 hour. Then, mPEG3k-SH dissolved in water w as added at 5 PEG molecules per nm2of the surface area of gold nanoparticles. The reaction was carried out at room temperature for another hour. Lastly, dibenzocyclooctyne (DBCO)-modified streptavidin proteins prepared in Example 4 below' were added and incubated with the gold nanoparticles at 500 molecules / nm2of the surface area. Finally, the reaction mixture was incubated overnight at room temperature.The excess unreacted mPEG3k-SH, N3-PEG5k-SH, and streptavidin proteins were removed by centrifugation washes. First, gold nanoparticles were centrifuged at 12,000 g centrifugal force for 30 minutes. This concentrated the gold nanoparticles into a small pellet at the bottom of the tube. Then the supernatant was discarded. A 1 mL solution of lx PBS + 0.02% Tween 20 was added to redisperse the gold nanoparticles. This centrifugation w ash was carried out 2 more times.Example 4. Dibenzocyclooctyne (DBCO)-modified streptavidin proteinsDBCO-Streptavidin proteins were prepared by reacting DBCO that is linked with aN- Hydroxy succinimide (NHS) group with native streptavidin proteins at 5: 1 molar coupling ratio. First, streptavidin proteins in solid form were weighed and dissolved in lx PBS to make a 1 mL of 10 mg / mL solution. DBCO-PEG4-NHS Ester (Vector laboratories Cat# CCT- A134-2) was prepared fresh and dissolved in DMSO to make a 5 mg / mL stock solution. 124 pL of the DBCO-PEG4-NHS Ester stock solution was added to the streptavidin solution, and the reaction was carried out for 1 hour at room temperature. To remove the unreacted reagents, the DBCO-modified streptavidin proteins were purified through a Zeba™ Spin Desalting according to the manufacturer’s protocol (ThermoFisher, Cat# 89882)Example 5. Anti-Fluorescein Biosensor CoatingQuartz biosensor, 1 mm in diameter and 2 cm in length were coated with aminopropylsilane (APS) using a chemical vapor deposition process (Yield Engineering Systems, 1224P) using manufacturer’s protocols. The APS coated biosensors were washed with lx PBS for 30 seconds, followed by coating with Ficoll-Anti-Fluoresceine conjugate at a concentration of 50 pg / mL in lx PBS buffer under orbital shaking of 1000 rpm for 10 minutes. Then the biosensors were washed again with lx PBS for 30 seconds followed by dipping in 15% sucrose solution for 60 seconds and drying at 37°C for 20 mins.Example 6. Gold growth solutionThe gold growth solution was composed of HAuCk and hydroxylamine and were freshly made for each use. The two reagents were kept separately and only mixed together before running the assay.Before the quantitation assay, 500 pL of hydroxylamine (20 mg / mL in DI water) and 500 pL H AuCh solution (2 mg / ml) were mixed at 1 : 1 ratio.Example 7. Biosensors coated with Anti-AAVXQuartz biosensor, 1 mm in diameter and 2 cm in length were coated with aminopropylsilane (APS) using a chemical vapor deposition process (Yield Engineering Systems, 1224P) using manufacturer’s protocols. The APS coated biosensors were washed with lx PBS for 30 seconds followed by coating with Ficoll-Anti-Fluoresceine conjugate at a concentration of 50 pg / mL in lx PBS buffer under orbital shaking of 1000 rpm for 10 mins.Then the biosensors were washed with coating buffer (0.5% BSA in PBST: 0.05% Tween 20 in IxPBS buffer) for 60 seconds followed by quick 20 seconds wash with PBST buffer (0.05% Tween 20 in IxPBS buffer) at 1000 rpm. The biosensors were coated with Fluorescein-Anti-AlexaFluor 647 antibody at 20 pg / mL in coating buffer for 300 seconds at 1000 rpm. The biosensors were washed with coating buffer for 60 seconds and then with PBST buffer for 20 seconds, both at 1000 rpm. Then the biosensor was coated with Captures elect™ AlexaFluor 647-Anti-AAVX (Thermo Fischer) at 7.5 pg / mL in coating buffer for 300 seconds at orbital shaking speed of 1000 RPM. The biosensors were washed with coating buffer for 60 seconds and with PBST buffer for 20 seconds at 1000 rpm. Then the biosensors were dipped in preservatives (15% sucrose in IxPBS buffer) for 60 seconds at 1000 rpm. The biosensors were then air dried at 37°C for 20 minutes.Example 8. Anti-IL6 Biosensors Coated with Anti-IL6Quartz biosensor, 1 mm in diameter and 2 cm in length were coated with aminopropylsilane (APS) using a chemical vapor deposition process (Yield Engineering Systems, 1224P) using manufacturer’s protocols. The APS coated biosensors were washed with lx PBS for 30 seconds followed by coating with Ficoll-Anti-Fluoresceine conjugate at a concentration of 50 pg / mL in lx PBS buffer under orbital shaking of 1000 rpm for 10 mins. Then the biosensors were washed with coating buffer (0.5% BSA, PBST) for 60 seconds followed by 20 seconds wash with PBST buffer at 1000 rpm. The biosensors were coated with Fluorescein Anti-IL6 Antibody at 20 pg / mL in coating buffer for 300 seconds at 1000 rpm. The biosensors were washed with PBST buffer for 20 seconds, both at 1000 rpm. The biosensors were dipped in preservatives (15% sucrose in lx PBS buffer) for 60 seconds at 1000 rpm. The biosensors were then air dried at 37°C for 20 minutes. The entire process is shown in Table 3 below .Example 9. Quantitation assay of single-stranded DNA (GFP oligonucleotide) OligonucleotidesAll oligonucleotides were purchased from Integrated DNA Technologies and HPLC purified. Oligonucleotide modification by fluorescein or biotin was requested during purchase.Oligo 1 (synthetic GFP positive stranded oligonucleotide, Region A is the first underlined nucleotides, and Region B is the second underlined nucleotides)5 '-CAA CGT CTA TAT CAT GGC CGA CAA GCA GAA GAA CGG CAT CAA GGT GAA CTT CAA GAT CCG CCA CAA CAT CGA GGA CGG CAG CGT GCA GCTCGC CGA CCA CTA CCA GCA GAA CAC CCC CAT CGG CGA CGG CCC CGT GCT GCT GCC CGA CAA CCA CTA CCT GAG CAC CCA GTC CGC CCT GAG CAA AGA CCC CAA CGA G-3’ (SEQ ID NO: 1)Oligo 2 (5 ’-fluorescein GFP hybridizing oligonucleotide, for capturing, See) hybridizes to Region B of Oligo 1.5’F*-TGC TCA GGG CGG ACT GGG TGC TCA GGT AGT GGT TGT CGG G-3’ (SEQ ID NO: 2)Oligo 3 (5 ’-biotinylated GFP hybridizing oligonucleotide, for signaling,) hybridizes to Region A of Oligo 1.5 B*-CTT GAA GTT CAC CTT GAT GCC GTT CTT CTG CTT GTC GG-3’ (SEQ ID NO: 3)The assay was started by hybridizing target single-stranded DNA (Oligo 1) at different concentrations to form double-stranded DNA. First, target DNA (Oligo 1) was incubated with two single-stranded DNA Oligo 1 and Oligo 2 for hybridizing into doublestranded DNA. Oligo 2 had a fluorescein molecule on one end. Oligo 3 had a biotin molecule on one end. The mixture was incubated in a solution with a final concentration of 5M NaCl, 50mM phosphate Buffer, 5mM ethylenediaminetetraacetic acid (EDTA), and 7.3% w / v Tween 20 for 5 minutes at 95 °C. The solution was cooled down by been incubated at 4 °C for 5 minutes. The hybridized double-stranded DNA was diluted 5 times in the Q buffer (PBS + 0.02% Tween 20 + BSA 0.02%).The assay was performed on a BLI system, and each step is shown in Table 1. Anti- Fluorescein Biosensors prepared in Example 5 were pre-wetted in a Max plate (Gator Bio. Cat# 130062) (Step 1) for 2 minutes at 1000 RPM.To start the quantitation assay, anti-Fluorescein Biosensors were incubated with the hybridized double-stranded GFP DNA for 10 minutes at 1000 RPM (Step 2). The biosensors were then washed 3 times in Q buffer at 1000 RPM (Step 3-5). Next, the biosensors were incubated with 100 pL of 50 pM of streptavidin-coated gold nanoparticles (15 nm) prepared according to Example 3 for 5 minutes (Step 6). The probes w ere w ashed four times in deionized w ater at 30 seconds each.Lastly, the biosensors were exposed to 100 pL of gold grow th solution prepared in Example 6 for 10 minutes at 1000 RPM shaking speed, and the interference phase was measured. The results of wavelength shift in nanometers are shown in FIG. 5. and the signals are summarized in Table 2. Table 1. Assay steps and experimental conditions for DNA quantitationTable 2. Results of GFP DNA quantificationExample 10. Quantitation of IL6 protein The assay was performed on a BLI system, and each step is shown in Table 3. All reagents were in Q buffer which is lx PBS + 0.02% Tween20 + BSA 0.02%. To set up the 384-well plate (Greiner Cat# 781900) for the assay, 100 pL of each sample was transferred into the appropriate wells. Q buffers were transferred to column 3 and 5. DI w ater was transferred to column 7-10. 10 pg / ml of Biotinylated-anti-IL6 was transferred to column 4. Streptavidin-coated gold nanoparticles were transferred to column 6. Gold growth solutionwas transferred to column 11. Anti-IL6 Biosensors prepared in Example 6 were pre-wet in a Max plate (Gator Bio. Cat#130062) (Step 1) for 2 minutes at 1000 RPM.To start the quantitation assay, 100 pL of target IL6 proteins at concentrations ranged from 214 to 25385 pg / mL were incubated with anti-IL6 biosensors for 10 minutes at 1000 RPM (step 2) followed by a 100 pL wash solution for 30 seconds at 1000 RPM (step 3). The biosensors were then incubated with 100 pL of 10 pg / ml of biotinylated anti-IL6 for 60 seconds at 1000 RPM. This was followed by one wash in 100 pL of wash solution at 1000 RPM. Next, the biosensors were incubated with a 100 pL of 50 pM of streptavidin-coated gold nanoparticles (15 nm) prepared according to Example 1 for 5 minutes. The biosensors were washed four times in 100 pL of deionized water at 30 seconds each.Lastly, the biosensors were exposed to 100 pL of gold growth solution containing 10 mg / ml of HAuCh and 1 mg / ml of Hydroxylamine in water for 10 minutes at 1000 RPM shaking speed, and the interference phase was measured. The results of wavelength shift in nanometers are shown in FIG. 6. and the signal is summarized in Table 4.Table 3. Assay steps and experimental conditions for IL6 quantitationTable 4. Results of IL6 quantificationExample 11. Quantitation assay of AAV5The assay was performed on a BLI system, and each step is shown in Table 5. All reagents were suspended in Q buffer which is lx PBS + 0.02% Tween20 + BSA 0.02%. 250 pL of wash buffer was transferred to a Max plate (Gator Bio inc. Cat#130062) from column 2 to column 10. To set up the 384-well plate (Greiner Cat# 781900) for the assay, 100 pL of each sample was transferred into the appropriate wells. AAV5 virus in concentrations from 6.86E6 per mL to 5E9 per mL were transferred to column 1. 2.5 pg / ml of Biotinylated-anti- AAV was transferred to column 2. DI water was transferred to column 3-6. Streptavidin- coated gold nanoparticles were transferred to column 7. Gold growth solution was transferred to column 8. AAVX Biosensors prepared in Example 7 were pre-wet in a Max plate (step 1) for 2 minutes at 1000 RPM.To start the quantitation assay, 100 pL of target AAV5 were incubated with AAVX Biosensors for 1500 seconds at 1000 RPM (step 2) followed by a 100 pL wash solution for 10 seconds at 1000 RPM (step 3-5). The biosensors were then incubated with 100 pL of 2.5 pg / ml of biotinylated anti-AAVX for 00 seconds at 1000 RPM (step 6). This was followed by three washes in 100 pL of wash solution at 1000 RPM (step 7-9). Next, the biosensors were incubated with a 100 pL of 50 pM of streptavidin-coated gold nanoparticles (15 nm) prepared according to Example 3 for 5 minutes (step 10). The biosensors were washed four times in 100 pL of deionized water at 30 seconds each (step 11-14).Lastly, the biosensors were exposed to 100 pL of gold growth solution containing 10 pg / ml of HAuCk and 1 mg / ml of Hydroxylamine in water for 10 minutes at 1000 RPM shaking speed, and the interference phase was measured (step 15). The results of wavelengthshift in nanometers are shown in FIG. 7, and the signals are summarized in Table 6.Table 5. Assay steps and experimental conditions for AAV5 quantitationTable 6. Results of AAV5 quantificationExample 12. Assay Results of Different Size of Starting Gold ParticlesGFP, IL6. and AAV5 assays were run according to Examples 9-11, except different starting gold particle size (15, 50, or 80 nm) were used. The assay results of 15 nm and 50 nm are shown in FIG. 8. Starting gold nanoparticle size of 80 nm generated small signals(wavelength shift), and the assay performance is inferior to that of 15 nm or 50 nmLimit of detection (sensitivity) is calculated from a standard curve as the lowest analyte concentration that can be accurately determined in an assay, which is the analyte concentration whose signal is 3 standard deviations from the signal of zero analyte.Table 7 shows the comparison of limit of detection of 15 nm vs. 50 nm of starting gold particle size in three assays. The results show that 15 nm provides a lower limit of detection.Table 7. Results of 15 nm vs. 50 nm of starting gold particle sizeThe invention, and the manner and process of making and using it, are now described in such full, clear, concise and exact terms as to enable any person skilled in the art to which it pertains, to make and use the same. It is to be understood that the foregoing describes preferred embodiments of the present invention and that modifications may be made therein without departing from the scope of the present invention as set forth in the claims. To particularly point out and distinctly claim the subject matter regarded as invention, the following claims conclude this specification.

Claims

WHAT IS CLAIMED IS:

1. A method of detecting an analyte in a liquid sample, comprising the steps of:(a) obtaining a solid support having a first antibody against an analyte immobilized on the surface of the solid support;(b) contacting the surface with a liquid sample comprising the analyte for a first period of time to bind the analyte to the surface;(c) washing the surface to remove unbound materials;(d) contacting the surface with a second antibody against the analyte conjugated with a first member of a binding pair for a second period of time;(e) washing the surface to remove unbound materials;(f) contacting the surface with gold nanoparticles coated with a second member of the binding pair for a third period of time;(g) washing the surface to remove unbound materials;(h) determining a baseline interferometry pattern;(i) contacting the surface with a solution comprising hydroxylamine and HAuCh to grow the size of gold nanoparticles for a fourth period of time, and determining a second interferometry pattern; and(j) determining the analyte concentration in the sample by measuring the interferometry phase shift between the second interferometry pattern and the baseline interferometry pattern, and quantitating the phase shift against a calibration curve.

2. The method of claim 1, wherein the gold nanoparticles of step (f) have a size of 7-50 nm.

3. The method of claim 1, wherein the binding pair is biotin and streptavidin, biotin and avidin, fluorescein and anti-fluorescein, or digioxigenin or anti-digioxigenin.

4. The method of claim 1, wherein the first member of the binding pair is biotin and the second member of the binding pair is streptavidin.

5. The method of claim 1, wherein the solution of step (i) comprises 1-10 mg / mL hydroxylamine and 5-10 mg / mL HAuCh.

6. The method of claim 1, wherein the first antibody and the second antibody are different.

7. A method of detecting a single-stranded (ss) DNA analyte in a liquid sample, comprising the steps of:(a) obtaining a first DNA probe that comprises a first member of a first binding pair;(b) obtaining a second DNA probe that comprises a first member of a second binding pair; wherein the first and the second DNA probes further comprise ssDNA complementary to different regions of the ssDNA analyte, and the first member of the first binding pair and the first member of the second binding pair are different haptens;(c) hybridizing the ssDNA analyte with the first and second DNA probes to form double-stranded (ds) DNA,(d) contacting the dsDNA of (c) with a solid support comprising a second member of the first binding pair immobilized on the surface of the solid support, to bind the dsDNA on the surface of the solid support through the first binding pair;(e) washing the surface to remove unbound materials;(f) contacting the surface with gold nanoparticles coated with a second member of the second binding pair for a first period of time;(g) washing the surface to remove unbound materials;(h) determining a baseline interferometry pattern;(i) contacting the surface with a solution comprising hydroxylamine and HAuC14 for a second period of time to grow the gold particles, and determining a second interferometry pattern; and(j) determining the analyte concentration in the sample by measuring the interferometry phase shift between the second interferometry patern and the baseline interferometry patern, and quantitating the phase shift against a calibration curve.

8. The method of claim 7, wherein the gold nanoparticles of step (f) have a size of 7-50 nm.

9. The method of claim 7 or 8, wherein the first and the second binding pairs are biotin and streptavidin, biotin and avidin, fluorescein and anti-fluorescein, or digioxigenin or anti- digioxigenin.

10. The method of claim 7, wherein the first member of the second binding pair is biotin and the second member of the second binding pair is streptavidin.

11. The method of claim 7, wherein the solution of step (i) comprises 1-10 mg / mL hydroxylamine and 5-10 mg / mL HAuCh.

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