Field of the Invention This invention relates to methods and devices for an assay of an analyte using analyte-specific binding partners. In addition, the invention relates to methods and devices for assays which respond to refractive index changes, such as, for example, surface plasmon resonance assays.

Background of the Invention A variety of methods have been developed to detect mass or refractive index changes in the presence of an analyte. Examples of such methods include internal and external reflection methods, such as, for example, ellipsometry, external Brewster angle reflectometry, evanescent wave reflectometry, Brewster angle reflectometry, critical-angle reflectometry, evanescent wave ellipsometry, surface plasmon resonance, scattered total internal reflection, optical waveguide sensing methods, refractometric optical fiber sensing methods, leaky waveguide sensing methods, resonance light scattering of particles, multilayered grating resonance, and diffraction anomaly grating methods. Surface plasmon resonance provides one example of these techniques that can be used to indicate the presence, amount, or concentration of an analyte in a sample based on a change in surface refractive index. In particular, as described, for example, in U. S. Patents Nos. 4,931,384; 4,828,387; 4,882,288; 4,992,385; 5,118,608; 5,164,589; 5,310,686; 5,313,264; 5,341,215; 5,492,840; 5,641,640; 5,716,854; 5,753,518; 5,898,503; 5,912,456; 5,926,284; 5,944,150; 5,965,456; 5,972,612; and 5,986,762, PCT Patent Applications Publication Nos. WO 88/07202 and WO 88/10418, and UK Patent Application Publication No. GB 2 202 045, all of which are incorporated herein by

reference, the shift of a notch in a wavelength modulated surface plasmon resonance spectrum can be correlated to the presence, amount, or concentration of an analyte in a sample. Other techniques listed above measure a change in a different property.

Such changes can include, for example, a change in the numerical value of a measured property (e. g., an increase or decrease in intensity of reflected, transmitted, or absorbed light, a change in polarization angle, or an increase or decrease in polarization) or a shift in frequency of a resonance or other spectral condition.

A number of these methods respond to a change in the refractive index on the surface of a test element. Typically, the analyte in a sample is bound to the surface of a testing element resulting in a refractive index change upon binding. In some instances, however, the analyte of interest is in such low concentration that direct refractive index changes at the surface by binding the analyte to the surface may be too small to be reliably detected. Accordingly, there is a need for the development of methods that permit detection of low concentration analytes, if needed.

Summary of the Invention Generally, the present invention relates to novel methods and devices for the assay of analytes. One embodiment is a method of determining a presence of an analyte in a sample. A first analyte-binding partner is disposed on a test portion of a surface of a test element. The test portion is then contacted with the sample to bind the analyte, if present in the sample, to the first analyte-binding partner. The test portion is subsequently contacted with a second analyte-binding partner to bind the second analyte-binding partner to the analyte. This second analyte-binding partner also includes a catalyst. A substrate is then brought into contact with the catalyst.

The catalyst converts the substrate into a product material that is then deposited on the test portion of the reflective surface. The presence of the product material on the test portion of the surface results in a change of refractive index of the test portion of the surface. The test portion of the surface is irradiated with light. The presence of the analyte is then determined by observing a characteristic of light that has irradiated the test portion of the surface and is modified by any change of the test portion of the surface. In one example, the presence of the analyte is determined by observing a surface plasmon resonance from the test portion of the reflective surface.

In at least some instances, the observation can also provide information regarding the amount or concentration of analyte in the sample.

In some embodiments of the method, the catalyst is an enzyme and the substrate is hydrophilic, but, in the presence of the catalyst, the substrate is converted into a hydrophobic product material. For example, the catalyst may cleave a hydrophilic component from the substrate to form a hydrophobic product material.

Another embodiment is a kit comprising a test element, a first analyte- binding partner, a second analyte-binding partner, a catalyst, and a catalyst substrate.

The test element has a reflective surface and a photon momentum altering structure.

The first analyte-binding partner is configured and arranged to be disposed on the reflective surface of the test element and to bind an analyte in a sample to the reflective surface. In some instances, the first analyte-binding partner of the kit is disposed on the reflective surface. In other instances, a user disposes the first analyte-binding partner on the kit. The second analyte-binding partner includes the catalyst attached thereto and is configured and arranged to bind to the analyte. The catalyst substrate is configured and arranged to produce a product material in the presence of the catalyst. When the product material is produced in the presence of analyte bound to the reflective surface (via the first analyte-binding partner), the product material is deposited on the reflective surface.

Another embodiment of the invention is a device which includes a test element, a first analyte-binding partner, a second analyte-binding partner, a substrate, a light source, and a detector. The first analyte-binding partner is configured and arranged to be disposed on a surface of the test element and to bind an analyte in a sample to the surface. The second analyte-binding partner includes a catalyst and is configured and arrange to bind to the analyte. The substrate is a substrate of the catalyst and produces a product material in the presence of the catalyst. The product material, when produced in the presence of analyte bound to the surface, is deposited on the surface of the test element to alter a refractive index of the surface. The light source is positioned to direct light toward the test element during operation of the device. The detector is positioned to detect light from the

light source which has interacted with the test element and to measure at least one characteristic of the light that is modified by any change of the surface.

The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The Figures and the detailed description which follow more particularly exemplify these embodiments.

Brief Description of the Drawings The invention may be more completely understood in consideration of the following detailed description of various embodiments of the invention in connection with the accompanying drawings, in which : Figure 1 is a schematic illustration of one embodiment of a surface plasmon resonance device, according to the invention; Figure 2 is a schematic illustration of an expanded view of a test element of the surface plasmon resonance device of Figure 1, according to the invention; Figure 3 is a schematic top view of one embodiment of a test element with multiple testing regions, according to the invention; Figure 4A is a schematic cross-sectional view of a test element with first analyte-binding partner disposed thereon, according to the invention ; Figure 4B is a schematic cross-sectional view of the test element of Figure 4A with analyte coupled to the first analyte-binding partner, according to the invention ; Figure 4C is a schematic cross-sectional view of the test element of Figure 4B with second analyte-binding partner coupled to the analyte, according to the invention; and Figure 4D is a schematic cross-sectional view of the test element of Figure 4C when a substrate of a catalyst coupled to the second analyte-binding partner is provided, according to the invention.

While the invention is amenable to various modifications and alternative forms, specifics thereof have been shown by way of example in the drawings and will be described in detail. It should be understood, however, that the intention is not to limit the invention to the particular embodiments described. On the contrary,

the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Detailed Description of the Preferred Embodiment The present invention is believed to be applicable to methods and devices for assay of an analyte using analyte-binding partners. The present invention is also directed to methods and devices for assays which respond to refractive index changes, such as, for example, surface plasmon resonance assays. While the present invention is not so limited, an appreciation of various aspects of the invention will be gained through a discussion of the examples provided below.

Testing Devices A variety of different methods can be used to determine the presence of an analyte based on a change in refractive index of a surface or other structure.

Examples of such methods include internal and external reflection methods, such as, for example, ellipsometry, external Brewster angle reflectometry, evanescent wave reflectometry, Brewster angle reflectometry, critical-angle reflectometry, evanescent wave ellipsometry, surface plasmon resonance, scattered total internal reflection, optical waveguide sensing methods, refractometric optical fiber sensing methods, leaky waveguide sensing methods, resonance light scattering of particles, multilayered grating resonance, and diffraction anomaly grating methods. In each of these methods, a characteristic of the observed light is modified in response to any change in the refractive index of materials that the light illuminates. The modification of the characteristic can be used to determine presence, amount, or concentration of an analyte in a sample being tested.

As an example of these methods, Figure 1 is a schematic illustration of one embodiment of a surface plasmon resonance device 100. The device 100 includes a light source 102 that shines light 104 toward a surface 106 of a test element 108.

The test element 108 includes a base 110 and a reflective metal layer 112 that defines the surface 106. Typically (except at or near the surface plasmon resonance frequency), the light 104 is substantially completely reflected (as reflected light 114) toward a detection device 116 and a processor 124. Typically, at least 75%,

preferably, at least 90%, and, more preferably, at least 95% of the light is reflected, except at or near the surface plasmon resonance frequency. In the illustrated embodiment, light 104,114 includes multiple wavelengths and the device includes a diffractive element 118 to separate the light by wavelength prior to reaching the detection device 116 so that a wavelength-dependent spectrum is obtained.

At or near the surface plasmon resonance frequency, the irradiating photons of light 104 interact with the conduction band electrons in the reflective metal layer 112 to generate surface plasmons. This substantially reduces or eliminates the intensity of reflected light 114 at that frequency. The conduction band electrons in the reflective metal layer act, at least in part, as a"plasma"with a fixed background of positive ions. The surface plasmon represents a quantum of oscillation of surface charges generated by the conduction band electrons that behave like a quasi-free electron gas.

Other methods and devices for surface plasmon resonance detection can be used, including, for example, methods and devices described in U. S. Patents Nos.

4,931,384; 4,828,387; 4,882,288; 4,992,385; 5,118,608; 5,164,589; 5,310,686; 5,313,264; 5,341,215; 5,492,840; 5,641,640; 5,716,854; 5,753,518; 5,898,503; 5,912,456; 5,926,284; 5,944,150; 5,965,456; 5,972,612; and 5,986,762, PCT Patent Applications Publication Nos. WO 88/07202 and WO 88/10418, and UK Patent Application Publication No. GB 2 202 045, all of which are incorporated herein by reference. As an example of an alternative detection scheme, some of the references cited above describe a device that observes transmitted light instead of reflected light. These alternative embodiments can be used to generate surface plasmon resonance signals for analysis. It will be recognized that other known devices can be used to make measurements according to the other methods listed above for observing an analyte via changes in refractive index.

Test Element Figure 2 schematically illustrates an expanded view of the cross-section of the test element 108 for use in surface plasmon resonance. Other test elements can be used with other measurement techniques to meet the requirements of those techniques. For light to interact with conduction band electrons in the reflective

metal layer 112 resulting in energy transfer from photons to surface plasmons, there must be a substantial matching between the energy and momentum of the photons and surface plasmons. For a flat metal surface, there is generally no wavelength of light that meets these conditions. However, if the metal surface is no longer flat, the momentum of the photons is altered. Although surface roughening can be used, two simple structures can be employed to alter photon momentum. These two structures are prisms and gratings.

Figure 2 illustrates a surface 106 that is altered by the formation of a sinusoidal grating. It will be recognized, however, that other gratings, including, for example, square well and triangular well gratings, can also be used. As an example, a sinusoidal grating can be prepared with peak-to-peak distances ranging from 200 to 800 nm and peak-to-valley distances ranging from 20 to 100 nm. It will be recognized that surfaces with prisms, instead of gratings, are also suitable, as described, for example, in U. S. Patents Nos. 5,164,589; 5,313,264; 5,341,215; 5,351,127; and 5,965,456, all of which are incorporated herein by reference.

The base layer 110 of the test element 108 is typically made from plastic or glass. Suitable plastics include, for example, polycarbonates, polymethylmethacrylate, polyethylene, and polypropylene. Typically, suitable plastics for the base layer are moldable and can sustain a stable shape. The grating can be formed in the surface of the base layer 110 by techniques, such as, for example, injection molding, etching, scoring, compression molding, and other known techniques. It can be advantageous to form the grating in the base layer because the base layer is a thicker bulk material, while the reflective metal layer is relatively thin. However, in some embodiments, the base layer is smooth and the grating is formed by modifying (e. g., etching or scoring) the reflective metal layer.

In at least some embodiments, the thickness of the base layer is 100 nm or less, although thicker base layers can be used.

The reflective metal layer 112 is disposed (e. g., deposited) on the base layer 110. The reflective metal layer can be formed by a variety of techniques including, for example, chemical or physical vapor deposition, sputtering, electroplating, or electroless plating. Preferably, if the base layer defines a grating, a technique is used that forms the reflective metal layer 112 as a conforma layer on the base layer 110.

Typically, the thickness of the reflective metal layer 112 ranges from 30 to 120 nm and is generally no more than about 100 nm.

Although the reflective metal layer 112 can be formed using any material that has conduction band electrons, the preferred materials are highly reflecting, do not form oxide, sulfide or other films upon atmospheric exposure, and are compatible with the chemistries used to perform the assays. Suitable metals include, for example, gold, indium, copper, platinum, silver, chrome, tin, and titanium. Gold is particularly suitable because it is resistant to oxidation and other atmospheric contaminants, but can still be reacted to bind with an analyte-binding partner. The surface plasmon resonance frequency and the coupling of the photons to the conduction band electrons in the reflective metal layer depend on a variety of factors including the nature of the material of the reflective metal layer, the structure of the reflecting surface of the reflective metal layer (including the peak-to-peak distance and peak-to-valley distance of the grating), and the presence of other materials on the surface. Peak-to-peak and peak-to-valley distances are dependent on the angle of incidence.

Referring again to Figure 1, the light source 102 is typically a multi- wavelength or single wavelength light source, such as, for example, a lamp (e. g., tungsten halogen lamp), light emitting diode (LED), or laser. Typically, the light 104 from the light source 102 is collimated and polarized prior to arriving at the surface 106 of the test element 108. The light is collimated to limit the range of angles at which the light intersects the surface 106 of the test element 108. The light is polarized because generally only p-polarized light interacts with the conduction band electrons of the reflective metal layer 112. Light sources that produce visible, infrared, or ultraviolet light, or a combination thereof can be used. As an example, a light source can be used that has wavelengths in the range of 300 to 900 nm. The bandwidth of the wavelength range for a particular assay can be, for example, 50 to 100nm.

In some embodiments, multiple light sources or multiple beams from a single light source (formed using, for example, multiple apertures in a screening element or multiple light fibers coupled to a single light source) are directed toward the test element 108. These beams 104a, 104b, 104c can be directed to different regions

126a, 126b, 126c of the test element 108, as illustrated in Figure 3. For example, the beams can be directed to regions (e. g., regions 126a, 126b) of the test element with different samples. To obtain a background signal, one beam may be directed to a region (e. g., region 126c) that does not produce a surface plasmon resonance due to, for example, the lack of a grating or other mechanism needed to couple the photons of the light with the conduction band electrons of the reflective metal layer.

In the illustrated embodiment, a diffraction element 118 is used to separate the reflected light into the component wavelengths. This light is then detected using a detection device 116, such as, for example, a CCD (charge-coupled device) array.

A CCD array includes an array of individual detectors arranged in columns and rows. The embodiment illustrated in Figure 1 illustrates the wavelengths of light being distributed along at least one column of the CCD array (in another embodiment, the diffraction element distributes the light along at least one row of the CCD array). When multiple beams of light are used, each beam of light will illuminate different column (s) of the CCD array. The signal (associated with a single beam of light from the light source) along the appropriate column (s) of the CCD array represents a surface plasmon resonance frequency spectrum of intensity versus wavelength. This and any other spectrum (e. g., a background spectrum or spectra for other samples) are provided to a processor 124 that analyzes the spectra.

As an alternative, a single wavelength light source can be used. In this embodiment, the angle that the light intersects the surface of the test element is varied and a signal corresponding to reflected light intensity versus incidence angle is generated. The presence of an analyte on the surface of the test element will change the angle at which the minimum reflection (or maximum absorbence) is obtained due to surface plasmon resonance coupling.

As another alternative, a monochromator can be used as a detection device.

The monochromator detects light of a particular wavelength and can be scanned across multiple wavelengths or multiple angles to obtain a spectrum.

The analysis of the spectrum is typically performed by a processor 124, with or without a storage medium, that is coupled to the detection device 116 to receive the signal. This analysis is performed by software, hardware, or a combination thereof. According to another embodiment, this same analysis is accomplished

using discrete or semi-programmable hardware configured, for example, using a hardware descriptive language, such as Verilog. In yet another embodiment, the analysis is performed using a processor having at least one look-up table arrangement with data stored therein to represent the complete result or partial results of equations based on a given set of input data, the input data corresponding to parameters used in the equations.

Assay Technique An assay for an analyte, according to the invention, includes disposing a first analyte-binding partner 120 onto the surface 106 of the reflective metal layer 112 (or other test element appropriate to any one of the techniques, listed above, for observing changes in index of refraction), as illustrated in Figure 4A. The first analyte-binding partner 120 is optionally disposed on the surface 106 and then provided to a user as part of a kit or the kit contains a generic test element without a first analyte-binding partner and the user can then bind the appropriate first analyte- binding partner to the surface of the test element.

The first analyte-binding partner 120 is generally bound to the surface of the test element by covalent, ionic, coordinative, or hydrogen bonding or combinations thereof. A variety of methods for bonding such materials to a reflective metal surface (or other appropriate surfaces) are known. For example, the first analyte- binding partner can include a reactive functional group that can bind to the surface or to a reactive functional group previously provided on the surface. As another example, the surface can be continuously or discontinuously coated with an organic material (e. g., a polymer or photoresist) to which the first analyte-binding partner can be reactively or otherwise attached. As yet another example, a first analyte- binding partner can be selected that does not dissolve in a solvent (e. g., water) that flows over or is disposed over the surface of the test element. For example, a hydrophobic first analyte-binding partner can be used; the first analyte-binding partner remaining on the surface because of its hydrophobicity. Alternatively, the first analyte-binding partner can be crosslinked on the surface to prevent solvation.

Other methods include self assembly of monolayers and reactive sulfur-containing compounds.

In an assay, a sample is brought into contact with the surface of the test element. As an example, the test element is disposed in a flow cell which is used to carry the sample (and other assay components, as described below) to the surface of the test element. As the sample is brought into contact with the surface of the test element, the first analyte-binding partner 120 selectively binds to a desired analyte 122, if present in the sample, as illustrated in Figure 4B. Preferably, at least 5%, more preferably, at least 20% and, most preferably, at least 60% of the analyte in the sample, if present, binds to the first analyte-binding partner 120.

The binding between the analyte and the first analyte-binding partner can include forming covalent, ionic, coordinative, hydrogen or van der Waals bonds or combinations thereof between the first analyte-binding partner and the analyte or adsorbing or absorbing the analyte on the first analyte-binding partner. Non-limiting examples of suitable pairs of first analyte binding partners and analytes are provided in Table 1: Table 1 First Analyte-Binding Partner Analyte antigen or hapten antibody or antibody fragment antibody or antibody fragment antigen or hapten hormone hormone receptor hormone receptor hormone polynucleotide strand (e. g., complementary polynucleotide DNA, RNA strands or strand fragments, oligonucleotides) avidin, streptavidin, biotin or biotinylated compound neutravidin, or avidin-or complex containing compound or complex biotin or biotinylated compound avidin, streptavidin, neutravidin, or complex or avidin-containing compound or complex protein A or G immunoglobulin immunoglobulin protein A or G enzyme enzyme cofactor or inhibitor enzyme cofactor or inhibitor enzyme lectin carbohydrate carbohydrate lectin

The particular analyte can be chosen to provide, for example, immunological, nucleic acid binding, enzymatic, chemical, or gas adsorption assays for use in fields such as, for example, agriculture, food testing, biological and chemical agent testing, drug discovery, monoclonal antibody detection, and chemical and biological process monitoring.

In the next step of the assay, a second analyte-binding partner 124 is brought into contact with the surface 106 and the analyte 122, if present, as illustrated in Figure 4C. The second analyte-binding partner 124 is selected using the same

considerations as the first analyte-binding partner 120. The first and second analyte- binding partners can be the same or different. Typically, the second analyte-binding partner is solvated or dispersed in a solution (generally water-based, although other solvents can be used) and brought into contact with the surface of the test element.

The second analyte-binding partner 124 includes a catalyst 126 or includes a linking group (e. g., an avidin or biotin group) to which a catalyst with the appropriate corresponding linking group (e. g., a biotin or avidin group, respectively) can be attached. The catalyst 126 can be, for example, an enzyme, metal or metal complex, or dye. Examples of suitable enzymes include alkaline phosphatases, glucose oxidase, and peroxidases (e. g., horseradish peroxidase). Preferably, the enzyme has one or more of the following features : rapid turnover, low non-specific binding, and ability to convert a hydrophilic substrate to a hydrophobic product.

After binding the second analyte-binding partner 124 to the analyte 122, if present, a substrate 128 of the catalyst 126 is introduced, as illustrated in Figure 4D.

The catalyst 126 converts the substrate 128 to a product material 130. If the catalyst 126 is bound to a second analyte-binding partner 124 which is bound to an analyte 122 which is, in turn, bound to a first analyte-binding partner 120, the product material 130 is typically deposited onto the surface 106 of the reflective metal layer 112. In contrast, if the catalyst is still in solution and is not coupled to the surface 106 by the second-binding partner, analyte, and first analyte-binding partner, the product material is not deposited onto the surface or is substantially less likely to be deposited on the surface. Thus, the presence or the amount of product material on the surface of the test element is indicative of the presence or amount of the analyte in the sample.

As an alternative, prior to introduction of the substrate, the surface is washed to remove any catalyst (and associated second analyte-binding partner) that is not bound to analyte. This will reduce the amount of product material formed by catalyst that is not bound to the test element via the analyte. However, this adds another step and, typically, additional time to the assay.

The catalyst (e. g., enzyme) and substrate are generally selected to promote the deposition of the product material onto the surface of the test element. In one embodiment, the substrate is a hydrophilic material and the product material is

hydrophobic. Thus, when using aqueous solutions to conduct the assay components over the test element, the product material is deposited on the surface of the test element due to its hydrophobicity which favors deposition on the hydrophobic metal surface.

As an example, alkaline phosphatase can be used as the catalyst. The substrate is typically a phosphate-containing compound. The alkaline phosphatase cleaves the phosphate group from the substrate to form a product material, which is either hydrophobic and interacts directly with the surface or is an active intermediate to form a hydrophobic condensation product with a second molecule and then this product interacts with the surface. In one embodiment, the alkaline phosphatase cleaves the phosphate group from the substrate to form an intermediate that undergoes dimerization producing an indigo dye. The dimerized product reduces a second substrate to a water insoluble product. This hydrophobic product has an affinity for the surface. Examples of suitable phosphate-containing compounds/second substrates include, for example, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium, fast red/naphthol-AS-BI-phosphate, and fast blue/naphthol-AS-BI-phosphate.

To further prevent deposition of product material formed using a catalyst not bound to the surface of the test element by analyte, the test element in one embodiment is positioned during the assay so that the gravitational force acts against the deposition of the product material on the surface of the test element. In other words, the test element is positioned"upside down". This positioning can further reduce the effect of the conversion of substrate by catalyst that is not bound to the surface of the test element via the analyte. The gravitational force will oppose the flow of product material to the surface of the reflecting metal layer. However, for catalyst disposed near the surface, because it is bound by the analyte, the product material generated by the catalyst will be sufficiently close to the surface to be deposited thereon as a consequence of, for example, the hydrophobicity of the product material.

The production of the product material and its deposition on the surface of the test element alters the surface plasmon resonance frequency (or angle of maximum adsorption of photons for a given wavelength of light) or some other

characteristic depending on the measurement technique. Generally, the magnitude of the change in position of the surface plasmon resonance reflects the amount of product material disposed on the surface. In at least some instances, the relationship between the position of the surface plasmon resonance and the amount of product material is linear or can be determined using a calibration curve generated using samples with known concentrations of analyte.

A shift in surface plasmon resonance frequency can be used to indicate the presence of an analyte in the sample. In some instances, the assay may require that the shift have a threshold magnitude to indicate the presence of the analyte.

Optionally, the surface plasmon resonance spectrum can be compared to a second spectrum obtained from a region on the test element where the sample is not brought into contact with the surface of the test element, but the second analyte-binding partner and catalyst are provided. This second spectrum can be used as a comparison to account for product material that is deposited on the surface from catalyst that is not bound to the surface via the analyte.

In some embodiments, the determination of the concentration or amount of an analyte in a sample can be made from the determination of the shift. Typically, the determination of concentration requires control of the amount of time that the substrate is in contact with the catalyst, the temperature of the substrate, and the concentration of the substrate. The shift in the surface plasmon resonance frequency is then indicative of the amount of product material deposited on the surface of the test element which, in turn, is indicative of the concentration or amount of. analyte in the sample. Optionally, the shift measured for the sample can be compared to the shift observed for a known concentration or amount of analyte in one or more calibration samples.

These assay techniques are particularly useful for samples that contain only a small amount of analyte. For these samples, the amount of analyte, when bound to the surface of the test element, may not be sufficient, even after adding the second analyte-binding partner, to provide a substantial shift in the surface plasmon resonance frequency. The present assays make the presence of the analyte measurable by catalytic generation of the product material. Thus, the assay is no longer limited to the mass change due to the analyte, but, instead, that mass change

can be multiplied by using a relatively large amount of catalyst substrate. It will be understood that the methods described above can be readily adapted to measurement techniques other than surface plasmon resonance.

Assay Example As one example of an assay, a sample, such as buffer, plasma, blood, or urine, containing an analyte, such as, for example, hCG (human choriagonadotropin), can be placed in a flow cell for SPR measurement. The flow cell includes a reflective metal surface (e. g., a gold surface) with a grating. A first antibody specific to the analyte is disposed on the reflective metal surface. The sample is flowed over the reflective metal surface at a flow rate (e. g., 200 IlL/min) for a period of time (e. g., 5 min.). The reflective metal surface can then be washed using, for example, a phosphate buffer having a surfactant, for a period of time (e. g., 4 min.) to remove substantially all of the sample except the analyte bound to the first antibody. Next, a second antibody specific to the analyte and coupled to an enzyme (e. g., alkaline phosphatase) is flowed through the cell at a flow rate (e. g., 200 tL/min) for a period of time (e. g., 4 min.) to bind to the analyte. The reflective metal surface is washed again for a period of time (e. g., 4 min.). A baseline SPR measurement is then made.

A substrate of the enzyme (e. g., 5-bromo-4-chloro-3-indolyl phosphate/ nitroblue tetrazolium) is flowed through the cell at a flow rate (e. g., 200, iiL/min) for a period of time (e. g., 5 min.). The substrate reacts in the presence of the enzyme to form a product material that is deposited on the reflective metal surface. After the completion of the period of time for flow of the substrate, another SPR measurement is made. The difference between the baseline SPR measurement and the second SPR measurement is then compared to a calibration curve to determine the amount of analyte in the sample.

To demonstrate the viability of this approach, a gold surface with a grating was coated with a monolayer of neutravidin. The surface was washed with a phosphate buffer containing a surfactant for 5 min. at a flow rate of 50 uL/min.

Biotinylated alkaline phosphatase from Pierce Chemical Co. (Rockford, IL) was flowed over the surface for 20 min. at 50 plL/min and a concentration of 100 ng/mL.

The surface was then washed with the same washing buffer for the same period of time and flow rate as in the previous washing step. The substrate, 5-bromo-4- chloro-3-indolyl phosphate/nitroblue tetrazolium from Pierce Chemical Co.

(Rockford, IL), was then flowed across the surface for 10 min. at a flow rate of 50 , ut. An SPR measurement was made. The process was repeated for 1: 10 and 1: 5 dilutions of the conjugate with the wash buffer. SPR measurements were made for each of these samples. There was an observable difference between the SPR resonance frequencies for these three measurements.

Table 2 SPR Response Concentration of SPR Shift Alkaline Phosphatase 0.1 ng/mL 1 nm 1 ng/mL 6 nm 10 ng/mL 25 nm The present invention should not be considered limited to the particular examples described above, but rather should be understood to cover all aspects of the invention as fairly set out in the attached claims. Various modifications, equivalent processes, as well as numerous structures to which the present invention may be applicable will be readily apparent to those of skill in the art to which the present invention is directed upon review of the instant specification.