WO2011014282A2

WO2011014282A2 - High magnification spectral reflectance biosensing with discrete light sources

Info

Publication number: WO2011014282A2
Application number: PCT/US2010/033397
Authority: WO
Inventors: Selim M. Unlu; Emre I. Ozkumur; George Daaboul; Rahul S. Vedula; Bennett B. Goldberg
Original assignee: Trustees Of Boston University
Priority date: 2009-05-01
Filing date: 2010-05-03
Publication date: 2011-02-03
Also published as: WO2011014282A3

Abstract

A spectral reflectance imaging device (100) for detecting biomolecular targets (126) includes an illumination source (101) that illuminates a substrate (122) with a plurality of separate wavelengths of incoherent light. The substrate includes an oxide layer (124) and a binding agent to selectively bind biomolecular targets to the substrate. An imaging device (130) captures the light reflected from or transmitted through the substrate and an image processing system (140) detects the biomolecular targets a function of the change in reflective properties of the substrate.

Description

HIGH MAGNIFICATION SPECTRAL REFLECTANCE BIOSENSING WITH

DISCRETE LIGHT SOURCES

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional

Application No. 61/174,816 filed May 1, 2009, the contents of which are incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

[0002] This invention was made with Government support under Contract No.

W911NF-06-2-0040 awarded by the Army Research Office. The Government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates generally to apparatuses and methods for the detection of biomolecules and particles in the absence of tunable laser illumination.

BACKGROUND OF THE INVENTION

[0004] The ability to detect biological target molecules, such as DNA, RNA, and proteins, as well as nanomolecular particles such as virions, is fundamental to our

understanding of both cell physiology and disease progression, as well as for use in various applications such as early and rapid detection of disease outbreaks and bioterrorism attacks. Such detection, however, is limited by the need to use labels, such as fluorescent molecules or radiolabels, which can alter the properties of the biological target, e.g., conformation, and which require additional, often time-consuming, steps. Thus, the ability to rapidly detect and directly visualize biological targets using label-free systems could have many practical applications and advantages. In addition, label-free systems would provide easier monitoring and quantification methods for detecting biomolecular interactions, such as antigen-antibody, receptor-ligand, virus-cell and protein-DNA binding interactions, as opposed to the various solid-phase immunoassays commonly used in both biological and medical research, such as enzyme-linked immunosorbent assays (ELISA) and Western blotting. Such techniques typically employ secondary probes that bind to captured analytes that are in turn detected with either fluorescent or enzyme-linked reagents, [0005] DNA and protein microarray technologies are actively being used by biologists and researchers today for high-throughput screening of biomarkers for drug discovery, disease research, and diagnosis, thereby converting the presence of target biomolecules to a measurable and quantifiable signal. The importance of high-throughput platforms has been demonstrated by the success of gene arrays in the analysis of nucleic acids, which has further provided impetus for the proposal that protein-peptide arrays could achieve similar utility for the analysis of multiple binding events. However, most of the detection systems available today for use in these high-throughput systems operate by the same guiding principle, whereby they scan the surface of a microarray and measure fluorescence from the biomolecules. Fluorescent labeling is a costly and time-consuming step that sometimes proves to be prohibitively difficult and expensive for use in these

technologies. In addition, detecting analytes through secondary probes is intrinsically complex, requiring multiple layers of interacting components that provide specificity without interfering with one another. Monitoring the primary bimolecular interaction (i.e., binding of analyte to its cognate probe) would greatly simplify many immunoassays.

SUMMARY OF THE INVENTION

[0006] High-throughput DNA and protein analysis technologies, such as microarray technologies, are actively being used by biologists and researchers today for high-throughput screening of biomarkers for drug discovery, disease research, and diagnosis. Most of the detection systems available today operate by the same guiding principle, whereby they scan the surface of a microarray and measure fluorescence, or some other label, from the biomolecules. Fluorescent labeling is a costly and time-consuming step that sometimes proves to be prohibitively difficult and expensive. State-of-the-art label-free detection systems such as imaging SPR are bulky, complex and expensive instruments, and thus they cannot be utilized for global health applications.

[0007] Substrate enhanced microarray imaging, as has been presented by Bergstein and UnIu (E. Ozkumur et al., 2008 PNAS 105: 23, 7988-7992) has the capability to detect the binding of biomolecular targets to a surface at tens of thousands of spots simultaneously in a label-free fashion. Current embodiments of substrate enhanced microarray imaging use tunable lasers as the primary illumination source. Again, tunable lasers are large, costly, and prohibit this technology from being used effectively in many clinical and field applications. The invention described herein operates under interferometric principles of detection, using non-laser light sources, such as LEDs, as the illumination source. LEDs are very low-cost, compact, and robust; and are ideal for large scale use and distribution for diagnostic and research applications. This invention incorporates the quantitative molecular binding measurements obtained through the traditional substrate enhanced microarray imaging system, but with the capability to use low-cost incoherent illumination sources that enable a high magnification embodiment for detection of single biomolecular targets found in a sample, such as a virus particle.

[0008] Thus, label-free measurement of binding events is a potentially powerful tool, simpler and more efficient than secondary probe-based systems. Some advances in label-free detection has been demonstrated by using electrical, electromechanical, and optical detection methods. One such method is surface plasmon resonance (SPR), which has been the basis for such commercial biosensors as the Biacore instruments (GE Healthcare Life Sciences). SPR has been shown to allows real-time, sensitive monitoring of an analyte binding to probes immobilized on a gold substrate. Although SPR has been the standard for label-free detection for single analytes, extension to large-format arrays has achieved limited success, due in large part to the bulkiness, complexity, and expense of the instruments, and thus they cannot be utilized for global health applications.

[0009] Substrate enhanced microarray imaging has the capability to detect the binding of biomolecules to a surface at tens of thousands of spots simultaneously in a label-free fashion. Current embodiments of substrate enhanced microarray imaging use tunable lasers as the primary illumination source. Again, tunable lasers are large, costly, and prohibit this technology from being used effectively in many clinical and field applications. Thus, there is a need in the art for alternate methods and apparatuses for label-free binding measurements of spectral reflectance that are amenable and cost-effective for use in high-throughput systems.

[0010] The inventors have discovered that multiple incoherent light sources, such as light-emitting diodes (LEDs), can be utilized as the illumination source for interferometric principles of detection and measurement. LEDs are very low-cost, compact, and robust, and are thus ideal for large-scale use and distribution for diagnostic and research applications. This invention improves upon quantitative molecular binding measurements obtained through the traditional substrate enhanced microarray imaging system, by utilizing low-cost incoherent illumination sources that enable a high magnification embodiment for detection and imaging of a single biomolecular target in an analyte or sample, where the biomolecular target ranges from about 50 nm to about 300 nm in length. [0011] The invention describes, in part, a method of using LED illumination for substrate enhanced detection of binding of biomolecular targets to a surface. Described herein, in one aspect, is a high-throughput spectroscopy method for simultaneously recording a response of an entire substrate surface, comprising sampling different wavelengths using an illumination light providing light in different spectral windows, and imaging the reflected or transmitted light by an imaging device.

[0012] In some embodiments of the invention, the light source comprises a narrowband light source. In one embodiment, the imaging device is a monochromatic charge coupled device (CCD) camera. In one embodiment, the narrowband light source comprises a standard bright-field microscope optical setup, wherein the substrate surface is illuminated by white light, and the reflected light is transmitted to an eyepiece. In one embodiment, the narrowband light source comprises three to six LEDs with different, discrete emission peak wavelengths. In one embodiment, the narrowband light source comprises two or more LEDs with different, discrete emission peak wavelengths. In one embodiment, the narrowband light source comprises three or more LEDs with different, discrete emission peak wavelengths. In one embodiment, the narrowband light source comprises four or more LEDs with different, discrete emission peak wavelengths. In one embodiment, the narrowband light source comprises five or more LEDs with different, discrete emission peak wavelengths. In one embodiment, the narrowband light source comprises six or more LEDs with different, discrete emission peak wavelengths. In some embodiments of the invention, each LED can be individually controlled and the reflectivity at each position on the surface of the substrate can be recorded as a function of position and wavelength.

[0013] In some embodiments of the aspects described herein, the imaging device is a monochromatic camera. In other such embodiments of the aspects described herein, the imaging device is a monochromatic CCD camera.

[0014] In some embodiments of the aspects described herein, the substrate is a layered substrate. In some embodiments, the layered substrate comprises 100 -1000 nm of SiO₂ layered on a Si wafer. In some embodiments, the layered substrate comprises at least 100 nm of SiO₂ layered on a Si wafer. In some embodiments, the layered substrate comprises at least 200 nm of SiO₂ layered on a Si wafer. In some embodiments, the layered substrate comprises at least 300 nm of SiO₂ layered on a Si wafer. In some embodiments, the layered substrate comprises at least 400 nm of SiO₂ layered on a Si wafer. In some embodiments, the layered substrate comprises at least 500 nm of SiO₂ layered on a Si wafer. In some embodiments, the layered substrate comprises at least 600 nm of SiO₂ layered on a Si wafer. In some embodiments, the layered substrate comprises at least 700 nm of SiO₂ layered on a

Si wafer. In some embodiments, the layered substrate comprises at least 800 nm of SiO₂ layered on a Si wafer. In some embodiments, the layered substrate comprises at least 900 nm of SiO₂ layered on a Si wafer. In some embodiments, the layered substrate comprises at least

1000 nm of SiO₂ layered on a Si wafer.

[0015] In one embodiment, the light source is a white light source and the imaging device is a color camera. In one embodiment, the color camera is a 3CCD camera. In one embodiment, the light source is a RGB (red green blue) LED light source and the imaging device is a color camera, such as a 3CCD camera. In one embodiment, the light source is a broadband light source. In one embodiment, the imaging device comprises a camera having a high magnification objective lens with a high numerical aperture. In one embodiment, the camera further comprises a spatial filter on the camera's optical axis.

[0016] In one aspect, the invention provides a spectral reflectance imaging system comprising: a substrate having a first reflective surface and a thin transparent layer providing a second reflective surface; a biolayer bound to the second reflective surface; an illumination source comprising at least three light sources, each providing light in one of three different narrow frequency bands and directing each frequency band of light at the substrate; and an imaging device directed at the second reflective surface of the substrate and adapted to produce imaging signals representative of light from the illumination source being reflected by the first reflective surface and the second reflective surface.

[0017] In some embodiments of the aspect, the first reflective surface is a silicon substrate and the transparent layer is silicon oxide (SiO₂).

[0018] In some embodiments of the aspect, the spectral reflectance imaging system further comprises an image acquisition and processing system, coupled to the imaging device and adapted to receive the imaging signals and under program control, produce an image of the biolayer on the second reflective surface.

[0019] In some embodiments of the aspect, the thickness of the biolayer is determined as function of the intensity of the reflected light received by the imaging device.

[0020] In some embodiments of the aspect, the transparent layer is less than 1 micron thick. In some embodiments of the aspect, the transparent layer has a thickness in the range from 100 nanometers to 1000 nanometers.

[0021] In some embodiments of the aspect, the illumination source includes four light sources. In some embodiments of the aspect, the illumination source produces light at 460 nanometer, 520 nanometer, 600 nanometer and 630 nanometer wavelengths. [0022] In some embodiments of the aspect, each light source produces light in a band that is 20 - 30 nanometers wide. In some embodiments of the aspect, each light source produces light in a band that is 10 - 20 nanometers wide. In some embodiments of the aspect, each light source produces light in a band that is 5 nanometers wide.

[0023] In some embodiments of the aspect, the illumination source produces white light and the system further includes a color wheel having at least three filters, each producing a beam of light in one of at least three narrow frequency bands that is directed at the substrate. In some embodiments of the aspect, the spectral reflectance imaging system further comprises at least one optical fiber and the light from each light source is directed through the at least one optical fiber toward the substrate. In some embodiments of the aspect, the spectral reflectance imaging system further comprises three optical fibers and each band of light from each light source is directed through a separate optical fiber toward the substrate.

[0024] In another aspect, the invention provides a method for detecting the binding of a particle to a surface of a substrate, the method comprising: providing a first specular reflecting interface surface of the substrate with a binding agent for binding a predefined particle to the surface of the substrate; providing a second specular reflecting interface that is substantially parallel to and underlies the first specular reflecting interface surface of the substrate; illuminating the surface with light substantially centered around one or more wavelengths; imaging light reflected or transmitted from the substrate using an imaging device; producing a spectral reflectance image of the surface of the substrate; and correlating the features on the image to discrete particles on the surface.

[0025] In some embodiments of the aspect, the imaging device comprises a camera having a high magnification objective lens with a high numerical aperture.

[0026] In some embodiments of the aspect, each wavelength of light is produced by a separate, narrow band light source.

[0027] In some embodiments of the aspect, the imaging device is a monochromatic

CCD camera.

[0028] In some embodiments of the aspect, the surface is illuminated by a light source from a standard bright-field microscope optical setup, and where the reflected light is transmitted to an eyepiece.

[0029] In some embodiments of the aspect, each wavelength of light is produced by a separate light emitting diode (LED), each having a different emission peak wavelengths, and where the imaging device is a monochromatic camera. [0030] In some embodiments of the aspect, the imaging device is a monochromatic

CCD camera.

[0031] In some embodiments of the aspect, the layered substrate comprises 500 nm of SiO₂ layered on a Si wafer.

[0032] In some embodiments of the aspect, the surface is illuminated with white light and the imaging device includes a color camera. In some embodiments of the aspect, the color camera is a 3CCD camera.

[0033] In some embodiments of the aspect, wherein the surface is illuminated by an

RGB (red green blue) LED and the imaging device includes a color camera. In some embodiments of the aspect, the color camera is a 3CCD camera.

[0034] In some embodiments of the aspect, the surface is illuminated by a broadband light source.

[0035] In some embodiments of the aspect, the camera further comprises a spatial filter on the camera's optical axis.

[0036] In another aspect, the invention provides a method for detecting a particle on a surface of a layered substrate comprising: providing the surface of the layered substrate; contacting a solution having at least one particle with the surface of the substrate;

illuminating the surface with at least three wavelengths of light; imaging the light reflected or transmitted from the substrate using an imaging device; and producing a spectral reflectance image of the surface of the substrate to detect the particle on the surface of the layered substrate.

[0037] In some embodiments of the aspect, the layered substrate comprises SiO₂ layered on a Si substrate.

[0038] In some embodiments of the aspect, the light is coherent or incoherent. In some embodiments of the aspect, each wavelength of light is produced by a separate, narrow band light source or by a broadband light source. In some embodiments of the aspect, each wavelength of light is produced by a separate laser, each having a different emission peak wavelength. In some embodiments of the aspect, each wavelength of light is produced by a separate light emitting diode (LED), each having a different emission peak wavelength. In some embodiments of the aspect, each wavelength of light is produced by a white light source. In some embodiments of the aspect, each wavelength of light is produced by an RGB

(red green blue) LED light source. In some embodiments of the aspect, each wavelength of light is produced by a standard bright-field microscope optical setup, and the reflected light is transmitted to an eyepiece. [0039] In some embodiments of the aspect, the imaging device is a monochromatic

CCD camera or a color camera. In some embodiments of the aspect, the color camera is a 3CCD camera. In some embodiments of the aspect, the imaging device comprises a camera having a high magnification objective lens with a high numerical aperture.

[0040] In some embodiments of the aspect, the camera further comprises a spatial filter on the camera's optical axis.

[0041] In some embodiments of the aspect, detecting the particle comprises detecting the binding of the particle on the surface of the layered substrate. In some embodiments of the aspect, the surface of the layered surfaces comprises a binding agent for binding a predefined particle and the solution comprises at least one predefined particle. In some embodiments of the aspect, the particle comprises one or more proteins, one or more salts, one or more polymers, one or more metals, or one or more microorganisms. In some embodiments of the aspect, the particle is a protein aggregate, a nanoparticle, a bead, or a virion particle.

BRIEF DESCRIPTION OF THE FIGURES

[0042] Figure 1 shows a diagrammatic view of a spectral reflectance imaging system for making interferometric measurements according to an embodiment of the present invention.

[0043] Figure 2 depicts how interference signature of reflected light changes upon adsorption of a layer of biomass or any other semi-transparent material to a surface.

[0044] Figure 3 demonstrates some properties desired in a substrate for enhanced microarray image composition.

[0045] Figure 4 demonstrates some properties desired for performing high magnification substrate enhanced microarray imaging.

[0046] Figure 5 depicts using a spatial filter as an option for performing high magnification substrate enhanced microarray imaging.

[0047] Figure 6A and 6B compare optical interferometry of the biosensors of the invention using lasers versus LEDs.

[0048] Figure 7 shows an exemplary optical setup of a biosensor of an embodiment of the invention.

[0049] Figure 8 depicts single -particle detection using an exemplary biosensor of the invention. [0050] Figure 9 depicts a 12-tile array format of a biosensor substrate surface of the invention.

[0051] Figures 10-lOC show an enlargement of a single tile of a 12-tile array format of a biosensor substrate surface of an embodiment of the invention. Figure 1OA shows the image of the 12-tile array. Figure 1OB is an enlarged image of a single tile. Figure 1OC shows data depicting the average diameter of the particles in the tile.

[0052] Figure 11 demonstrates the ability of an exemplary biosensor of the invention to discriminate between particles of different sizes present in a sample.

[0053] Figure 12 depicts how an image of a particle is taken using a biosensor of the invention.

[0054] Figure 13 depicts how an exemplary biosensor of the invention is used to distinguish between particles of different shapes.

[0055] Figure 14 shows the SRB image of half of the sample showing individual viruses as bright dots.

[0056] Figure 15 shows the one-to-one correspondence between the SEM image and

SRB image. In Figure 15, SEM image (left) is compared to the optical image obtained on

SRB (right). The virus particles appear as dark dots on the SEM image due to charging.

[0057] Figure 16 further illustrates the SRB detection and comparison to SEM results by focusing on part of the image from Figure 15 and drawing arrows of equal length to correlate the virus particles imaged by both modalities.

DETAILED DESCRIPTION

[0058] Described herein are label-free biosensors and methods of use thereof for the detection of a variety of biomolecular targets. In some aspects, the label-free biosensors and methods described herein provide a high-throughput method for simultaneously recording a response of an entire substrate surface, comprising sampling different wavelengths using a light source providing incoherent light in different spectral windows, and imaging the reflected or transmitted light using an imaging device. The inventors have discovered that light-emitting diodes (LEDs) can be utilized as the illumination source for interferometric principles of detection. Interferometric measurements can provide enormous sensitivity and resolution using optical path length differences (OPD), with the best current systems achieving <10^"10 rad resolution.

[0059] Accordingly, described herein are label-free biosensors and methods of using such biosensors for substrate enhanced detection of binding of molecules to a surface. The invention provides significant improvements by eliminating the need for a tunable laser. The claimed invention, in part, samples the reflectance spectrum by illuminating the substrate sequentially with different wave lengths of light, using, for example, LEDs that are spectrally separated, and recording the reflectance by an imaging device, such as a 2-D arrayed pixel camera. In this way, the reflectance spectrum for the whole field-of-view is recorded simultaneously, providing improved performance over tunable laser illumination which can only provide a relatively narrow band of illumination. Using this method, label-free, high- throughput or microarray imaging can be accomplished. The invention can also provide high- magnification imaging for detection of biomolecular targets in the 15nm to 150nm range. Such high-magnification detection can be used, for example, for the detection of single particle on a surface.

[0060] The invention describes, in part, a high-throughput spectroscopy technique where sampling at different wavelengths is realized by using narrowband light sources, such as LEDs, covering different spectral windows, and the reflected or transmitted light is imaged to an imaging device, such as a monochromatic CCD camera, thus allowing the response of the entire imaged surface to be recorded simultaneously. In one aspect, the bright field optical setup is modified in the following way: the white light source is replaced with two or more LEDs with different emission peak wavelengths, and the eyepiece is replaced with a monochromatic camera. The microarray is fabricated on a layered substrate (for example: 500 nm of SiO2 layered on a Si wafer). Each LED is turned on one at a time and the reflectivity at each position on the surface is recorded as a function of position and wavelength. As molecules bind to the microarray surface, the reflectivity vs. wavelength curve for each point of the surface will change in an observable way.

[0061] In some embodiments of this aspect, three to six LEDs with different emission peak wavelengths are used as the light source. In some embodiments of this aspect, two LEDs with different emission peak wavelengths are used as the light source. In some embodiments of this aspect, three LEDs with different emission peak wavelengths are used as the light source. In some embodiments of this aspect, four LEDs with different emission peak wavelengths are used as the light source. In some embodiments of this aspect, five LEDs with different emission peak wavelengths are used as the light source. In some embodiments of this aspect, six LEDs with different emission peak wavelengths are used as the light source. In some embodiments of this aspect, seven or greater LEDs with different emission peak wavelengths are used as the light source. [0062] In preferred embodiments, the light sources used in the biosensors and methods described herein have a narrow range of wavelength, and the width between the wavelengths of each individual light source is small.

[0063] In one embodiment, the range of the wavelength of an individual light source used is less than 5 nm. In one embodiment, the range of the wavelength of an individual light source used is less than 10 nm. In one embodiment, the range of the wavelength of an individual light source used is less than 15 nm. In one embodiment, the range of the wavelength of an individual light source used is less than 20 nm. In one embodiment, the range of the wavelength of an individual light source used is less than 25 nm. In one embodiment, the range of the wavelength of an individual light source used is less than 30 nm. In one embodiment, the range of the wavelength of an individual light source used is less than 35 nm. In one embodiment, the range of the wavelength of an individual light source used is less than 40 nm. In one embodiment, the range of the wavelength of an individual light source used is less than 45 nm. In one embodiment, the range of the wavelength of an individual light source used is less than 50 nm. In one embodiment, the range of the wavelength of an individual light source used is less than 100 nm.

[0064] In one embodiment, the separation between the wavelengths of each individual light source used is less than 10 nm. In one embodiment, the separation between the wavelengths of each individual light source used is less than 15 nm. In one embodiment, the separation between the wavelengths of each individual light source used is less than 20 nm. In one embodiment, the separation between the wavelengths of each individual light source used is less than 25 nm. In one embodiment, the separation between the wavelengths of each individual light source used is less than 30 nm. In one embodiment, the separation between the wavelengths of each individual light source used is less than 35 nm. In one embodiment, the separation between the wavelengths of each individual light source used is less than 40 nm. In one embodiment, the separation between the wavelengths of each individual light source used is less than 45 nm. In one embodiment, the separation between the wavelengths of each individual light source used is less than 50 nm. In one embodiment, the separation between the wavelengths of each individual light source used is less than 100 nm.

[0065] In some embodiments of the aspects described herein, the microarray is fabricated on a layered substrate comprising 100 nm -1000 nm of SiO₂ layered on a Si wafer. In some embodiments of this aspect, the microarray is fabricated on a layered substrate comprising at least 100 nm of SiO₂ layered on a Si wafer. In some embodiments of this aspect, the microarray is fabricated on a layered substrate comprising at least 200 nm of SiO₂ layered on a Si wafer. In some embodiments of this aspect, the microarray is fabricated on a layered substrate comprising at least 300 nm of SiO₂ layered on a Si wafer. In some embodiments of this aspect, the microarray is fabricated on a layered substrate comprising at least 400 nm of SiO₂ layered on a Si wafer. In some embodiments of this aspect, the microarray is fabricated on a layered substrate comprising at least 500 nm of SiO₂ layered on a Si wafer. In some embodiments of this aspect, the microarray is fabricated on a layered substrate comprising at least 600 nm of SiO₂ layered on a Si wafer. In some embodiments of this aspect, the microarray is fabricated on a layered substrate comprising at least 700 nm of SiO₂ layered on a Si wafer. In some embodiments of this aspect, the microarray is fabricated on a layered substrate comprising at least 800 nm of SiO₂ layered on a Si wafer. In some embodiments of this aspect, the microarray is fabricated on a layered substrate comprising at least 900 nm of SiO₂ layered on a Si wafer. In some embodiments of this aspect, the microarray is fabricated on a layered substrate comprising at least 1000 nm of SiO₂ layered on a Si wafer.

[0066] The biosensors and methods of the invention described herein, can be used, in part, for high magnification interferometric measurements, for example, but not limited to, detecting a single biomolecular target or particle, such as a virus, in a given sample.

A "particle," as defined herein, refers to any target to be detected by the biosensors and methods described herein that has a radius of up to 150 nm. Exemplary radii include at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 125, or 150 nm. It is to be understood that a particle may not have a perfectly spherical shape, but can also be ellipsoid, rod- shaped, hexahedral, polyhedral, cuboid, or any such shape in which at least one dimension corresponds to the measurements described herein. It can be also understood that the target can include any biological or chemical material, such as one or more proteins (e.g., in a protein aggregate, such as an amyloid aggregate or a thrombus), one or more salts (e.g., a calcium carbonate nanop article), one or more polymers (e.g., a labeled or an un-labeled polystyrene bead or polysaccharide bead), one or more metals (e.g., gold particles, gold nanoparticles, or gold beads), or one or more microorganisms (e.g., a virion particle).

[0067] The use of high-magnification interferometric measurements is an approach to detection of single biomeolcular targets and particles. The methods can be modified by imaging through a high magnification objective lens with a high numerical aperture and placing a spatial filter on the camera's optical axis. The high numerical aperture objective lens will allow imaging at high magnifications and the spatial filter is used to maintain the contrast of the interference cause by the layered substrate by only collecting light from high angles of incident light. The optical setup described will allow for resolving sub-wavelength structures without losing contrast or lateral resolution.

[0068] Another approach to simplify the system further involves using a braodband source and a colored CCD camera in which the spectral sampling is done by the camera. Pixels of the camera dedicated for detection of separate colors can be used to extract the intensity of light included in a given spectral band, thus enable a spectral detection scheme.

[0069] LED-based high magnification substrate enhanced imaging offers a high reduction of cost from the more conventional laser based spectral reflectance imaging biosensor (SRIB). This reduction in cost is estimated to be about 20,000 dollars which is the cost of the laser. An LED based illumination source allows this imaging biosensor to be more robust and portable, thus allowing field applications. Another advantage is the high magnification capability of the invention. High magnification will allow for the detection of single biomolecular targer on the biosensor surface (e.g., > 50nm in length or diameter). In some embodiments, two or more LEDs, three or more LEDs, four or more LEDs, five or more LEDs, six or more LEDs, or three to six LEDS, can be used to perform high-throughput substrate enhanced imaging. In some embodiments, a white light source or an RGB LED with a 3CCD or other color camera can be used to capture spectral information at three distinct wavelengths to increase temporal resolution. This is beneficial in studying dynamic biological interactions, for example.

[0070] The invention describes, in part, a method of using LED illumination for substrate enhanced detection of binding of molecules to a surface. The invention provides in one aspect a high-throughput spectroscopy method for simultaneously recording a response of an entire substrate surface, comprising sampling different wavelengths using a light source covering different spectral windows, and imaging the reflected or transmitted light to a imaging device. The methods of the invention can be used, in part, for use in any high- throughput and label-free application.

[0071] One aspect of the invention provides a platform or a system for label-free and high-throughput optical sensing of solid substrates, comprising an illuminating source and a imaging device. In all embodiments, the illuminating source is not a tunable laser. In some embodiments, the light source is an LED. In some embodiments, the light source comprises two or more LEDs, three or more LEDs, four or more LEDs, five or more LEDs, six or more LEDs, or three to six LEDS. [0072] In some embodiments, the imaging device is a camera. The platform of the invention can be used for multiplexed and dynamic detection of biological material on a substrate. In some embodiments, the platform is used to detect nucleic acid-nucleic acid interactions. In some embodiments, the platform is used to detect nucleic acid-protein interactions. In some embodiments, the platform is used to detect protein-protein interactions. In some embodiments, the protein is a peptide. In some embodiments, the protein is an antibody. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises RNA.

[0073] In some embodiments of all aspects of the invention, the substrate comprises a silicon dioxide (SiO₂) surface. In some embodiments, the silicon dioxide surface is 100 nm - 1000 nm. In some embodiments, the silicon dioxide surface is less than 100 nm. In some embodiments, the silicon dioxide surface is less than 200 nm. In some embodiments, the silicon dioxide surface is less than 300 nm. In some embodiments, the silicon dioxide surface is less than 400 nm. In some embodiments, the silicon dioxide surface is less than 500 nm. In some embodiments, the silicon dioxide surface is less than 600 nm. In some embodiments, the silicon dioxide surface is less than 700 nm. In some embodiments, the silicon dioxide surface is less than 800 nm. In some embodiments, the silicon dioxide surface is less than 900 nm. In some embodiments, the silicon dioxide surface is less than 1000 nm.

[0074] All embodiments of the invention can be described through functional modules, which are defined by computer executable instructions recorded on computer readable media and which cause a computer to perform method steps when executed. The modules have been segregated by function for the sake of clarity. However, it should be understood that the modules need not correspond to discrete blocks of code and the described functions can be carried out by the execution of various code portions stored on various media and executed at various times.

[0075] In some embodiments, the invention provides a system for obtaining data regarding optical sensing of a solid substrate comprising a) a determination module configured to determine optical information, wherein the optical information comprises sampling different wavelengths using a light source covering different spectral windows; b) a storage device configured to store data output from the determination module; c) a comparison module adapted to compare the data stored on the storage device with a control data, the comparison being a retrieved content; and d) a display module for displaying a page of the retrieved content for the user on the client computer, wherein the retrieved content is a light absorption profile of the solid substrate, wherein a certain light absorption profile is indicative of binding of an analyte. In some embodiments, the light source is an LED. In all embodiments, the light source does not comprise a tunable laser.

[0076] In some embodiments, the invention provides a computer program comprising a computer readable media or memory having computer readable instructions recorded thereon to define software modules including a determination module and a comparison module for implementing a method on a computer, said method comprising a) determining with the determination module optical information, wherein the optical information comprises sampling different wavelengths using a light source covering different spectral windows; b) storing data output from the determination module; c) comparing with the comparison module the data stored on the storage device with a control data, the comparison being a retrieved content, and d) displaying a page of the retrieved content for the user on the client computer, wherein the retrieved content is a light absorption profile of the solid substrate, wherein a certain light absorption profile is indicative of binding of an analyte. In some embodiments, the light source includes one or more LEDs. In all embodiments, the light source does not comprise a tunable laser.

[0077] The "computer readable medium" can include data and computer-executable instructions for performing the steps of the method of the invention. Suitable computer readable media include floppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc. The computer executable instructions can be written in a suitable computer language or combination of several languages. Basic computational biology methods are described in, e.g. Setubal and Meidanis et al.,

Introduction to Computational Biology Methods (PWS Publishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics: Application in Biological Science and Medicine (CRC Press, London, 2000) and Ouelette and Bzevanis

Bioinformatics: A Practical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc., 2^nd ed., 2001).

[0078] In some aspects, the function modules of embodiments of the invention include a determination module, a storage device, a comparison module and a display module.

[0079] The determination module can include computer executable instructions to determine and provide optical information using an optical instrument. As used herein, an "optical instrument" refers to any instrument that either processes light waves to enhance an image for viewing, or analyzes light waves (or photons) to determine one of a number of characteristic optical properties.

[0080] Known determination modules for determining optical properties include, for example, but are not limited to, microscopes, cameras, interferometers (for measuring the interference properties of light waves), photometers (for measuring light intensity);

polarimeters (for measuring dispersion or rotation of polarized light), reflectometers (for measuring the reflectivity of a surface or object), refractometers (for measuring refractive index of various materials), spectrometers or monochromators (for generating or measuring a portion of the optical spectrum, for the purpose of chemical or material analysis),

autocollimators (used to measure angular deflections), and vertometers (used to determine refractive power of lenses such as glasses, contact lenses and maginfier lens).

[0081] A "spectrograph" or "spectrometer", as defined herein, is an optical instrument used to measure properties of light over a specific portion of the electromagnetic spectrum, typically used in spectroscopic analysis to identify materials. The variable measured is most often the light's intensity but could also, for instance, be the polarization state. The independent variable is usually the wavelength of the light, normally expressed as a fraction of a meter, but sometimes expressed as a unit directly proportional to the photon energy, such as wavenumber or electron volts, which has a reciprocal relationship to wavelength. A spectrometer is used in spectroscopy for producing spectral lines and measuring their wavelengths and intensities. Spectrometer is a term that is applied to instruments that operate over a very wide range of wavelengths, from gamma rays and X-rays into the far infrared. If the region of interest is restricted to near the visible spectrum, the study is called

spectrophotometry.

[0082] Spectrophotometry involves the use of a spectrophotometer. As defined herein, a "spectrophotometer" is a photometer (a device for measuring light intensity) that can measure intensity as a function of the color, or more specifically, the wavelength of light. There are many kinds of spectrophotometers. Among the most important distinctions used to classify them are the wavelengths they work with, the measurement techniques they use, how they acquire a spectrum, and the sources of intensity variation they are designed to measure. Other important features of spectrophotometers include the spectral bandwidth and linear range. There are two major classes of spectrophotometers; single beam and double beam. A double beam spectrophotometer measures the ratio of the light intensity on two different light paths, and a single beam spectrophotometer measures the absolute light intensity. Although ratio measurements are easier, and generally more stable, single beam instruments have advantages; for instance, they can have a larger dynamic range, and they can be more compact. Historically, spectrophotometers use a monochromator to analyze the spectrum, but there are also spectrophotometers that use arrays of photosensors. Especially for infrared spectrophotometers, there are spectrophotometers that use a Fourier transform technique to acquire the spectral information quicker in a technique called Fourier Transform InfraRed. The spectrophotometer quantitatively measures the fraction of light that passes through a given solution. In a spectrophotometer, a light from the lamp is guided through a

monochromator, which picks light of one particular wavelength out of the continuous spectrum. This light passes through the sample that is being measured. After the sample, the intensity of the remaining light is measured with a photodiode or other light sensor, and the transmittance for this wavelength is then calculated. In short, the sequence of events in a spectrophotometer is as follows: the light source shines through the sample, the sample absorbs light, the detector detects how much light the sample has absorbed, the detector then converts how much light the sample absorbed into a number, the numbers are e are transmitted to a comparison module to be further manipulated (e.g. curve smoothing, baseline correction). Many spectrophotometers must be calibrated by a procedure known as "zeroing." The absorbency of some standard substance is set as a baseline value, so the absorbencies of all other substances are recorded relative to the initial "zeroed" substance. The

spectrophotometer then displays % absorbency (the amount of light absorbed relative to the initial substance). The most common application of spectrophotometers is the measurement of light absorption, but they can be designed to measure diffuse or specular reflectance. Strictly, even the emission half of a luminescence instrument is a kind of spectrophotometer.

[0083] The optical information determined in the determination module can be read by the storage device.

[0084] As used herein the "storage device" is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of storage devices suitable for use with the present invention include stand-alone computing apparatus; communications networks, including local area networks (LAN), wide area networks (WAN), Internet, Intranet, and Extranet; and local and distributed processing systems. Storage devices also include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as compact disc; electronic storage media such as RAM, ROM, EPROM, EEPROM and the like; general hard disks and hybrids of these catagories such as

magnetic/optical storage media. The medium is adapted or configured for having recorded there on sequence information or expression level information. The data is typically provided in digital form that can be transmitted and read electronically, e.g., via the Internet, on diskette, or any other mode of electronic or non-electronic communication.

[0085] As used herein, "stored" refers to a process for storing information on the storage device such that it can be read back from the device. Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising the sequence information or expression level information.

[0086] A variety of software programs and formats can be used to store the optical information on the storage device. Any number of data processor structuring formats (e.g., text file or database) can be employed to obtain or create a medium having the information recorded thereon.

[0087] By providing optical information in computer-readable form, one can use the optical information in readable form to compare a specific optical profile with the optical information stored within a database of the comparison module. For example, direct comparison of the determined optical information from a given analyte can be compared to the control data optical information (e.g., data obtained from a control analyte). The comparison made in computer-readable form being the retrieved content from the comparison module, which can be processed by a variety of means.

[0088] A page of the retrieved content can then be displayed through a "display module".

[0089] As defined herein, a "light emitting diode (LED)" is an electronic light source based on the semiconductor diode. When the diode is forward biased (switched on), electrons are able to recombine with holes and energy is released in the form of light. This effect is called electroluminescence and the color of the light is determined by the energy gap of the semiconductor. The LED is usually small in area (less than 1 mm ) with integrated optical components to shape its radiation pattern and assist in reflection. Like a normal diode, the LED consists of a chip of semiconducting material impregnated, or doped, with impurities to create a p-n junction. As in other diodes, current flows easily from the p-side, or anode, to the n-side, or cathode, but not in the reverse direction. Charge-carriers— electrons and holes— flow into the junction from electrodes with different voltages. When an electron meets a hole, it falls into a lower energy level, and releases energy in the form of a photon. The wavelength of the light emitted, and therefore its color, depends on the band gap energy of the materials forming the p-n junction. In silicon or germanium diodes, the electrons and holes recombine by a non-radiative transition which produces no optical emission, because these are indirect band gap materials. The materials used for the LED have a direct band gap with energies corresponding to near-infrared, visible or near-ultraviolet light. LEDs are usually built on an n-type substrate, with an electrode attached to the p-type layer deposited on its surface. P- type substrates, while less common, occur as well. Many commercial LEDs, especially GaN/InGaN, also use sapphire substrate. Most materials used for LED production have very high refractive indices. This means that much light will be reflected back in to the material at the material/air surface interface. LEDs of use for the present invention, include but are not limited to:

[0090] As defined herein, a "tunable laser" is a laser whose wavelength of operation can be altered in a controlled manner. While all laser gain media allow small shifts in output wavelength, only a few types of lasers allow continuous tuning over a significant wavelength range.

[0091] As defined herein, a substrate surface can include a "specular reflecting interface." Such specular reflecting interfaces refer to those surfaces upon which incoming light undergoes "specular reflection," i.e., the mirror- like reflection of light (or sometimes other kinds of wave) from a surface, in which light from a single incoming direction (a ray) is reflected into a single outgoing direction. Such specular reflecting behavior of a surface, substrate or interface, is described by the law of reflection, which states that the direction of incoming light (the incident ray), and the direction of outgoing light reflected (the reflected ray) make the same angle with respect to the surface normal, thus the angle of incidence equals the angle of reflection; mathematically defined θi = θr. A second defining

characteristic of specular reflection is that the incident, normal, and reflected directions are coplanar. Specular reflection can be accurately measured using, for example, a glossmeter. The measurement is based on the refractive index of an object. In some embodiments of the aspects described herein, a specular reflecting interface comprises a substrate having a transparent dielectric layer, for example a layer of Silicon Oxide (SiO2) on a Silicon substrate.

[0092] In some embodiments, an alternative transparent dielectric layer, such as an indium tin oxide layer, is used as a thin transparent or specular reflecting interface layer. In some embodiments of the aspects described herein, the indium tin oxide layer is less than or equal to 1000 nm, less than or equal to 900 nm, less than or equal to 800 nm, less than or equal to 700 nm, less than or equal to 600 nm, less than or equal to 500 nm, less than or equal to 400 nm, less than or equal to 300 nm, less than or equal to 200 nm, less than or equal to 100 nm, less than or equal to 90 nm, less than or equal to 80 nm, less than or equal to 70 nm, less than or equal to 60 nm, less than or equal to 50 nm, less than or equal to 40 nm, less than or equal to 30 nm, less than or equal to 20 nm, or less than or equal to 10 nm.

Applications of the Sensors and Methods of the Invention

[0093] The ability to detect biological target molecules, such as DNA, RNA, and proteins, as well as nanomolecular particles such as virions, is fundamental to our

understanding of both cell physiology and disease progression, as well as for use in various applications such as the early and rapid detection of disease outbreaks and bioterrorism attacks. Such detection, however, is limited by the need to use labels, such as fluorescent molecules or radiolabels, which can alter the properties of the biological target, e.g., conformation, and which can add additional, often time-consuming, steps to a detection process.

[0094] Described herein are rapid, sensitive, simple to use, and inexpensive biosensors that are useful for a variety of applications involving the detection of biomolecular targets and analytes, ranging from research and medical diagnostics, to detection of agents used in bioterrorism. Such targets and analytes include, but are not limited to,

polynucleotides, peptides, small proteins, antibodies, viral particles, and cells. Furthermore, the biosensors described herein have the ability to simultaneously quantify many different biomolecular interactions with high sensitivity for use in pharmaceutical drug discovery, proteomics, and diagnostics. Such biomolecular complexes include, for example,

oligonucleotide interactions, antibody- antigen interactions, hormone -receptor interactions, and enzyme-substrate interactions.

[0095] The direct detection of biochemical and cellular binding without the use of a fluorophore, a radioligand or a secondary reporter, using the biosensors and methods described herein, removes the experimental uncertainty induced by the effect of a label on, for example, molecular conformation, the blocking of active binding epitopes, steric hindrance, inaccessibility of the labeling site, or the inability to find an appropriate label that functions equivalently for all molecules in an experiment. The sensors and detection methods described herein greatly simplify the time and effort required for assay development, while removing experimental artifacts that occur when labels are used, such as quenching, shelf life, and background fluorescence.

Detection of Sub-Cellular Biomolecular Targets

[0096] Described herein are biosensors suitable for the detection of a wide variety of biomolecular targets present in a sample. Such biomolecular targets include, but are not limited to, sub-cellular molecules such as polynucleotides, peptides, polypeptides, and proteins present in a sample. Binding of one or more of these molecules to the substrate surface of the biosensors described herein causes a change in the interference pattern of the substrate surface, relative to the interference pattern of the substrate surface in the absence of binding, that can be measured by the biosensor, thus allowing the biosensor to detect the presence of one or more binding events. In addition, the biosensors described herein can be designed to have immobilized molecules bound to the substrate surface, such that a change in interference pattern is detected by the biosensor upon binding of one or more biomolecular targets present in a sample to one or more of the immobilized molecules present on the substrate surface. Such biosensors are useful for the detection of a variety of biomolecular interactions, such as, for example, oligonucleotide-oligonucleotide, oligonucleotide-protein, antibody-antigen, hormone-hormone receptor, and enzyme- substrate interactions.

[0097] Accordingly, in one aspect, the biosensors described herein are used to detect binding of a biomolecular target to a biosensor substrate layer, wherein binding of a biomolecular target present in a sample contacted with the biosensor substrate layer changes an optical path length relative to an optical path length in the absence of the sample, resulting in a phase-shifted interference pattern that is detected and measured by the biosensor. In some embodiment of this aspect, the sample that contacts the biosensor has a plurality of biomolecular targets, such that multiple biomolecular targets bind to the biosensor substrate layer and are detected by the biosensor.

[0098] The biosensors of the invention can be used to study one or a number of specific binding interactions in parallel, i.e., multiplex applications. Binding of one or more specific binding substances to their respective binding molecules can be detected, without the use of labels, by applying a sample comprising one or more biomolecular targets to a biosensor that has one or more specific binding molecules immobilized on its surface. The biosensor is illuminated with light, and if one or more biomolecular targets in the sample specifically bind one or more of the immobilized molecules, a phase-shift in the interference pattern occurs relative to the interference pattern when one or more specific biomolecular targets have not bound to the immobilized binding molecules. In those embodiments where a biosensor substrate surface comprises an array of one or more distinct locations comprising one or more specific immobilized binding molecules, then the interference pattern is detected from each distinct location of the biosensor.

[0099] Thus, in some embodiments of the invention, a variety of specific binding molecules, for example, antibodies, can be immobilized in an array format onto the substrate surface of a biosensor of the invention. The biosensor is then contacted with a test sample of interest comprising potential biomolecular target binding partners, such as proteins. Only the proteins that specifically bind to the antibodies immobilized on the biosensor remain bound to the biosensor. Such an approach is essentially a large-scale version of an enzyme-linked immunosorbent assay; however, the use of an enzyme or fluorescent label is not required. For high-throughput applications, biosensors can be arranged in an array of arrays, wherein several biosensors comprising an array of specific binding molecules on the substrate surface are arranged in an array.

[0100] Accordingly, in other embodiments of this aspect and all such aspects described herein, biosensors are used to detect binding of one or more of a plurality of biomolecular targets present in a sample to a biosensor substrate layer comprising one or more of a plurality of immobilized molecules attached to the substrate layer. For example, one or more specific immobilized molecules can be arranged in an array of one or more distinct locations on the surface of the biosensor. The one or more distinct locations can define microarray spots of about 50-500 microns, or about 150-200 microns in diameter.

[0101] In such embodiments, the immobilized molecules can be a DNA

oligonucleotide, RNA oligonucelotide, a peptide, a protein, such as a transcription factor, antibody or enzyme, a small organic molecule, or any combination therein. Such biosensors are useful for the detection of biomolecular interactions, including, but not limited to, DNA- DNA, DNA-RNA, DNA -protein, RNA-RNA, RNA-protein, and protein-protein interactions. For example, a biosensor having a plurality of DNA oligonucleotides immobilized on the substrate surface can be used to detect the presence of a protein, such as a transcription factor, present in a sample contacted with the substrate layer, that binds to one or more of the oligonucleotides. Further, as the biosensors described herein do not require labeling of the biomolecular targets, conformational changes that occur on binding of the biomolecular target to the immobilized molecule can be more precisely detected and measured.

[0102] Thus, the novel technology described herein is useful in applications where large numbers of biomolecular interactions are measured in parallel, particularly when molecular labels will alter or inhibit the functionality of the biomolecular targets under study. High-throughput screening of pharmaceutical drug compound libraries with protein biomolecular targets, and microarray screening of protein-protein interactions for proteomics are non-limiting examples of applications that require the sensitivity and throughput afforded by this approach. [0103] The methods described herein can also be used to determine kinetic and affinity constants for molecular interactions between a biomolecular target in a sample and an immobilized molecule attached to the substrate, including association constants, dissociation constants, association rate constants, and dissociation rate constants. The method of the present invention can also be used to determine the concentration of one or more

biomolecular targets in a sample.

[0104] Thus, in one embodiment, a biosensor is used to detect binding of one or more of a plurality of biomolecular targets present in a sample to a biosensor substrate layer comprising one or more of a plurality of immobilized DNA oligonucleotides attached to the substrate layer. In another embodiment, a biosensor is used to detect binding of one or more of a plurality of biomolecular targets present in a sample to a biosensor substrate layer comprising one or more of a plurality of immobilized RNA oligonucleotides attached to the substrate layer. In another embodiment, a biosensor is used to detect binding of one or more of a plurality of biomolecular targets present in a sample to a biosensor substrate layer comprising one or more of a plurality of immobilized peptides attached to the substrate layer. In another embodiment, a biosensor is used to detect binding of one or more of a plurality of biomolecular targets present in a sample to a biosensor substrate layer comprising one or more of a plurality of immobilized proteins attached to the substrate layer. In one such embodiment, the protein is an antigen. In another such embodiment, the protein is a polyclonal antibody, monoclonal antibody, single chain antibody (scFv), F(ab) fragment, F(ab')₂ fragment, or an Fv fragment. In another such embodiment, the protein is an enzyme.

[0105] Some embodiments of the invention provide a method of detecting whether a biomolecular target inhibits the activity of an enzyme or binding partner, i.e., "inhibition activity" of the biomolecular target. In one such embodiment, a sample comprising one or more biomolecular targets to be tested for having inhibition activity is contacted with a biosensor comprising one or more immobilized molecules. This is followed by adding one or more enzymes known to act upon at least one of the immobilized molecules on the biosensor substrate. Where the one or more enzymes have altered the one or more immobilized molecules on the substrate surface of the biosensor, for example, by cleaving all or a portion of an immobilized molecule from the surface of a biosensor, a shift in the interference pattern is detected by the biosensor. Thus, a sample comprising a biomolecular target having no inhibition activity allows the enzyme activity to occur unabated, such that the interference pattern changes upon addition of the enzyme(s); a biomolecular target with substantially complete inhibition activity halts the reaction substantially completely, such that no change in interference pattern is detected by the biosensor upon addition of the enzyme(s); and a biomolecular target with partial inhibition halts the reaction partially, resulting in an intermediate shift in the interference pattern upon addition of the enzyme(s).

[0106] Further, in some embodiments, the biosensors described herein can be used to detect a change in interference pattern at one or more distinct locations on a biosensor substrate. For example, when the biosensor is used to identify biomolecular targets having enzymatic inhibition activity, the samples comprising one or more biomolecular targets is contacted with one or more distinct locations on the biosensor substrate surface, and then one or more enzymes are contacted at these distinct locations. The optical interference pattern of the one or more distinct locations is then detected and compared to the initial optical interference pattern. In other embodiments, the sample comprising one or more biomolecular targets being tested for inhibitory activity is mixed with the one or more enzymes, which can be contacted to the one or more distinct locations, and the optical interference pattern is compared to the optical interference pattern obtained when no biomolecular targets are present in the sample.

[0107] In other embodiments of this aspect, and all such aspects described herein, the biosensors are used to detect binding of one or more of a plurality of biomolecular targets present in a first sample applied to a substrate layer of the biosensor to one or more of a plurality of biomolecular targets present in a second sample, such that the second sample is added to the first sample, and causes changes in optical path length relative to an optical path length in the absence of the second sample, resulting in a phase-shifted interference pattern that is detected and measured by the biosensor.

[0108] As used herein, a molecule immobilized on the substrate surface of a biosensor can be, for example, an organic molecule, such as a nucleic acid, oligonucleotide, peptide, polypeptide, antigen, polyclonal antibody, monoclonal antibody, single chain antibody (scFv), F(ab) fragment, F(ab') ₂ fragment, Fv fragment, small organic molecule, polymer, compounds from a combinatorial chemical library, inorganic molecule, or any combination therein.

[0109] A sample refers to any sample containing a biomolecular target, such as, for example, blood, plasma, serum, gastrointestinal secretions, homogenates of tissues or tumors, synovial fluid, feces, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid, tears, prostatitc fluid, or cellular lysates. A sample may also be obtained from an environmental source, such as water sample obtained from a polluted lake or other body of water, or a liquid sample obtained from a food source believed to contaminated.

Detection of Viral Biomolecular Targets

[0110] The development of the biosensors described herein is useful for a variety of applications in which it was not previously possible, feasible, or practical to perform frequent or rapid testing for viruses, such as the fields of pharmaceutical discovery, diagnostic testing, environmental testing, bioterrorism, and food safety. A virus is a small infectious agent that can replicate only inside the living cells of other organisms. Most viruses are too small to be seen directly with a light microscope. Early and rapid detection of viruses or viral particles is important for detecting contaminations in food supplies, and in protection against

bioterrorism threats, as current detection methods, such as electron microscopy, are time- consuming, non-portable, and expensive.

[0111] Accordingly, in one aspect, the biosensors described herein are used to detect binding of a viral biomolecular target to a biosensor substrate layer, wherein binding of a viral biomolecular target present in a sample contacted with the biosensor substrate layer changes an optical path length relative to an optical path length in the absence of the sample, resulting in a phase-shifted interference pattern that is detected and measured by the biosensor. In some embodiment of this aspect, the sample that contacts the biosensor has a plurality of viral biomolecular targets, such that multiple viral biomolecular targets bind to the biosensor substrate layer and are detected by the biosensor. The different viral biomolecular targets can be differentiated on the basis of, for example, size, shape, or a combination therein.

[0112] The biosensors of the invention can be used for multiplex applications whereby one or a number different viruses are studied in parallel. Binding of one or more specific binding viral biomolecular targets can be detected, without the use of labels, by applying a sample comprising one or more biomolecular targets to a biosensor that has one or more specific binding molecules, such as virus-specific antibodies or fragments thereof, immobilized on its surface. The biosensor is illuminated with light, and if one or more viral biomolecular targets in the sample specifically binds one or more of the immobilized molecules, a phase-shift in the interference pattern occurs relative to the interference pattern when one or more specific viral biomolecular targets have not bound to the immobilized virus-specific binding molecules. In those embodiments where a biosensor substrate surface comprises an array of one or more distinct locations comprising the one or more specific immobilized virus-specific binding molecules, then the interference pattern is detected from each distinct location of the biosensor.

[0113] Thus, in some embodiments of the invention, a variety of specific binding molecules, for example, antibodies, can be immobilized in an array format onto the substrate surface of a biosensor of the invention. The biosensor is then contacted with a test sample of interest comprising potential viral biomolecular targets. Only the viruses that specifically bind to the antibodies immobilized on the biosensor remain bound to the biosensor. Such an approach is essentially a large-scale version of an enzyme-linked immunosorbent assay, without using an enzyme or fluorescent label. For high-throughput applications, biosensors can be arranged in an array of arrays, wherein several biosensors comprising an array of specific binding molecules on the substrate surface are arranged in an array.

[0114] In one such embodiment of the aspect, a biosensor substrate surface comprises one or more antibodies specific for different viruses, whereby different locations on the substrate surface comprise antibodies specific for distinct viral species, such that changes in the optical interference pattern at different locations on the surface, upon contacting a sample with the substrate surface, is indicative of the presence of distinct viral species in the sample (e.g., smallpox, Ebola and Marburg viruses). Such a biosensor is useful, for example, in the rapid identification of agents used during a bioterrorist attack.

[0115] In one such embodiment of the aspect, a biosensor substrate surface comprises one or more antibodies specific for different influenza hemagglutinins, whereby different locations on the substrate surface comprise antibodies specific for distinct hemagglutinins, such that changes in the optical interference pattern at different locations upon contacting a sample with the substrate surface is indicative of the presence of distinct influenza species (e.g., Influenza A, Influenza B, and Influenza C) in the sample. Such a biosensor can distinguish, for example, between the presence of different influenza serotypes in a sample, such as HlNl, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, and H10N7.

[0116] Exemplary viruses and viral families that can be detected using the biosensors and methods described herein include, but are not limited to: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-I (also referred to as HTLV-III), HIV-2, LAV or HTLV-III/LAV, or HIV-III, and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses,

echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); adenovirus; Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses);

Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses, i.e., Rotavirus A, Rotavirus B. Rotavirus C); Birnaviridae; Hepadnaviridae (Hepatitis A and B viruses); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, Human herpes virus 6, Human herpes virus 7, Human herpes virus 8, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Epstein-Barr virus; Rous sarcoma virus; West Nile virus; Japanese equine encephalitis, Norwalk, papilloma virus, parvovirus B19; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); Hepatitis D virus, Hepatitis E virus, and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class l=enterally transmitted; class

2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astro viruses). Detection of Sub-Cellular and Cellular Changes

[0117] The biosensors described herein are also useful for applications involving the detection of changes in cellular and sub-cellular functions in a sample. Such applications include, but are not limited to, testing of pharmaceutical drug candidates on cellular functions, morphology, and growth.

[0118] Accordingly, in one aspect, the biosensors described herein are used in a method of conducting a cell-based assay of a sample comprising one or more cells, whereby a cellular function being measured by the cell-based assay results in a phase-shift in the optical interference pattern detected and measured by the biosensor. The interference pattern detected and measured by the biosensor provides a high-resolution image that can be used to identify and detect, for example, internal and external changes to a cell or cells present in a sample. In some embodiments, the cell-based assay measures a cellular function. In some embodiments, the cellular function is selected from the group consisting of cellular viability, cellular growth or changes in size, phagocytosis, channel opening/closing, changes in intracellular components and organelles, such as vesicles, mitochondria, membranes, structural features, periplasm, or any extracts thereof, and protein distribution.

Other Applications [0119] The biosensors described herein can also be used in a variety of other applications. These applications include, but are not limited to, environmental applications (e.g., the detection of pesticides and river water contaminants); detection of non-viral pathogens; determining the presence and/or levels of toxic substances before and following bioremediation; analytic measurements in the food industry (e.g., determination of organic drug residues in food, such as antibiotics and growth promoters; detection of small molecules, such as water soluble vitamins; detection of non-organic chemical contaminants), and the detection of toxic metabolites such as mycotoxins.

Definitions

[0120] As defined herein, a "biomolecular target" refers to a biological material such as a protein, an oligonucleotide, a cell, a virus particle, and a bacterium. Other types of biomolecular target which can be detected by the biosensors described herein include low molecular weight molecules (i.e., substances of molecular weight <1000 Daltons (Da) and between 1000 Da to 10,000 Da), and include amino acids, nucleic acids, lipids,

carbohydrates, nucleic acid polymers, viral particles, viral components and cellular components. Cellular components that can serve as biomolecular targets can include, but are not limited to, vesicles, mitochondria, membranes, structural features, periplasm, or any extracts thereof.

[0121] As

[0122] As used herein the terms "sample" or "biological sample" means any sample, including, but not limited to cells, organisms, lysed cells, cellular extracts, nuclear extracts, components of cells or organisms, extracellular fluid, media in which cells are cultured, blood, plasma, serum, gastrointestinal secretions, homogenates of tissues or tumors, synovial fluid, feces, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid, tears and prostatic fluid. In addition, a sample can be a viral or bacterial sample, a sample obtained from an environmental source, such as a body of polluted water, an air sample, or a soil sample, as well as a food industry sample.

[0123] "Tissue" is defined herein as a group of cells, often of mixed types and usually held together by extracellular matrix, that perform a particular function. Also, in a more general sense, "tissue" can refer to the biological grouping of a cell type result from a common factor; for example, connective tissue, where the common feature is the function or epithelial tissue, where the common factor is the pattern of organization.

[0124] A "nucleic acid", as described herein, can be RNA or DNA, and can be single or double stranded, and can be, for example, a nucleic acid encoding a protein of interest, a polynucleotide, an oligonucleotide, a nucleic acid analogue, for example peptide- nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acid (LNA) etc. Such nucleic acid sequences include, for example, but are not limited to, nucleic acid sequence encoding proteins, for example that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example, but not limited to, RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc.

[0125] As used herein, the term "DNA" is defined as deoxyribonucleic acid. The term

"polynucleotide" is used herein interchangeably with "nucleic acid" to indicate a polymer of nucleosides. Typically a polynucleotide of this invention is composed of nucleosides that are naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) joined by

phosphodiester bonds. However the term encompasses molecules comprising nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. Where this application refers to a polynucleotide it is understood that both DNA, RNA, and in each case both single- and double-stranded forms (and complements of each single- stranded molecule) are provided. "Polynucleotide sequence" as used herein can refer to the polynucleotide material itself and/or to the sequence information (i.e. the succession of letters used as abbreviations for bases) that biochemically characterizes a specific nucleic acid. A polynucleotide sequence presented herein is presented in a 5' to 3' direction unless otherwise indicated.

[0126] The term " polypeptide" as used herein refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide is a relatively short polypeptide, typically between about 2 and 60 amino acids in length. Polypeptides used herein typically contain amino acids such as the 20 L- amino acids that are most commonly found in proteins. However, other amino acids and/or amino acid analogs known in the art can be used. One or more of the amino acids in a polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc. A polypeptide that has a nonpolypeptide moiety covalently or noncovalently associated therewith is still considered a "polypeptide". Exemplary modifications include glycosylation and palmitoylation.

Polypeptides may be purified from natural sources, produced using recombinant DNA technology, synthesized through chemical means such as conventional solid phase peptide synthesis, etc. The term "polypeptide sequence" or "amino acid sequence" as used herein can refer to the polypeptide material itself and/or to the sequence information (i.e., the succession of letters or three letter codes used as abbreviations for amino acid names) that biochemically characterizes a polypeptide. A polypeptide sequence presented herein is presented in an N- terminal to C-terminal direction unless otherwise indicated.

[0127] "Receptor" is defined herein as a membrane-bound or membrane-enclosed molecule that binds to, or responds to something more mobile (the ligand), with high specificity.

[0128] "Ligand" is defined herein as a molecule that binds to another; in normal usage a soluble molecule, such as a hormone or neurotransmitter, that binds to a receptor. Also analogous to "binding substance" herein.

[0129] "Antigen" is defined herein as a substance inducing an immune response. The antigenic determinant group is termed an epitope, and the epitope in the context of a carrier molecule (that can optionally be part of the same molecule, for example, botulism neurotoxin A, a single molecule, has three different epitopes. See Mullaney et al., Infect Immun October 2001; 69(10): 6511-4) makes the carrier molecule active as an antigen. Usually antigens are foreign to the animal in which they produce immune reactions.

[0130] As used herein "antibodies" can include polyclonal and monoclonal antibodies and antigen-binding derivatives or fragments thereof. Well known antigen binding fragments include, for example, single domain antibodies (dAbs; which consist essentially of single VL or VH antibody domains), Fv fragment, including single chain Fv fragment (scFv), Fab fragment, and F(ab')₂ fragment. Methods for the construction of such antibody molecules are well known in the art. As used herein, the term "antibody" refers to an intact immunoglobulin or to a monoclonal or polyclonal antigen-binding fragment with the Fc (crystallizable fragment) region or FcRn binding fragment of the Fc region. Antigen-binding fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. "Antigen-binding fragments" include, inter alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single domain antibodies, chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. The terms Fab, Fc, pFc', F(ab') 2 and Fv are employed with standard

immunological meanings [Klein, Immunology (John Wiley, New York, N. Y., 1982); Clark, W. R. (1986) The Experimental Foundations of Modern Immunology (Wiley & Sons, Inc., New York); Roitt, I. (1991) Essential Immunology, 7th Ed., (Blackwell Scientific

Publications, Oxford)]. [0131] "Polyclonal antibody" is defined herein as an antibody produced by several clones of B-lymphocytes as would be the case in a whole animal. Usually refers to antibodies raised in immunized animals. "Monoclonal antibody" is defined herein as a cell line, whether within the body or in culture, that has a single clonal origin. Monoclonal antibodies are produced by a single clone of hybridoma cells, and are therefore a single species of antibody molecule. "Single chain antibody (Scfv)" is defined herein as a recombinant fusion protein wherein the two antigen binding regions of the light and heavy chains (Vh and Vl) are connected by a linking peptide, which enables the equal expression of both the light and heavy chains in a heterologous organism and stabilizes the protein. "F(Ab) fragment" is defined herein as fragments of immunoglobulin prepared by papain treatment. Fab fragments consist of one light chain linked through a disulphide bond to a portion of the heavy chain, and contain one antigen binding site. They can be considered as univalent

antibodies. "F(Ab')₂ Fragment" is defined herein as the approximately 90 kDa protein fragment obtained upon pepsin hydrolysis of an immunoglobulin molecule N-terminal to the site of the pepsin attack. Contains both Fab fragments held together by disulfide bonds in a short section of the Fe fragment. "Fv Fragment" is defined herein as the N-terminal portion of a Fab fragment of an immunoglobulin molecule, consisting of the variable portions of one light chain and one heavy chain.

[0132] As used herein, the term "small molecule" refers to a chemical agent including, but not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0133] As used herein, the term "drug" or "compound" refers to a chemical entity or biological product, or combination of chemical entities or biological products, administered to a person to treat or prevent or control a disease or condition. The chemical entity or biological product is preferably, but not necessarily a low molecular weight compound, but may also be a larger compound, for example, an oligomer of nucleic acids, amino acids, or carbohydrates including, without limitation, proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications and combinations thereof.

[0134] "Inhibition activity" is defined herein as the ability of a molecule or compound to slow or stop another molecule from carrying out catalytic or other functional activity. For example, a compound that has inhibition activity of a protease inhibits the protease from cleaving a protein. Such inhibition activity is carried out "against" the catalytic molecule. "Inhibition activity" also means the ability of a molecule or compound to substantially inhibit or partially inhibit the binding of a binding partner to a specific binding molecule.

[0135] The terms "label" or "tag", as used herein, refer to a composition capable of producing a detectable signal indicative of the presence of the target in an assay sample. Suitable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.

[0136] The articles "a" and "an" are used herein to refer to one or to more than one

(i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. Thus, in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a pharmaceutical composition comprising "an agent" includes reference to two or more agents.

[0137] As used herein, the term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment. As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages can mean +1%.

[0138] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references cited throughout this application, as well as the figures and tables are incorporated herein by reference. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Other features and advantages of the invention will be apparent from the following Detailed Description, the drawings, and the claims.

[0139] The present invention can be defined in any of the following numbered paragraphs:

1. A spectral reflectance imaging system comprising: a substrate having a first reflective surface and a thin transparent layer providing a second reflective surface; a biolayer bound to the second reflective surface; an illumination source comprising at least three light sources, each providing light in one of three different narrow frequency bands and directing each frequency band of light at the substrate; and an imaging device directed at the second reflective surface of the substrate and adapted to produce imaging signals representative of light from the illumination source being reflected by the first reflective surface and the second reflective surface.

2. A spectral reflectance imaging system according to paragraph 1, wherein the first reflective surface is a silicon substrate and the transparent layer is silicon oxide (SiO₂).

3. A spectral reflectance imaging system according to paragraph 1, further comprising an image acquisition and processing system, coupled to the imaging device and adapted to receive the imaging signals and under program control, produce an image of the biolayer on the second reflective surface.

4. A spectral reflectance imaging system according to any of paragraphs 1-3, wherein the thickness of the biolayer is determined as function of the intensity of the reflected light received by the imaging device.

5. A spectral reflectance imaging system according to paragraph 1, wherein the transparent layer is less than 1 micron thick.

6. A spectral reflectance imaging system according to paragraph 1, wherein the transparent layer has a thickness in the range from 100 nanometers to 1000 nanometers.

7. A spectral reflectance imaging system according to paragraph 1, wherein the illumination source includes four light sources.

8. A spectral reflectance imaging system according to paragraph 7, wherein the illumination source produces light at 460 nanometer, 520 nanometer, 600 nanometer and 630 nanometer wavelengths. 9. A spectral reflectance imaging system according to paragraph 1, wherein each light source produces light in a band that is 20 - 30 nanometers wide.

10. A spectral reflectance imaging system according to paragraph 1, wherein each light source produces light in a band that is 10 - 20 nanometers wide.

11. A spectral reflectance imaging system according to paragraph 1, wherein each light source produces light in a band that is 5 nanometers wide.

12. A spectral reflectance imaging system according to paragraph 1, wherein the illumination source produces white light and the system further includes a color wheel having at least three filters, each producing a beam of light in one of at least three narrow frequency bands that is directed at the substrate.

13. A spectral reflectance imaging system according to paragraph 1, further comprising at least one optical fiber and wherein the light from each light source is directed through the at least one optical fiber toward the substrate.

14. A spectral reflectance imaging system according to paragraph 13, further comprising three optical fibers and wherein each band of light from each light source is directed through a separate optical fiber toward the substrate.

15. A method for detecting the binding of a particle to a surface of a substrate, the method comprising: providing a first specular reflecting interface of the surface of the substrate with a binding agent for binding a predefined particle to the first specular reflecting interface surface of the substrate; providing a second specular reflecting interface that is substantially parallel to and underlies the first specular reflecting interface; illuminating the surface with light substantially centered around one or more wavelengths; imaging light reflected or transmitted from the substrate using an imaging device; producing a spectral reflectance image of the surface of the substrate; and correlating the features on the image to discrete particles on the surface.

16. The method of paragraph 15, wherein the imaging device comprises a camera having a high magnification objective lens with a high numerical aperture.

17. The method of paragraph 15, wherein each wavelength of light is produced by a separate, narrow band light source.

18. The method of any one of paragraphs 15-17, wherein the imaging device is a monochromatic CCD camera.

19. The method of any one of paragraphs 15-18, wherein the surface is illuminated by a light source from a standard bright-field microscope optical setup, and wherein the reflected light is transmitted to an eyepiece. 20. The method of any one of paragraphs 15-19, wherein each wavelength of light is produced by a separate light emitting diode (LED), each having a different emission peak wavelengths, and wherein the imaging device is a monochromatic camera.

21. The method of paragraph 20, wherein the imaging device is a monochromatic CCD camera.

22. The method of any one of paragraphs 15-21, wherein the layered substrate comprises 500 nm of SiO₂ layered on a Si wafer.

23. The method of any one of paragraphs 15-22, wherein the surface is illuminated with white light and the imaging device includes a color camera.

24. The method of paragraph 23, wherein the color camera is a 3CCD camera.

25. The method of any one of paragraphs 15-24, wherein the surface is illuminated by an RGB (red green blue) LED and the imaging device includes a color camera.

26. The method of paragraph 25, wherein the color camera is a 3CCD camera.

27. The method of any one of paragraphs 15-26, wherein the surface is illuminated by a broadband light source.

28. The method of any one of paragraphs 15-27, wherein the camera further comprises a spatial filter on the camera's optical axis.

29. A method for detecting a particle on a surface of a layered substrate comprising: providing the surface of the layered substrate; contacting a solution having at least one particle with the surface of the substrate; illuminating the surface with at least three wavelengths of light; imaging the light reflected or transmitted from the substrate using an imaging device; and producing a spectral reflectance image of the surface of the substrate to detect the particle on the surface of the layered substrate.

30. The method of paragraph 29, wherein the layered substrate comprises SiO₂ layered on a Si substrate.

31. The method of any of paragraphs 29-30, wherein the light is coherent or incoherent.

32. The method of paragraph 29, wherein each wavelength of light is produced by a separate, narrow band light source or by a broadband light source.

33. The method of paragraph 32, wherein each wavelength of light is produced by a separate laser, each having a different emission peak wavelength.

34. The method of paragraph 32, wherein each wavelength of light is produced by a separate light emitting diode (LED), each having a different emission peak wavelength. 35. The method of paragraph 32, wherein each wavelength of light is produced by a white light source.

36. The method of paragraph 32, wherein each wavelength of light is produced by an RGB (red green blue) LED light source.

37. The method of paragraph 32, wherein each wavelength of light is produced by a standard bright-field microscope optical setup, and wherein the reflected light is transmitted to an eyepiece.

38. The method of any of paragraphs 29-37 ', wherein the imaging device is a monochromatic CCD camera or a color camera.

39. The method of paragraph 38, wherein the color camera is a 3CCD camera.

40. The method of any of paragraphs 29-39, wherein the imaging device comprises a camera having a high magnification objective lens with a high numerical aperture.

41. The method of any of paragraphs 38-40, wherein the camera further comprises a spatial filter on the camera's optical axis.

42. The method of any of paragraphs 29-41, wherein detecting the particle comprises detecting the binding of the particle on the surface of the layered substrate.

43. The method of paragraph 42, wherein the surface of the layered surfaces comprises a binding agent for binding a predefined particle and the solution comprises at least one predefined particle.

44. The method of any of paragraphs 29-43, wherein the particle comprises one or more proteins, one or more salts, one or more polymers, one or more metals, or one or more microorganisms.

45. The method of paragraph 44, wherein the particle is a protein aggregate, a nanoparticle, a bead, or a virion particle.

[0140] It is understood that the foregoing detailed description and the following examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those, skilled in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.

EXAMPLES

[0141] In one or more embodiments, the invention is directed to an apparatus that can detect binding of biomolecules to capture agents on a surface. The capture agents can be immobilized on a layered surface that has a spectral reflectance signature that is altered upon immobilization of said biomolecules on the surface. The apparatus allows for the

simultaneous imaging of the entire field of view of a surface for high-throughput application, and thus eliminates the need for spectrometers. The invention has several advantages over the current methods that utilize detection systems incorporating a tunable laser illumination and imaging of the reflectance to a CCD. Such systems are not ideal when low-cost and portable detection systems are required.

[0142] Figure 1 shows a diagrammatic view of a spectral reflectance imaging system

100 according to an embodiment of the present invention. The system 100 can include an illumination source 101, directing light onto the substrate 122, the oxide layer 124 and the particles 126 being detected, and an imaging system 130 for capturing images of the light reflected by the substrate 122, the oxide layer 124 and the particles 126. The system 100 can also include a computer system 140 for controlling the illumination source 101 and receiving imaging signals from the imaging system 130. The illumination source 101 provides incoherent light in at least two different center frequencies and having a substantially narrow frequency band. In some embodiments, the illumination source 101 can produce incoherent light in at least three different center frequency and as many as seven different center frequencies. The illumination source 101 can include a plurality of illumination elements, such as Light Emitting Diodes (LEDs) or equivalent light sources, each providing incoherent light in one of the plurality of center frequencies. In some embodiments, the illumination source 101 can include an array of illumination elements, including one or more illumination elements providing light in one of the plurality of center frequencies and being arranged in a geometric (e.g., circular or rectangular), random, or spatially displaced array with other illumination elements providing incoherent light in other center frequencies. The light from the illumination source 101 can be directed through a focusing lens 112 and other optical elements (e.g., polarizing lens, filters and light conditioning components, not shown) to a beam splitter 114 that directs the light onto the substrate 122, the oxide layer 124 and the particles 126. Optical components can be provided to condition the light to uniformly illuminate substantially the entire surface of the layered substrate 122. The light reflected by the substrate 122, the oxide layer 124 and the particles 126 can be directed through the beam splitter 114 and imaging lens 134 into a camera 132 to capture images of the substrate surface. The camera 132 can be, for example, a CCD camera (color or monochromatic) and produce image signals representative of the image. The image signals can be transferred over a cable from the camera 132 to the computer system 140.

[0143] Computer system 140 can include one or more central processing units

(CPUs) and associated memory (including volatile and non-volatile memory, such as, RAM, ROM, flash, optical and magnetic memory) and a display 146 for presenting information to a user. The memory can store one or more computer programs that can be executed by the CPUs to store and process the image data and produce images of the substrate surface.

Additional computer programs can be provided for analyzing the image data and the images to detect interference patterns and the particles 126 on the surface of the oxide layer 124 of the substrate 122.

[0144] The computer programs can be executed by the computer to implement a method according to one or more embodiments of the present invention whereby

interferometric measurements can be made. The computer programs can control the one or more LEDs that can be used to illuminate layered substrate. The computer programs can control the LEDs in order to illuminate them sequentially (by frequency) or all at the same time. The optical path difference (OPD) between the bottom and top surface causes an interference pattern. The interference patterns can be imaged as intensity variations by the CCD camera 132 across the whole substrate at once.

[0145] In an alternative embodiment, each of the illumination elements 102, 104, 106 can be coupled to an optical fiber (not shown) that directs the light at the layered substrate 122. Optical components can be provided to condition the light to uniformly illuminate substantially the entire surface of the layered substrate 122

[0146] Figure 2 depicts how the interference signature of reflected light changes upon adsorption of a layer of biomass or any other semi-transparent material to a surface. The reflections off different layers cause a wavelength-dependant spectral interference pattern, such that addition or adsorption of a biomass or any other semi-transparent material to the surface changes the optical path length, causing a phase- shifted interference pattern, as illustrated in the plot to the right. This change can be characterized by sampling the spectral reflectivity of the substrate at different wavelengths and fitting curves to the acquired intensity data points (as illustrated by black dots). The figure also depicts how the phase shift is proportional to the "thickness" of the substrate layer.

[0147] Figure 3 demonstrates the properties desired in a substrate for enhanced microarray image composition. The substrate is composed of a semi-transparent top layer (1) on a reflective bottom layer (2). The thickness of the top layer should be chosen so the LED sources sample at least half period covering a peak or trough of the reflectivity curve. For example, if a silicon bottom substrate and a silicon dioxide for the top substrate are chosen and a red green blue (RGB) LED is used to sample the curves at the following peak emission wavelength: 455 nm, 598 nm and 635nm, then an appropriate silicon dioxide thickness would be 500 nm.

[0148] Figure 4 demonstrates the properties desired for performing high

magnification substrate enhanced microarray imaging. For making high magnification imaging, objectives with higher numerical apertures (NA) should be used. Because the light is collected at a high range of angles, most of the light averages out (as illustrated in the figure to the right). Also the use of thin oxide increases the limit for spatial resolution because of less dispersion in light as it passes through it.

[0149] Figure 5 depicts using a spatial filter as an option for performing high magnification substrate enhanced microarray imaging. To maintain the lateral resolution for single particle detection and the contrast of the reflectivity curve, it may be desirable to place a spatial filter on the collection path that will reject a range of angles of the reflected light. Simple observation of interference can be seen on the colors on soap bubbles. One of the ultimate examples of high precision measurements using optical interference is the LIGO with attometer capability.

[0150] Figures 6A and 6B compare optical interferometry of the biosensors of the invention using lasers versus LEDs. The optical thickness of the SiO₂ substrate layer affects the curve periodicity of the incident light. As lasers have a narrow tuning range (20 nm), to use a laser a thick oxide layer is required (Figure 6A, left). However, LEDs have a wider tuning range, and thus a much thinner oxide layer is required (Figure 6 A, right). Figure 6B shows that using multiple LEDs as the light sources to illuminate a thinner oxide layer permits the detection of a higher resolution optical interference pattern.

[0151] Figure 7 shows an exemplary optical setup of a biosensor of the invention.

[0152] Figure 8 depicts single -particle detection using a biosensor of the invention.

Fluorescent lOOnm carboxyl modified beads were immobilized on a lysine surface, and incubated for 15 minutes. The left panel depicts the image resulting from the use of the optical interferometric biosensors described herein. The right panel is an image based on detection of the fluorescence of the beads using conventional fluorescent scanning methods.

[0153] Figure 9 depicts a 12-tile array format for use in biothreat VHF applications, wherein antibodies specific against different viruses, such as Ebola and Marburg, are used to coat specific regions of the substrate layer of a biosensor, such that contact with a sample containing the viruses the antibodies are specific for results in detection of those viruses by the biosensor. In other embodiments, such tiled/regional arrays can be used to differentiate between different influenza strains. For example, antibodies specific for different hemagglutinins can be used as the immobilized molecules on the biosensor to distinguish between the common flu, HlNl, and avian flu in a sample. Such arrays allow for shape and size discrimination of single particles present on the biosensor substrate surface.

[0154] Figures 10A- 1OC show an enlargement of a single tile of a 12-tile array format. Figure 1OA shows the image of the 12-tile array. SRB height profile image of a sensor chip with an array of 50μm tiles. lOOnm diameter (R=50nm) polystyrene beads are captured on the surface, and individually detected. Figure 1OB is an enlarged image of a single tile and (Figure 10B) demonstrates that single particles are detected at each diffraction limited spot independently using the biosensor of the invention. Automated bead (or virus) detection over one sensing tile, identified by circles. Figure 1OC shows the average width of the particles in the tile. Histogram of particle size distribution demonstrating the ability to distinguish particles with a variance of < 7nm in diameter (σ <3.5nm for radius). Such arrays can have, in some embodiments, 10⁶ sensors with < 0.1 fg sensitivity with W/cm2 power density.

[0155] Figure 11 demonstrates the ability of a biosensor of the invention to discriminate between particles of different sizes present in a sample. The upper panel shows an image of particles of mixed sizes on an array, and the lower graph depicts the average and standard deviation of the radii (nm) of the particles in the image. In the example shown, particles of 50 nm, 75 nm, and 100 nm radii are clearly distinguished by the biosensor.

[0156] Figure 12 depicts how an image of a particle is taken, such that in some embodiments, the sensor is designed such that the distance between the power monitor and the SiO₂ substrate is 2 times the wavelength of the incident plane wave, and that the depth of the SiO₂ substrate is the same as that of the incident wavelength.

[0157] Figure 13 depicts how, in some embodiments, the biosensor is used to distinguish between particles of different shapes. In the example shown, the biosensor distinguishes between an ellipsoid and spherical particle. Such biosensors are useful for a variety of applications, including, but not limited to, discriminating between viral particles having different shapes, but similar sizes.

[0158] Spectral Reflectance Biosensing (SRB) uses optical interferometry for label- free, high throughput, high sensitivity and dynamic detection of molecular binding on a solid surface. SRB has demonstrated protein-protein binding and DNA-protein binding in real time, label-free, and in a high-throughput format with exquisite sensitivity and reproducibility [I]. SRB is capable of quantifying a biomaterial binding sensitivity of -10 pg/mm [2] and it is superior to state-of-the-art label-free techniques such as SPR since it is immune to temperature and analyte concentration fluctuations (bulk effect). We have significantly advanced SRB beyond the original tunable-laser configuration and implemented a multi-LED discrete wavelength system that allows for high spatial resolution imaging with the demonstrated ability to detect single nanoscale particles and characterize their diameter from the phase signature (3,4).

[0159] In our current system with ~700nm spatial resolution, we have demonstrated sensitivity of 0.05fg with a potential of 10-fold further improvement. Data in Figure 10 shows lOOnm diameter (R=50nm) polystyrene beads readily individually imaged in a platform easily multiplexed by functionalizing each sensing region against a different target while maintaining 3 orders of magnitude higher sensing area than other techniques (5,6,7). We have successfully detected 70 nm and lOOnm particles (equivalent to HlNl virus) with an SNR better than 20. A critical advantage of our technique is that the sensitivity and response is independent of the position on the sensor surface, and thus a single particle on the entire sensor surface of typically 0.25mm size can be detected. Furthermore, the measurements yield a quantifiable signature proportional to the size of the particle allowing for size discrimination. Figure 1OC shows the histogram of automatically measured bead diameter of 200 particles with a variance of σ=6.8 nm, close to the 5nm specified by the manufacturer, implying that our actual σ may be much less. These data demonstrate detection of single virus particles with unprecedented accuracy in a high-throughput, rapid, deployable, and manufacturable platform.

[0160] In additional embodiments, we can improve the speed, throughput, and phase sensitivity and implement an additional capability: automatic shape recognition. Imaging biodetection with SRB has a great advantage in that we can employ both polarization and pupil function engineering to characterize the shape, size and orientation of particles with resolution beyond the classical diffraction limit. In the Rayleigh and Rayleigh-Gans regimes (8), spherical particles have polarizability as a function of their size regardless of the polarization according to Mie theory. On the other hand, in addition to their size dependency, aspherical particles have polarizability that is a function of their shape, aspect ratio and polarization state of the incident light (9). We rigorously studied (10) the effect of the polarization of incident light and pupil function engineering on improving resolution in integrated circuit imaging. Therefore using the established scattering theory of particles and advance techniques for polarization control we can advance the current virus detection schemes further to detect viruses with different size and shape. Shape recognition is a truly unique advantage of the SRB technique and has significant impact in virus detection especially for VHF pathogens.

[0161] Our work on deactivated HlNl viruses and our experiments demonstrate that we are able to successfully detect single HlNl viruses on our SRB platform. Experiments were performed on sample surface with 1% Poly- L- Lysine chemistry providing non-specific binding. A 200μl solution containing of HlNl particles was placed on the chip for 2 minutes and washed in DI. The samples were similar to those described above. This experiment was to test proof-of-principle of optical detection. Surfaces prepared with HlNl specific antibodies will be carried out in the next several weeks. Figure 14 shows the SRB image of half of the sample showing individual viruses as bright dots. Figure 14 shows a sensor chip with an array of 50μm regions or tiles that were incubated with HlNl viruses. Whole chip with 12 square patterns was imaged simultaneously at single particle sensitivity. We expand on the results from the lower left square.

[0162] To confirm the detected particles are individual viruses, we performed SEM characterization of the sample. Due to the limited field of view of the SEM and difficulty of SEM on an insulating sample, we studied only one of the square regions (lower left on Figure 15). As shown in Figure 15, there is a one-to-one correspondence between the SEM image and SRB image. SEM images verified that HlNl particles were dispersed on the surface as individual particles varying in diameter from 90nm to 160nm, with majority of them at approximately 120nm (radius ~60nm). As predicted from our earlier experiments, this size is well within our sensing capability and the results clearly show that we are able to detect individual HlNl viruses and count them. Our results demonstrate that, we can detect a single HlNl virus captured on the entire detector surface, thus yields 3 orders of magnitude higher sensing area than other techniques. In Figure 15, SEM image (left) is compared to the optical image obtained on SRB (right). The virus particles appear as dark dots on the SEM image due to charging. The inset shows a higher resolution image verifying that we are observing single HlNl particles. Virus sizes vary from 90nm to 160nm in diameter with the majority at around 120nm diameter. All viruses that appear in the SEM Images are present in the SRB intensity Image.

[0163] In Figure 16, to further illustrate the SRB detection and comparison to SEM results, we focus on part of the image from Figure 15 and draw arrows of equal length to correlate the virus particles imaged by both modalities. Our results show qualitative size discrimination and the quantitative size discrimination ability of our system.

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Claims

1. A spectral reflectance imaging system comprising:

a substrate having a first reflective surface and a thin transparent layer providing a second reflective surface;

a biolayer bound to the second reflective surface;

an illumination source comprising at least three light sources, each providing light in one of three different narrow frequency bands and directing each frequency band of light at the substrate; and

an imaging device directed at the second reflective surface of the substrate and adapted to produce imaging signals representative of light from the illumination source being reflected by the first reflective surface and the second reflective surface.

2. A spectral reflectance imaging system according to claim 1, wherein the first reflective surface is a silicon substrate and the transparent layer is silicon oxide (SiO₂).

3. A spectral reflectance imaging system according to claim 1, further comprising an image acquisition and processing system, coupled to the imaging device and adapted to receive the imaging signals and under program control, produce an image of the biolayer on the second reflective surface.

4. A spectral reflectance imaging system according to any of claims 1-3, wherein the thickness of the biolayer is determined as function of the intensity of the reflected light received by the imaging device.

5. A spectral reflectance imaging system according to claim 1, wherein the transparent layer is less than 1 micron thick.

6. A spectral reflectance imaging system according to claim 1, wherein the transparent layer has a thickness in the range from 100 nanometers to 1000 nanometers.

7. A spectral reflectance imaging system according to claim 1, wherein the illumination source includes four light sources.

8. A spectral reflectance imaging system according to claim 7, wherein the illumination source produces light at 460 nanometer, 520 nanometer, 600 nanometer and 630 nanometer wavelengths.

9. A spectral reflectance imaging system according to claim 1, wherein each light source produces light in a band that is 20 - 30 nanometers wide.

10. A spectral reflectance imaging system according to claim 1, wherein each light source produces light in a band that is 10 - 20 nanometers wide.

11. A spectral reflectance imaging system according to claim 1, wherein each light source produces light in a band that is 5 nanometers wide.

12. A spectral reflectance imaging system according to claim 1, wherein the illumination source produces white light and the system further includes a color wheel having at least three filters, each producing a beam of light in one of at least three narrow frequency bands that is directed at the substrate.

13. A spectral reflectance imaging system according to claim 1, further comprising at least one optical fiber and wherein the light from each light source is directed through the at least one optical fiber toward the substrate.

14. A spectral reflectance imaging system according to claim 13, further comprising three optical fibers and wherein each band of light from each light source is directed through a separate optical fiber toward the substrate.

15. A method for detecting the binding of a particle to a surface of a substrate, the method comprising:

providing a first specular reflecting interface of the substrate with a binding agent for binding a predefined particle to the first specular reflecting interface of the substrate;

providing a second specular reflecting interface that is substantially parallel to and underlies the first specular reflecting interface;

illuminating the surface with light substantially centered around one or more wavelengths; imaging light reflected or transmitted from the substrate using an imaging device; producing a spectral reflectance image of the surface of the substrate;

and correlating the features on the image to discrete particles on the surface.

16. The method of claim 15, wherein the imaging device comprises a camera having a high magnification objective lens with a high numerical aperture.

17. The method of claim 15, wherein each wavelength of light is produced by a separate, narrow band light source.

18. The method of any one of claims 15-17, wherein the imaging device is a

monochromatic CCD camera.

19. The method of any one of claims 15-18, wherein the surface is illuminated by a light source from a standard bright-field microscope optical setup, and wherein the reflected light is transmitted to an eyepiece.

20. The method of any one of claims 15-19, wherein each wavelength of light is produced by a separate light emitting diode (LED), each having a different emission peak wavelengths, and wherein the imaging device is a monochromatic camera.

21. The method of claim 20, wherein the imaging device is a monochromatic CCD camera.

22. The method of any one of claims 15-21, wherein the layered substrate comprises 500 nm of SiO₂ layered on a Si wafer.

23. The method of any one of claims 15-22, wherein the surface is illuminated with white light and the imaging device includes a color camera.

24. The method of claim 23, wherein the color camera is a 3CCD camera.

25. The method of any one of claims 15-24, wherein the surface is illuminated by an RGB (red green blue) LED and the imaging device includes a color camera.

26. The method of claim 25, wherein the color camera is a 3CCD camera.

27. The method of any one of claims 15-26, wherein the surface is illuminated by a broadband light source.

28. The method of any one of claims 15-27, wherein the camera further comprises a spatial filter on the camera's optical axis.

29. A method for detecting a particle on a surface of a layered substrate comprising: providing the surface of the layered substrate;

contacting a solution having at least one particle with the surface of the substrate; illuminating the surface with at least three wavelengths of light;

imaging the light reflected or transmitted from the substrate using an imaging device; and

producing a spectral reflectance image of the surface of the substrate to detect the particle on the surface of the layered substrate.

30. The method of claim 29, wherein the layered substrate comprises SiO₂ layered on a Si substrate.

31. The method of any of claims 29-30, wherein the light is coherent or incoherent.

32. The method of claim 29, wherein each wavelength of light is produced by a separate, narrow band light source or by a broadband light source.

33. The method of claim 32, wherein each wavelength of light is produced by a separate laser, each having a different emission peak wavelength.

34. The method of claim 32, wherein each wavelength of light is produced by a separate light emitting diode (LED), each having a different emission peak wavelength.

35. The method of claim 32, wherein each wavelength of light is produced by a white light source.

36. The method of claim 32, wherein each wavelength of light is produced by an RGB (red green blue) LED light source.

37. The method of claim 32, wherein each wavelength of light is produced by a standard bright-field microscope optical setup, and wherein the reflected light is transmitted to an eyepiece.

38. The method of any of claims 29-37, wherein the imaging device is a monochromatic CCD camera or a color camera.

39. The method of claim 38, wherein the color camera is a 3CCD camera.

40. The method of any of claims 29-39, wherein the imaging device comprises a camera having a high magnification objective lens with a high numerical aperture.

41. The method of any of claims 38-40, wherein the camera further comprises a spatial filter on the camera's optical axis.

42. The method of any of claims 29-41, wherein detecting the particle comprises detecting the binding of the particle on the surface of the layered substrate.

43. The method of claim 42, wherein the surface of the layered surfaces comprises a binding agent for binding a predefined particle and the solution comprises at least one predefined particle.

44. The method of any of claims 29-43, wherein the particle comprises one or more proteins, one or more salts, one or more polymers, one or more metals, or one or more microorganisms.

45. The method of claim 44, wherein the particle is a protein aggregate, a nanoparticle, a bead, or a virion particle.