CA2331787A1 - Enumeration method of analyte detection - Google Patents

Abstract

This invention is directed to an optically-based method and system for analyte detection using solid phase immobilization, specific analyte labels adapted for signal generation and corresponding processes for the utilization thereof. The enumeration detection method disclosed herein narrows the area for signal observation, thus, improving detectable signal to background ratio. The system is comprised of a platform/support for immobilizing a sample stage having a labeled sample (analyte complex) bound thereto, a radiation source, an optical apparatus for generating and directing radiation at said sample and a means for data collection and analysis. Upon engagement of the system, the sample generates a signal capable of differentiation from background signal, both of which are collected and imaged with a signal detector that generated a sample image to a data processing apparatus. Said apparatus receives signal measurements and, in turn, enumerates individual binding events. Generated signal may be increased via selected mass enhancement. The invention, enumeration assay methodology detecting individual binding events, may be used, for example, in analyses to detect analyte or confirm results in both research, commercial and point of care applications.

Description

WO 99/58948 PCTIL~S99/14917 ENUMERATION METHOD OF ANAILYTE DETECTION
FIELD OF THE INVENTION
This invention relates to the general fields of molecular biology, biochemistry, microbiology and biological researclh, specifically, to detection of analytes, and more specifically, to an enumeration assay method and system for the detection of individual binding events. The present invention enables the detection of low concentrations of specific molecules of interest (analytes) using solid phase immobilization and optical signals capable of generating, detecting and measuring mass changes.
BACKGROUND AND PRIOR ART
Improving the lower limit of detection -- t:he threshold of detection of chemical sensitivity -- has been a primary objective of ligand binding assay development since its inception. It has long been recognized that optical detection methods defined by the relationship between various optical interactions with mass on a solid phase, in particular ellipsometry, are capable in principle of providing a high level of sensitivity for standard binding reactions when compared to alternative signal generation methods, for example, enzymelsubstrate interaction, fluorescent emission, radioactive emission and color emission. It has also been recognized that mass could be added to the binding complex in order to amplify the optical siignal generated. It has been demonstrated that large amounts of mass can be successfully conjugated to the binding complex to this end. An example of this method is provided by the optical ellipsometric immunoassay (OpTestTM, I)L)x, Inc.), a detection system for molecular and microscopic scale events, that measures interactions between biological samples and light.
The prior art discloses several imaging methods for the detection of analytes. U.S. Patent no. 5,599,668 to Stimpson et ai., entitled Light Scattering Optical Waveguide Method for Detecting Specific Binding Events, discloses a DNA-hybridization imager that detects the scattering of light directed into a waveguide, using labeled microspheres (beads) and visually monitors binding by video imaging. The waveguide device is required as a solid phase and imaging is achieved with a CCD camera and frame grabber software.
Allen et al., U.S. Patent no. 5,488,567, entitled Digital Analyte Detection System is directed to the digital detectia~n of the presence of analyte particles based upon illumination thereof. Distinct pixel regions of the sample are illuminated and the emitted signal detected.
A novel optical biosensor system is taught in A Biosensor Concept Based on Imaging Eldipsometry for Visualization c f Biomolecular Interactions (Jin et al. (1995) Anal. Biochem. 232:69. The biosensor system utilizes specificities of biomolecular interactions in combination with protein patterned surfaces and imaging ellipsometry and a CCD camera to collect data.
The general use of imaging ellipsometry ir!i conjunction with a CCD
camera and framegrabber board is disclosed in Performance of a Microscopic Imaging Ellipsometer {Beaglehole (1988) Rev. Scii. lnstrum. 59(12):2557. No type of life science or biological system application of the imaging is suggested.
A Method for Detecting the Presence of Antibodies using Gold-Labeled Antibodies and Test Kit are taught in U.S. Patent no. 5,079,172 to Hari et al.
This methodology is directed to detecting labeled microparticles using microscopy, for example, an electron microscope imaging system.
Chemical and biochemical analysis involving the detection and quantitization of Light occurs in a variety of situations. One application is the detection of zuialytes for the determination of the presence or amount of a particular analyte. In many assays for analytes, the: concern Lies with either absorption or emission of Light radiation (e.g., fluorescence or chemiluminescence). In such cases, a sample is irradiated and the effect of the sample on the transmitted or emitted light is detected. In the case of emitted light resulting from irradiation, non-analyte molecules may also emit light creating relatively high background noise and resulting in the introduction of substantial error in measurement. Additional systE;matic errors may also collectively contribute to the noise associated with measurement.
The quality of chemical measurements involving light can be defined in terms of the ratio of a suitable measurement of the optical signal from a sample due to the presence of analyte to the noise variation inherent within the system.
The source of noise that may affect the results may come from anywhere within the optical path, including the sample, the signal source, detector variation and environmental interference. However, these variations are not necessarily inherent, and may also include externally imposedl or induced variations. In general, efforts to augment this signal to noise (SiN) ratio have centered on improving the sensitivity of a measurement apparatus so as to reduce the "detection limit" associated with a particular anal~rte. The detection limit refers to the analyte concentration within a sample above which the signal attributable to the presence of zmalyte is such that a desired S./N ratio is achieved. In practice, this detection limit is ascertained by conducting an experimental procedure designed to elicit an optical signal related to analyte concentration.
Specifically, data relating to signal and noise intensity is plotted in the form of a calibration .curve for a range of analyte concentrations, thereby enabling straightforward determination of the detection limiit.
The determination of concentration in unknown samples is then effected by comparing the signal obtained experimentally from the unknown with the calibration curve. A typical unit of concentration i.n chemical measurements is moles/liter [i.e., Molarity (M)], where a mole is defined as Avogadro's number (6.0225 X 1023). Unfortunately, even the most sen:>itive conventional experimental techniques have detection limits on the order of about one femtomolar (fM), or nearly one billion analyte particles per liter.
Measurements in which concentration is determined by reference to a calibration curve may be characterized as being inherently "analog" rather than "digital". That is, a signal correlated with analyte concentration is initially produced by the measurement device. The calibration curve is then consulted to obtain an approximation of the analyte concentratiion. Since the calibration curve is continuous as a function of concentration, the concentration derived from the calibration curve generally is not an integer. In contrast, digital measurement data are often embodied in binary (i.e., two-level) signals that unequivocally represent specific integers. Accordingly, a fundamental difference between analog and digital modes of measurement is that the addition of a single additional analyte to a sample analyzed using analog means cannot be unambiguously detected. Although dramatic improvements have been made in the accuracy of chemical measurements, such advancements have been based on the fundamentally analog concepts of increasing signal and reducing noise.
In molecular samples involving low levels of au~alyte concentration a 5 digital measurement methodology affords at least two advantages: na calibration curve reference and detection of single molecules :in a sample. Enumeration methodologies are useful in samples where the an~~lyte concentration is sufficiently low that statistical noise accompanyin~; each binary measurement value remains less than the difference between successive integers.
Accordingly, it is an object of the present invention to provide an optical technique for determining low levels of analyte concentration by means of an intrinsically digital measurement scheme adapted for individual binding event detection.
To date, development in the prior art has been directed to imaging of an area of binding, as opposed to distinct video pixels {an array of digitized picture elements) or individual binding sites. The various. problems of the prior art are overcome by the present invention. Shortcomings of the prior art includes for example, limitation to emission based reaction detection, averaging and/or detecting reactions over an area or plurality of pixels and the necessity of both signal producing and non-producing areas and distribution determination. The present invention overcomes these drawbacks by providing an integrated system and methodology for analyte detection through enumeration of individual binding events. While prior art is suitable for qualitative and limited quantitative determination, none of the prior art can be easily and efficiently used in the accurate enumeration of individual analyte binding events, nor does it teach the enhanced performance characteristics disclosed herein. The present invention provides improved enumeration sensitivity and accuracy, thereby obviating the herein-described prior art.
A prior art search failed to reveal any references disclosing the present invention or making it obvious to one of ordinary skill in the art.
Furthermore, combinations of the disclosures of the referenced prior art would not teach the present invention nor would such a combination make the invention obvious.
No reference teaches, or suggests, the novel characteristics or combinations employed in the instant detection of solid-phase tround analyte on a molecule-by-molecule basis. The methods disclosed herein. are useful, for example, for the solid phase detection of biological markers where the frequency, density or distribution of binding events is below the detectable threshold of conventional immunoassay, DNA probe and immuno-chromatographic detection methodologies.
SUMMARY OF THE INVENTION
The instant invention is based on novel methods of analyte detection as a means for detection of specific molecules using; solid phase immobilization and optical signal generation. More specifically, this invention comprises the use of optical signals and detectors capable of detecting and measuring mass changes resulting in analyte detection. This method further relates to commercial applications for automating detection and interfacing with existing assay methodologies, therefore lending itself to commercial applications, for example, high throughput pharmaceutical screening and point-of care detection.
That is, this invention is directed to the solid pha;>e, optical detection and enumeration of individual binding events mediated by specific binding interactions.
This invention is defined by analyte solid phase immobilization, a signal generator, a signal carrier including optical pathways, a means of signal detection and novel data analysis. It encompasses a method for improving the detectability of individual binding events by utili~~ing a narrow optical beam size or by parsing or dividing a larger beam into smaller virtual beams using a diode array or a charged-coupled device (CCD) detector. The use of various optical signals and physical amplification elemenia is discussed herein.
In its broadest embodiment, the invention is directed to a method and system for solid phase, optical detection and enumeration of individual target analyte binding events comprising the steps of: irnmobilizing an analyte 1 S complex on a reflective or transmissive substrate directly from solution, said complex comprising a target analyte complexed vvith at least one signal generator element conjugated to at least one secondary analyte specific binding element; reflecting or transmitting electromagnetic radiation from or through the substrate having the analyte complex immobilized thereon; capturing a signal generated from said reflecting or transmitting of electromagnetic radiation;
and, analyzing the signal far the presence and/or amount of analyte present.
More specifically, a system and method for digitally detecting the presence of analyte particles within a sample is disclosed herein. Each analyte complex is disposed to generate an optically detectable response upon stimulation (e.g., illumination) in a known manner. Furthermore, signal generators may be passive or active. Passive signal generators include those that interact with, but do not process, illumination,, e.g., absorption, scattering.
Active signal generators are those that actively transform photonic energy S through a change in state, i.e., fluorescence, chemiiluminescence and plasmon resonance. For stimulation or illumination, the dil;ital analyte detection system includes optical apparatus for illuminating a multiplicity of distinct pixel regions within the sample so as to induce each of the analyte complexes included therein to generate an optical signal, i.e., photons. As discussed herein, Stimpson et al. and Allen et al. employ the; use of CCDs and pixels for detection purposes. In the instant invention, the pixel regions are dimensioned such that the number of analyte complexes includc;d within each region is sufficiently small that the aggregate optical signal generated by each region is less than a maximum detection threshold, preferably, 1 particle per pixel or multiple pixels per particle.
The digital detection system further includc;s apparatus for measuring the optical signal generated from each pixel region. A data processing network-receives the optical signals, quantifies the signals, and based on the measurements, counts the number of analyte particles within each pixel region so as to determine the number of analyte particles within the sample.
The detection techniques of the present invention can be used for detecting a wide variety of analytes. As used herein, the term "optical response" is intended to collectively refer to the signal generation from a single analyte complex, however induced. In addition, the term "generated signal" as used herein carresponds to a measurement of the optical responses detected from a particular pixel or pixel region. The assay sample medium is preferably a solid phase bound analyze complex in which detectable label not bound to an analyte may be removed through conventional washing procedures.
In a preferred embodiment the analyte particles within each pixel region are measured individually based on discrete signal units providing optical responses substantially above a background noise level. The magnitude of each optical response is required to be large enough to~ allow the particular photodetection apparatus employed to discriminate between optical responses and ambient background noise. One or more optical responses of a signal unit may be associated with a single analyte particle, 'but the number of units will be substantially identical for each analyte particle. For the mast part, the number of signal units per analyze complex will be more than one.
The assay sample medium often has low <;oncentrations of analyte, generally at picomolar or less, frequently femtom.oiar or less. Assay volumes are usually less than about 100 ~l, frequently less than 10 ~1 and may be 1 ~1 or less. It is desirable to match the CCD pixels to the signal generator label, ranging in size from 50 nm to 5 microns, such that the labels can be individually detected. The actual size of the CCI) pixels is irrelevant in that this is accomplished through magnifying optics.
Assays normally involve specific binding :pairs, where by specific binding pairs it is intended that a molecule has a complementary molecule, where the binding of the elements of the specific binding pair is at a substantially higher aff pity than random complex: formation. The elements of a WO 99!58948 PCT/US99/10917 specific binding pair can be referred to as "ligands" and "receptors."
Generally receptors are immobilized to the solid phase to capture, or immobilize, the analyte of interest (the "ligand") from a fluid sample. Thus, specific binding pairs may involve haptens and antigens (referred to as "ligands") and their 5 complementary binding elements, such as antibodies, enzymes, surface membrane protein receptors, lectins, etc. (generall~~ known as "receptors") Specific binding pairs may also include complementary nucleic acid sequences, both naturally occurring and synthetic, either RNA, or DNA, where for convenience nucleic acids will be included within the concept of specific 10 binding elements comprising ligands and receptors.
In carrying out the assay, a conjugate of a specific binding element and a detectable and discrete label is involved. Methods of preparing these conjugates are well known, and are, therefore, not discussed herein. Depending upon the analyte, various protocols may be employed, which may be associated with commercially available reagents or such reagents which may be modified.
Other features and advantages of the instant invention will become apparent from the following detailed description, taken in conjunction with the accompanying figures, that illustrate by way of e~:ample, the principles of the instant invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the determination of rn~ass per unit volume or equivalent thereof in standard immunoassay methodology;

WO 99158948 PC'1'/I3S99/10917 Figure 2 depicts optical averaging occurring over an assay area;
Figure 3 depicts the highly non-homogeneous assay area integration;
Figure 4 illustrates the statical reduction to insignificance when low numbers of binding events are averaged over a laarge assay area;
Figure 5 shows small bean ellipsometry or scatterometry provide higher relative signal for discreet binding events;
Figure 6 illustrates the methodological approach for surface resolution, thereby approximating discreet binding event identification;
Figure 7 illustrates laser determination of ;~.ggregate response;
Figure 8 depicts scanning micro-laser configuration for the determination of individual cellular scale readings;
Figure 9 illustrates relative size in relation to detection;
Figure 10 depicts CCD andlor diode array beam employed to parse the laser beam into discreet signals;

Figure 11 illustrates the variability of optical signals useful for detection and resolution purposes;
Figure 12 shows examples of optical signal formats: past, current and prophetic;
Figure 13 illustrates the scale of potential :.canning micro-Iaser configurations; and Figure 14 depicts optical enhancement potential.
Figure 15 depicts the preferred instrumentation embodiment of the instant invention.
DETAILED DESCRIPTION OF THE PREFEF~RED EMBODIMENT
It is understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. The general principles and conditions for analyte detection, manipulations (hybridization and amplification), and optics (lasers and ellipsometry) are well known in the art. The instant invention describes a novel method of detection for individual binding events.
One skihed in the art recognizes that the instant invention, as disclosed herein, may be performed in a broad range of samples. Such samples include, for example, biological samples derived from agriculture sources, bacterial and viral sources, and from human or other animal so~xrces, as well as other samples such as waste or drinking water, agricultural products, processed foodstuff and air. The present invention is useful for the detection of low numbers of immobilized specific molecules.
The present invention is embodied in a method employing optical signals and detectors capable of detecting and me<~suring mass changes in a sample assay area. Regardless of the specific application of the instant invention, the methodology details are calculated .according to protocols well known in the art, as well as those disclosed herein. Further, the refinement of said necessary calculations is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them without undue experimentation.
This application references and specifically discusses the use of ellipsametry as the optical method; this convention is for convenience only.
It is understood that this methodology applies to a range of optical signal types, including those referenced in Figure 12. It is specifically envisioned that the performance of a variety of optical methods will be substantially improved by adopting the general approach described herein. I:n particular, scattering methods form the basis of one class of instruments that is distinct from ellipsometry. Other effects such as absorption, refractive index change, and diffraction are used within an essentially similar optical configuration, and may provide particular result benefits. In application, the defining of the optical signal format drives the choice of appropriate imnnobilization surfaces and suitable data analysis methods for the purpose of .distinguishing individual binding events. Thus, the attributes of the immobilization system and data analysis system are contingent upon the attributes of the selected optical signal format. The purpose of the optical signal format (the conjunction of a signal carrier, signal generator and signal detector) is to cause and detect a signal.
The ability to distinguish the signal caused by the signal generator label from the signal caused by the background platform upon which the system is run, the solid phase, is fundamental to the optical signal format.
Definitions helpful in understanding the specification and claims are included throughout the instant disclosure. The definitions provided herein should be borne in mind when these terms are used in the following examples and throughout the instant application. The disclosures made herein are limited, for simplicity and convenience, to assays directed to the addition of mass (e.g.
ligand binding assays), and reference is made to immunoassay methods.
However, the same principles of optical signal defection generally apply to systems where mass is removed from the system I;e.g. lytic or dissociation assays), and this invention is, thus, applicable to assays measuring mass change and derivatives thereof. Furthermore, this invention is directed to both transmission- and reflection-based solid phase assays.
Those skilled in the art readily recognize the present invention is broadly applicable in the areas of art described herein. The following examples and detailed descriptions serve to explain and illustrate the present invention.
laid examples are not to be construed as limiting of the invention in anyway.
Various modifications are possible within the scope of the invention.
The advent of small bead conjugation, beads ranging in diameter from 25 nm to 20 microns, opened the way to a new form of signal detection. That signal detection is described in the present applicavtion, and hereinafter referred to as the enumeration method. The instant invention enables the detection of individual binding events. The principle being to narrow the size, actual or 5 virtual, of the area observed for signal, thereby improving the ratio of true signal to background signal, while concurrently using selected mass enhancement elements to increase the signal generated. Only strong signal generators are able to be detected for individual events occurring at the molecular level. On the scale of detectability typical for optical thin film 10 systems, the addition of mass ranging 50 nm to 5 microns generate a high signal, allowing individual binding event detection. Further, certain macromolecules or cellular bodies are large enou~;h that they may be detected without additional mass enhancement, i.e., without secondary labels or reagents.
The present invention, thus, solves the problem oP detection of low 15 concentrations of specific molecules of interest (analytes) using solid phase immobilization and optical signals capable of detecting and measuring mass changes.
In one embodiment, such mass changes are additively achieved or mediated by analyte camplexing or binding via steric, shape mediated or other non-covalent, interactions with a ligand binding pair. Examples of such interactions include antigen-antibody binding, nucleic acid (DNA, RNA, PNA) binding, and other specific macromolecular (prote;in, giycoprotein, or carbohydrate binding) interactions. Alternatively, mass change is subtractively achieved through specific enzymatic, chemical or other specific dissociating or w0 99/5894$ PCT/US99/10917 lytic agents. Examples of assay systems utilizing specific binding or lytic interactions suitable for mass change analysis inchude, for example, immunoassay, hybridization assay, protein binding; assay and enzyme activity assay.
Alternate embodiments of this invention include secondary reagents used to amplify or differentiate the optical signal associated with the binding or lytic event through specific enhancement or alteration of that signal. Such enhancement involves the addition of simple mass to a complexing event, or the generation of a differentiable type of signal from a specific species or process.
Alternatively, such enhancement involves the alteration of one or more of the elements of the binding or lytic event generating ;~ differentiable optical signal, or the enhancement initiates a detectable self assembly or aggregation process.
In solid phase assay of the type described herein, results are typically derived from a statistical distinction between the assay signal and the background noise. This type of assay is typically performed utilizing macro-scale volumes (> 1 p,l) of a liquid sample or suspension. Similarly, the immobilization area typically used for this type oiP assay is also at the macro-scale (> 1000 microns). These assays detect and/or quantify the target analyte through detection and measurement of signal generated by large numbers of binding or lytic events. The signals generated by tens of thousands to hundreds of millions of discrete binding or lytic events are aggregated, typically through the interaction of all of the events with a single optical signal path providing a single result. One reason for this traditional approach is that the binding or iytic events to be detected occur on a molecular scale, and thus large numbers of events are required to create a detectable signal., Additionally, this large number of events creates a statistically meaningful basis for the result.
A clear limitation of this traditional approach is evidenced in the case of very low concentrations of analyte. The signal generated by sparse binding events must be great enough to be distinguished against the background noise.
Alternatively, the signal generated must be differentiable against the field of negative signal caused by averaging the change in signal over the entire surface area of the reaction zone. In solid phase assays, tlae signal strength of this field is, thus, a function of the volume of sample or the area of the reactive surface.
In these cases, the signal generated by sparse binding or Iytic events incorporates the signal generated by the much larger unaffected region of the test area. In the case of very low concentration artalytes this has the effect of creating a very small difference between a positive; and a negative signal, in turn, limiting the lower level of detection that is achievable.
The instant invention is a solid phase detection method and system for biological markers where the frequency, density or distribution of the binding events is far below that which is detectable by traditional immunoassay, I~NA
probe, immuno-chromatographic or other ligand Minding methods.
Immobilization Solid phase methods are well known in the art of assay development as a means of separating, or capturing, an analyte of interest {"ligand" or "analyte") from a mufti-component fluid sample. Solid phase assays require a capture material ("receptor"} that is immobilized onto the solid phase that binds specifically to the analyte of interest, forming a Iil;and-receptor complex.
The ligand and receptor bind specifically to each other, generally through non-covalent means such as ionic and hydrophobic interactions, Vanderwaal's forces and hydrogen bonding. Certain ligand-receptor combinations are well known in the art and can include, for example, immunological interactions between an antibody or antibody Fab fragment and its antigen, hapten, or epitope; biochemical binding of proteins or small molecules to their corresponding receptors; complementary base pairing between strands of nucleic acids.
Solid phase immobilization of receptor material is well known in the are. General classes of immobilization include, for example, but are not limited to adsorption, covalent attachment, and linker-mediated. Adsorptive binding is generally non-specific and relies on the non-covalent interactions between the solid phase and the captuxe material. Covalent binding refers to linking of the capture material to the solid phase via the formation of a chemical bond.
Linker mediated immobilization involves the specific use of secondary molecules and/or macromolecules attached to the surface and capture material that interact specifically to form a bound structure. Immobilization methods are generally chosen so that the capture material retains its specificity for binding to the analyte of interest.
Once the capture material is immobilized to the solid phase, the solid support is reactive to analyte binding ("reactive surface"). Before the addition of a fluid sample containing the analyte of interest, it may be necessary to treat the reactive surface with additional materials to prevent ("block") the non-specific binding ("NSB") of non-analyte components of the fluid sample to be tested. Typical blacking materials include, far ex~unple, proteins such as casein and bovine serum albumin, detergents, and long-chain polymers.
Typically, the chosen receptor is immobili2;ed to a solid phase. A test solution containing the analyte of interest comes in contact with the immobilized receptor whereby a ligand-receptor complex is formed on the solid phase. Once this complex is formed, all other components of the test solution are removed, usually by rinsing the solid phase. 'lChe analyte bound to the solid phase may be additionally complexed with a mass amplifying agent through a secondary specific receptor binding to form an an<~lyte complex. This complex may be formed either in the fluid sample containing the analyte before the sample contacts the reactive surface, or after the analyte is bound to the reactive surface. After binding of the analyte or analyte complex to the reactive surface is complete, this binding can be measured by any of several means.
Substrates useful for creating the disclosed solid phase binding platform include all reflective and transmissive materials suitable for optical or "near optical" wavelength reading. Suitable substrates include, for example, those substrates that provide sufficiently consistent or precise interactions with light in order to yield consistent and meaningful results. 'To that end, the use of highly absorptive surfaces or attachment layers may create optical contrast in the scattering applications disclosed herein.
Optical Signal Format The Optical Signal Format of the instant invention is comprised of at WO 99/58948 PCT/ilS991I0917 least a signal carrier, a signal generator and a signal detector.
Optical Sisnal Format: Sid Generator The present invention specifically relates to a method for altering the 5 ratio of signal to non-signal surface area, allowing; for more sensitive results.
Also, this invention uses specific labels selected to interact with specific optical beam types to create an enhanced, differentiable or amplified signal.
The traditional goal of a binding assay method is the determination of mass per unit volume (e.g., ng/ml) or equivalent (e.g., IU). See Figure 1. A
10 solid phase is typically used as a separation platform to isolate an analyte from other elements of a sample and from excess reagents. For certain types of assays, the signal generator remains attached to the binding complex, and thus is read from the solid phase (e.g., optical methods as discussed infra, or fluorescence). The mass of analyte found in the volumetric sample is converted i 5 to mass immobilized on the solid phase in a proportional manner.
The signal generator, as used herein, is that component of the invention that interacts with a signal carrier to create a signal. Key to this concept is the known, specific and predictable interaction betwef,n the two. A signal generator element includes material which may be used to specifically label, amplify, 20 distinguish, mark or generate a detectable signal associated with the immobilized target analyte, thus differentiating binding from the absence thereof.
Limitations on selection of a signal generator are driven by the selection of signal carrier, secondary reagent conjugation specificity, target analyte, and physical, chemical and/or electrical reactions. Within these limitations, a plethora of signal generators exists. These include, for example, material adding significant mass to the analyte complex, self assembling, aggregating, enzymatic or chemically active materials, film-forming materials, materials generating optical signatures or distinctive optical lproperties, i.e., high refractive index, chiral properties, high absorption, high levels of scatter.
Furthermore, multiple signal generators may be employed to create discrete signals for different binding events.
Light Scattering Labels The signal generator component of the scattering embodiments disclosed herein may be referred to as a light-scattering Iabea. A light scattering label is a molecule or a material, often a particle, which causes incident light to be scattered elastically, i.e. substantially without absorbing the Iight energy.
Exemplary labels include metal and non-metal labels such as colloidal gold or selenium; red blood cells; and dyed polymer particles and microparticles (beads) made of latex, polystyrene, polymethylacrylate, po:lycarbonate or similar materials. The size of such particulate labels ranges from 10 nm to 10 pm, typically from SO to 900 nm, and preferably 50 nm - a microns. The larger the particle, the greater the light scattering effect, but this is true of both bound and bulk solution particles. Suitable particle labels are ~avazlable from Bangs Laboratories, Inc and Fishers.
In the present invention, the label is attached to either a secondary receptor ("labeled secondary receptor") that binds specifically to the analyte of WO 99/58948 PCTlLTS99l10917 interest, or to an analog of the analyte ("labeled analog"),. depending on the format of the assay. For a competitive assay fornnat, the labeled analog specifically binds with the reactive surface in corr~petition with the analyte of interest. For a direct sandwich assay format, the labeled secondary receptor is specific for a second epitope on the analyte. This permits the analyte to be "sandwiched" between the immobilized receptor amd the labeled secondary receptor. In an indirect sandwich assay format, the secondary receptor is also specific for a second epitope on the amalyte and is labeled with a material that specifically binds an additional light scattering lat>eI. For example, once an analyte is captured by the reactive surface, a biotinylated antibody may be used to sandwich the analyte, and an avidinated light scattering label is used for signal generation.
Regardless of the assay format, the receptor or analog must be attached to the light scattering label to form a "labeled conjugate." As with the immobilization of the capture ligands to the solid phase, the light scattering labels may be covalently bonded to the receptor on analog, but this is not essential. Physical adsorption is also suitable. In such case, the attachment to form the labeled conjugate needs only to be strong enough to withstand forces in certain subsequent assay steps, such as washing or drying.
In the preferred embodiment, signal generators are conjugated to binding reagents, which in turn, allow specific interaction with the target analyte, analyte complex or immobilized capture material. Such signal generators include, for example, beads and microparticles and colloidal metals, as discussed previously. Signal generators may also include self assembling and synthetic polymers, glass, silica, silial compounds, silanes, liquid crystals or other optically active materials, macromolecules, nucleic acids, catalyzed, auto-catalyzed or initiated aggregates, and endogenous or exogenous sample components. Useful banding reagents generally include antibodies, antigens, specific binding proteins, carbohydrates, lectins, lipids, enzymes, macromolecules, nucleic acids and other specific binding molecules.
Optical Si~~nal Format: Sign;~l Carrier Signal carriers useful in the instant invention are optical and near-optical pathways. These pathways interact with a signal l;enerator such that single event detection is possible. Either monochromatic or multiple wavelength electromagnetic radiation reflected from or transmitted through the sample may be used to detect a change in signal.
Outical Si~~nal Format: Signal Detection Historically, the effect of the use of a single optical beam for reading the surface, e.g., a laser beam, is the production of a single result representing the mass change effects of all binding events within the assay area. Where a large beam is presented to the immobilized mass and the result is integrated by a single detector, the effective result is the same.
As shown in Figure 2, the historically idealized model for this method is the optical averaging occurring over a statistically significant or an entire assay area; represented by an approximately normal distaribution of binding events over the assay area. In virtually all actual cases, the binding distribution over the assay area is highly non-homogeneous. See Figure 3. An advantage of the current optical ellipsometric read method employing a single large beam and single detector, hereinafter referred to as OTERTM (DI~x, Inc.}, is that.it inherently integrates all of the binding events within the assay area without regard to distribution, aggregating countless individual binding events into a single average result.
A disadvantage of this method derives fronn that same optical averaging effect. As depicted in Figure 4, in those cases in which the target analyte is comprised of small molecular size particles or in which there are sparse binding events, this method tends to cause results to be sta~tistica~lly reduced to insignificance when averaged over this relatively large assay area Consequently, results that involve very low concentration positives are indistinguishable from negative results against background noise or variability of the assay system.
1 S One embodiment of the instant invention involves a novel microbiological use of ellipsometric methodologies, that is, the determination of individual binding events via enumeration. This method solves the signal averaging problem by dividing the surface being analyzed into a Iarge number of discrete "local" detection areas. Any signal generated within such a local reading zone is averaged over a much smaller areas or field, and thus is "diluted" against an otherwise negative background to a much smaller extent.
For low concentration analytes this method generates numerous local results for any given test surface, most of which report negative results.
However, in those cases where positive binding ha.s occurred, the local reaction zone reports a very high positive signal; the averaging over the entire area has not diluted the positive signal. Thus, a non-integrated result profile is generated thereby reporting discrete positive results over a total test area that may be by in large negative, while allowing for much larger individual signals to be 5 generated for local positive events.
The enumeration methodology, thus, allows for extremely sensitive assay procedures, including the determination of individual binding events. An obvious application of this method (as referenced in Figure 5) is in microbiology for the detection of law numbers of microorganisms. The ability 10 to detect individual cells or clusters of cells (colony forming units) enables the elimination of time consuming culture steps. This is particularly important for those pathological organisms for which the presence of even a single organism must be considered a positive result. That is, a zero-tolerance level. Another useful application of the instant invention is in hybridization assays, wherein the 15 reaction product exists in extremely small quantities. In this case, individual binding event detection eliminates the need for cwmbersome amplification techniques, for example, PCR, NASBA and SDA. All assay systems having clinically relevant thresholds of detection below those readily achieved by traditional assay methods benefit from this invention.
20 The enumeration principle is illustrated in :Figure S using a small beam diameter, to provide a local reading area. This beam provides a vastly higher relative signal for discrete binding events, as averaged over a much smaller spot area. lVlore specifically, a collimated beam of light is scanned over a test piece in a raster (X-Y) fashion. The beam, outside diameter (DD) approximately 20 WO 99158948 PCT/US99/1091'7 microns, scans over a cell or group of cells evidencing drastic changes in the reflected light properties as received at the detector. The amplitude of those changes depends on, for example, the size of the optical beam andlar the size of the cell or cell groups. In particular, a cell that is small in comparison to the beam will be difficult to detect above general noiise associated with background light and detector amplification. The closer the beam OD and cell size approach each other, the larger the optical property changes. Practical light sources for application of the instant invention include a beam having an OD
ranging approximately from 0.650-1.550 microns, i.e., laser diodes. Laser diodes are compact in size and utilize small diamieter lenses to manipulate light, thus, facilitating variable equipment dimensions, for example, bench top, lap top and hand held equipment. Moreover, a CCD detector could result in a significant improvement in sensitivity and shorten assay run time. A
fundamental difference between the OTER and enumeration approaches, thus, is the optical pathway employed.
A signal detector, in general, must be receptive at the wavelength of the signal carrier and must be configured to receive l:he system information.
Signal detectors may include CCD cameras, single silicon detectors and diode array detectors. An ellipsometer in conjunction with C'.CD looks at the entire reaction zone and breaks it up into areas. Thus, there is a need to eliminate the negative areas and sum the positive areas. The invention disclosed herein magnifies a spot on the reaction zone and breaks that spot ini:o areas, looking for individual binding events, e.g., beads, cells, colony forming units. Figure 6 depicts topological resolution of the surface evidencing enumeration of individual binding events.
It is, in fact, because the binding events are not integrated over the surface that this method is used to approximate individual or discrete binding event identification. K.ey to practicing the enumeration method, is the ability to segment, parse or segregate discrete areas of sign;~l for highly focused readings, thereby, increasing the ability to discriminate a positive from a negative result.
Signal parsing may take place either within the carrier aspect or the detector aspect of the invention. These results are displayed as a series of discrete signal values and compared to a predetermined cut-off point, thereby determining positive binding events within any local read zone. In this manner individual binding events are enumerated on the surface, with a resolution determined by the size of the read zone. To change the relative aspect ratios of the true signal versus background signal or noise involves changing the amount of background over which any true signal is averaged. A constant signal, averaged over a progressively smaller background signal becomes progressively more distinct, until individual signal generators are readily enumerated.
Figures 7 and $ compare the differences between the current OTER
instrument configuration and one of the enumeratiion capable instrument configurations. The intersecting beam in the OTE:EZ configuration has a surface area of approximately 13 square millimeters (Pi*r2=S.A (mm2) = 3.14159 x ~ _ 12.6566 mm 2) over which any positive binding events are averaged. Signal parsing by the use of a much smaller diameter beam is illustrated in Figure $
(i.e., 20 pm). The beam is scanned across the surface, taking discrete local readings over the same total surface area. In this example, the reaction zone is 2 mm in diameter, and the scanning beam is 20 ~m in diameter. Using standard conversions (see Figure 9), the total reaction zone surface area is 3,141,590 ~.m2, while the small scanning beams reads 314.159 p,~ at each local zone. With 100 discrete measurements along irhe diameter, a 20 pm beam makes 10,000 discrete readings withing the reaction zone.
An inherent signal is generated by each. binding event. That signal is not altered by the reduction of the reading zone. Each event generates the same response locally as it would in the OTER configuration. However, the area over which this signal is averaged is reduced 10,00() times, thus, effectively amplifying the signal against the background b~y 10,000 times in the enumeration system. This change represents an enormous increase in the ability to differentiate a positive result from a negative result, effectively improving the lower limit of detection (chemical sensitivity or threshold of detection) of the assay method by 10,000 times.
Figure 9 represents preliminary calculations as to the limits of detection possible using the OTER and the enumeration approaches. The specific number and examples chosen are not significant to the disclosure, and should not be interpreted as limiting its scope. Rather, they .are included herein as an example of the sensitivity differences possible between the two systems. Enumeration is able to detect a single binding event, and as few as 100 binding events generate a clearly enumerable positive result over the system and biological noise. The probable limit of detection for an unamplified ~OTER system under comparable circumstance is 2 x 106 cfu/ml. The addition of mass to the system via amplification does not result in substantial improvement of sensitivity due to the pervasive effect of area averaging.
Signal parsing may also take place at the detector. Through the detector system, an aggregate signal may be divided into discrete information pathways correlating to discrete areas on the test-piece using a broad or Iarge beam width.
For example, a CCD or diode array detector may be used in this manner. In cases such as this, the parsed signals must be kept discrete and proportional through the detection and reporting process; magnification, focus and carrier:detector position control are methods fo:r keeping information commensurate throughout the system. The use of a monolithic or single crystal I0 diode detector requires signal to be divided into suitable small units within the signal carrier.
An alternative embodiment to the small beam scanning approach is the use of a CCD or diode array to read and parse the laser beam into smaller discrete signals. The object of this embodiment remains the determination of small spot response within the large beam spot area. However, in this case the definition of the small read zone (local result) its not provided by the diameter of the intersecting beams, but by the arrangement of the detector receiving the beam. Further, the detector, such as a photo diode array, CCD or other non-integrating signal receiver; receives the information contained in the large beam, and preserves this information as smaller local results for processing. This effectively creates a large number of virtual be~~ns, defined by the path that the light intersecting the array as a specific detection point has taken, all operating simultaneously. The aggregate signal for alI virtual beams equals the large beam signal -- each virtual beam references only a limited surface area -- and the results are not integrated together.
An advantage of this method is that it is rapid (parallel signal processing). The scanning approach is a serial process in which each reading is made in sequence. Additionally, the technical challenges of producing this embodiment are substantially less than those involved in the development of a small beam laser and an accurate scanning control mechanism.
As discussed supra, a variety of optical signals may be used within this system. The specific optical signal is selected to provide the appropriate level of information, based upon the nature of the material to be detected, and the 10 resolution desired. The examples provided herein use ellipsometry and scatterometry, see Figure 11. However, a variety of optical methods will be substantially improved by adopting the general concepts and methodologies described herein. In particular, effects such as absorption, refractive index change; chiral effects and diffraction may be used within essentially similar 15 optical configurations. Figure 12 lists possible; optical signal types, thus, displaying the range of methods amenable to the enumeration approach. It is neither limiting nor intended to comprise a complete listing thereof Mass enhancement Labels can play a central role in the practice of the enumeration method at high sensitivities. Figures 13 and 14 illustrate, 20 proportionally, the aspect ratio or relative heig;ht:width:breadth of various size materials that may be used as signal generators. As is diagramed in these figures, organisms at the cellular scale generate very significant signal without amplification within the system. In comparison, the thin attachment layer represented along the bottom of the reading zone surface creates a clearly WO 99158948 PCTlUS99/10917 distinguishable signal with the current OTER format. The signals generated by mass contained in the much larger objects used as labels significantly improve sensitivity.
Additionally, for either the scanning (small beam) or the array (virtual beam) approach as discussed, a substantial improvement in signal detectability is possible using unique characteristics of opti<;ally based mass detection systems. Particular properties of any given mess enhancement label may be used to alter the optical signal based upon its physical characteristics, including its effect on optical characteristics: refractive index, scatter, chiral effect, general adsorption, wavelength specific adsorption and diffraction.
Use of selected labels to induce unique or distinct optical effects creates an improved ability to discriminate the signal ;generated by the binding of label to the complex from that created by surface background or in the absence of specific binding events. This operates through. the creation of an enhanced or 1 S attenuated apparent signal over that which would be created by normal materials.
Figure 14 specifically provides an example of this type of effect through the use of high refractive index material in an ellipsometric format. Because the change in polarization state detected by ellipsometry is caused by two distinct factors (absolute mass and refractive index) the use of a high refractive index material as the mass enhancement label effectively increases the apparent mass detected by the ellipsometer, thus, further amplifying the signal from the binding event.
Any number of optical interactions with specific types of material Wp gg/ggg~g PCT/US99/10917 designed to amplify or enhance the strength of the signal, or to create a unique signal type, are envisioned and are included herein by reference.
Detection of scattered light (scatterometry) may occur visually or by photoelectric means. For visual detection the e:ye and brain of an observer perform the image processing steps that result :in the determination of scattering or not at a particular sites. The terms "sites" and "site" refer, herein, to the area covered by one ligand. Scattering is observed when the sites appears brighter than the surrounding background. If the number of sites are small, perhaps a dozen or less, the processing steps can be effected essentially simultaneously. If the number of sites is large (a few hundred or more) a photoelectric detection system is desired.
Photoelectric detection systems include any system that uses an electrical signal which is modulated by the light intensit~~ at the sites. For example, photodiodes, charge coupled devices, photo transistors, photoresistors and photomultipliers are suitable photoelectric dete~;,tion devices. Preferably, detector arrays (pixels) correspond to the array of sites on the reactive surface for signal parsing, some detectors corresponding to non-sites portions. More preferred, however, are digital representations of the reactive surface such as those rendered by a charge coupled device (CCD) camera in combination with available frame grabbing and image processing software. The image processing techniques preferred in the instant invention are derived from Image-Pro~ Plus for WindowsTM (Media Cybermetrics).
A CCD camera or video camera forms an image of the entire reactive surface, including alI label and non-label areas., and feeds this image to a frame grabber card of a computer. The image is converted by the frame grabber to digital information by assigning a numerical value to each pixel. The digital system may be binary (e:g. bright=1 and dark-=0) but an 8-bit gray scale is preferred, wherein a numerical value is assigned to each pixel such that a zero (0) represents a black image, and two hundredl and fifty-five (255) represents a white image, the intermediate values representing various shades of gray at each pixel.
Data Analysis The digital information may be displayed on a monitor, or stored in RAM or any storage device for further manipulation, such as imaging printing and archiving. Image processing software, such as Image Pro Plus fox Windows (IPP), is used to analyze the digital information and determine the boundaries or contours of each situs, and the value of intensity at each situs.
IPP is commercially available software for digital image acquisition, processing and analysis. IPP automatically counts and measures objects within an image, after which it sorts and classified the objects by specific characteristics, including, far example: angles, area, length, v~~idth, diameter radius perimeter, area or aspect ratios, color, position, optical density and hole areas. IPP is also ZO able estimate the number of objects contained within a cluster of objects.
IPP may be programmed to perform a specific series of functions and analyses in order to differentiate true aanalyte complex particles form other particles or optical features, e.g., dust, non-specific binding, solid phase anomolies, masking. That is to say, the object mearurement characteristics discussed herein may be used to create signal:non-signal f lters.
Often, the image will require ehnhancement to improve the software's ability to enumerate individual binding events. Enhancement techniques may include, for example, brightness:contract adjustment and spatial:morphological filtering. More specifically, there are three basic categories of image enhancement: intensify index modification, spatial filtering and image frequency manipulation.
Modification of the intensity index is directed to a change in the way intensity values of each pixel are interpreted. Aspects of the intensity index include, for example, birghtness, contract, gam~rna correction, thresholding, background flattening, background subtraction and intensity equalizatoin.
Spatial filtering techniques analyze and process an image in small regions of pixels. Specifically, by reducing or' increasing the rate of change that occurs in the intesntiy transitons within an image. This filtering includes convolution (linear) and non-convolution (non-linear).
Manipulation of the image frequencies is directed to the elimination of periodic or coherent noise in an image by converting the image to a set of frequencies, and editng out the frequencies causing the noise problem. A
common technique used for this is the Fourier Transform.
It is envisioned that the digigtal image processing funcions necessary may be consolidated into a laboratory-basded iintrument adapted for and capable of semi- and/or automatically performing all sofware-based steps of enumeration. It is not an essential element of the invention to display the ssurface image. It is essential only that the software image processing is performed entirely with the datea provided by the digitization of the image.
The inventive clustering process as described in U.S. Patent no.
5,329,461 may be adapted for utilization in a variety of applications to spatially resolve and count discrete analyte particles or individual binding events in 5 conjunction with the instant invention. For example, detection of analyte particles comprising a molecule and a labor or for rapid scanning to locate areas of interest within an image of a sample.
Instrumentation 10 Generally, a prepared test piece is secured to the sample stage and manually positioned such that the center of a test spot is aligned with the center of the objective lens. The test piece may be prepared to contain multiple test spots, therefore, to begin the test spot designated as 1, or first, is centered.
Using the sample sta.ge's translational capabilities, the detector is manually I S focused on the scattering particles. Next, the image produced by the light scattering is collected and saved. Finally, the sample stage is translated to two alternate locations, one each to the left and right of center, and image acquisition repeated at each location. Each generally herein-described step in the detection process may be repeated for any number of test spots contained on 20 a test piece.
The instrument employed for the enumeration methodology disclosed herein consists of 3 defining modules: a sample stage, an optical signal format corresponding to the immobilized analyte complex, and a means for data collection and analysis. Each module is adapted for independent translation on WO 99!58948 PCT/U599110917 at least 2 axises, thereby facilitating optimal optical effect, alignment and focus.
The instrument and its modules, in toto, are fixed and stationary in relation to one another by standard attachment means to, :Por example, a solid, planar, horizontal platform. More specifically, as shown in Figure 15, the enumerator 100 is comprised of a means for data collection and analysis 85 consisting essentially of a computer 80 and video display terminal 60 functionally combined with a sample stage 10 and optical signal format consisting essentially of a signal carrier 40 and a signal detector 25 configured such that when a signal generator, such as a light scattering label, is irradiated, it is able to be detected by the enumerator 100.
The sample stage 10 may be any planar stage or platform adapted for receiving and securing thereon a mounting jig 15 onto which a test piece 70 is secured to the mounting jig 15. The test piece: 70 rnay be secured by any suitable means, such as, double sided adhesive tape or a mechanical mounting means. Said stage 10 translates on at least an X-Y axis basis, and in the preferred embodiment, also possesses additional rotational and angle control.
The test piece 70 is further comprised of test spots, prepared as described herein.
The optical signal format is comprised of a signal generator such as a light scattering label bound to a test spot as described herein, a signal carrier 40 and a signal detector 2S. In the preferred embodiment the signal carrier 40 is an electromagnetic radiation source, and more preferably, a laser diode adaptively mounted to possess both rotational sand angular control. The signal detector 25, an integrally combined microscop,~ focus tube 30 and objective 20 WO 99158948 PCTlUS99l10917 functionally combined with a photodetector, and preferably a CCD, camera 50 are disposed, by any standard mounting means, vertically above the sample stage 10. The signal detector 25 is functionally combined by standard means with the data collection and analysis means 85 comprised of a PC 80 and video display terminal 60, each of which is accordingly appointed with appropriate software and electronics.
In use, the PC 80 and video display terminal 60, and signal carrier 40 are powered on and allowed to warm up for at: least 30 minutes. While the unit is warming up, the test piece 70 is adhered to the mounting jig 15, which in turn, is secured to the sample stage 10 directly and vertically below the signal detector 25. The test spot on the test piece 70 that has the target analyte bound thereto is then centered, aligned and focused between the signal detector 25 and the signal carrier 40. The enumerator 100 is engaged, an image acquired and exhibited and/or stored accordingly. The test piece 70 is realigned for additional image capture to the left and right o~f the test spot, as described herein. Engagement of the enumerator 100 and image capture is repeated in a similar manner for each of the test spots on the test piece 70.
Prior to engagement of the enumerator 100, the appropriate software preparation is performed. For example, subfolders, default settings and macros are setup.
Generally, Iight scattered by surface-bound microspheres is collected and magnified by a microscope objective lens and focused onto a CCD array, e.g.
640 x 480 pixels. CCD signal output is fed to both a black and white monitor and a data translation frame grabber such as Data Translation DT3155 high accuracy scientific frame grabber (Data Translation, Inc.): Image acquisition and analysis of the image formed by scattered light is accomplished with software adapted for and/or specifically directed to such function, for example, Image Pro Plus (Media Cybernetics).
Data analysis that includes discrimination and counting of scattering objects within an image is performed by software designed for such a purpose.
Customized functions adapted into such softw~~re via, for example, macro programs, include exclusion of non-binding events from the object count by filtering, image intensity averaging and binary filtering. An example of a macro adapted far use in the preferred embodiment of the invention includes:
transformation of bright scattering objects into a standard 3 x 3 cross;
application of a watershed filter to the resulting cresses to separate scattered objects; determination of mean image intensity and the standard deviation of that mean; determination of a Lower limit intensity threshold fox a binary filter based on the mean image intensity; application of binary filter with threshold values of Lower limit; and, automatic count of resulting objects having a mean diameter, for example, less than 10 pixels. The number of objects counted for each image is averaged aver the three images produced for each test spot --center, left and right.
Example 1: Specific Binding Assay Preparation of Whole Wafer Test Pieces. The test pieces used are commercially available 5' silicon (Si) wafers. Thin layer polyurethane coated wafers are produced using standard spin-coating procedures to lay the polyurethane on the reflective surface of the wafer. Briefly, the wafers are prepared by addition of 500 ~1 of a thoroughly mixed 1.25% solution of Polymedica M1020 Polyurethane (Polymedica, Inc.) in N,N-dimethylacetamide (DMAC) (Sigma Chemical Co.) To the center of a silicon wafer (Addison Engineering) spinning at 5000 rpm. The wafer is air dried and then baked at 70° C for 16-20 hours. Next, a 10 circle by 10 circle pattern is applied to the non-reflective wafer surface using a 3.5" x 3.5" rubber stamp coated with RTE
108 silicone rubber adhesive sealant (GE Silicones, inc.). The resulting circular outlines serve as a means to isolate each circuh~r polyurethane coated test spot 00.25" diameter). The adhesive is cured at ambient room temperature for approximately 24 hours prior to use in assay.
Adsorption of Streptavidin Coated Microspheres to a Biotin,1 Surface. Each of the polyurethane coated wafer test spots are coated with 20 ~l of a 1 lzg/ml of biotinylated bovine serum albwmin {BSA) (Sigma Chemical Co.), or alternatively a non-biotinylated BSA for use as a negative control.
The wafer is incubated at 37° C for one hour in a 100% humidity chamber.
After incubation, the wafers are rinsed 3 times with deionized water and dried with compressed air. Following BSA immobilization, the test spots are blocked with 30 ~.l of 3% BSA for 1 hour at 37° C, then rinsed 3 times with deionized water and dried with compressed air.
Streptavidin coated polystyrene microsplheres (350 nm diameter) (Bangs Laboratories) are serially diluted in borate buffi~r (0.1 M, pH 8.5 + 0.01 Tween-20), for resulting dilution ranging between 1:10 and 1:10,000. Next, 20 ~,1 of each dilution is applied to the biotinylated and non-biotinylated test spots WO 99158948 PCT/t1S99/10917 and the wafer incubated at 37° C for 1 hour, rinsed for 10 seconds with deionized water, compressed air dried and analyzed with the invention disclosed herein, the results of which are shown in Table; I. These data show that light scattering labels bound to a surface can be detected and enumerated using the 5 present invention; that streptavidin coated microspheres bind specifically to a biotinylated surface; and that the number of rnicrospheres counted on the surfaces is dependent on the number applied to the surface.
Example 2: Staphylococcal Enterotoxin B ('SEB) Detection Assay 10 Preparation of Whole Wafer Test Pieces. The test pieces used are commercially available 5' silicon (Si) wafers. Thin layer polyurethane coated wafers are produced using standard spin-coating procedures to lay the polyurethane on the reflective surface of the wafer. Briefly, the wafers are prepared by addition of 500 ~,l of a thoroughly mixed 1.25% solution of 15 Polymedica M1020 Polyurethane (Polymedica, Inc.) In N,N-dimethylacetamide (DMAC) {Sigma Chemical Co.) To the center of a silicon wafer (Addison Engineering} spinning at 5000 rpm. The wafer is air dried and then baked at 70° C for 16-20 hours. Next, a 10 circle by 10 circle pattern is applied to the non-reflective wafer surface using a 3.5" on 3.S" rubber stamp coated with RTV
20 108 silicone rubber adhesive sealant (GE Silicones, Inc.). The resulting circular outlines serve as a means to isolate each circular Polyurethane coated test spot 00.25" diameter). The adhesive is cured at ambient room temperature for approximately 24 hours prior to test spot mounting on test piece and use in assay.

SEB Detection. A full sandwich assay is used for the detection of SEB
in a sample buffer. The general protocol consists of coating capture antibody to individual test spots, blocking, adding different; concentrations of SEB to the coated test spots, applying a biotinylated secondary reporting antibody, and labeling the bound secondary antibody with avidinated polystyrene microspheres.
Test wafers are coated with polyclonal a-SEB capture antibody by applying 20 pl of a 30 ~,g/ml (in 0.1 M PBS, pH 7.2) solution to each assay test spot. The wafer is incubated at 37° C for 1 hour to allow passive adsorption of the capture antibody to the polyurethane. After incubation, the wafer is rinsed 3 time with deionized water and dried with compressed air Following capture antibody immobilization, each test spot is blocked with 40 ~,l of a 3% BSA solution (0.1 M PBS., pH 7.2) to reduce non-specific protein adsorption from subsequent assay steps.. The wafer is incubated at 37°
C for 1 hour and subsequently rinsed 3 times with deionized water and dried with compressed air.
SEB samples are prepared by serial dilution of a 1 mglml stock into sample buffer (0.1 M PBS + 1% BSA + 0.01°~o Tween-2-, pH 7.2), with final toxin concentrations ranging from 0.1 ng/ml to 100 mg/ml. Buffer with no SEB is used as a negative control. Twenty ~l of each of the dilutions and the negative control are applied to separate test spots across the wafer surface.
The water is incubated at 37° C for 30 minutes then rinsed 3 times with deionized water and dried with compressed air Biotinylated «-SEB antibody is diluted to 4 pg/ml in sample buffer.

Each test spot is coated with 20 ul of this secondary antibody dilution. The wafer is incubated at 37° C for 30 minutes them rinsed 3 times with deionized water and dried with compressed air.
Test spots are coated with 20 p.i of a 1:100 dilution of streptavidin coated 350 nm diameter polystyrene microspheres in borate buffer (0.1 M, pH
8.5 + 0.01 % Tween-20). The wafer is incubated at 37° C for 30 minutes then each test spot is rinsed for 10 seconds, dried with compressed air and analyzed.
The results of such analysis are shown in Table II. These data show that the present invention can be used to enumerate the binding of an antigen to a solid phase in a specific and quantitative manner. The lower limit of detection for this method is 550 pg/mI.
Data acquisition and analysis are performed as generally described herein. The wafer or test piece is mounted on a stage, positioned, focussed and images captured. Data analysis includes employing a macro program within 1 S Image Pro Plus.
While the above description contains many specificities, these specificities should not be construed as limitations on the scope of the invention, but rather exemplification of the preferred embodiment thereof.
That is to say, the foregoing description of the invention is exemplary for purposes of illustration and explanation. Without departing from the spirit and scope of this invention, one skilled in the are can make various changes and modifications to the invention to adapt it to various usages and conditions.
As such, these changes and modifications are properly, equitably, and intended to be within the full range of equivalence of the claims. Thus, the scope of the invention should be determined by the appended claims and their legal equivalents, rather than by the examples provided herein.

TABLE I
Specific Adsorption of Beads to Biotinylated Surfaces Bead Dilution # Objects: # Object:
Biotinylated Surface non-Biotinylated Surface 1:10 2263 201 l:lo0 2019 2~

1:500 1375 9 1:1000 849 13 1:10,000 115 8 WO 99!58948 PCT/US99/10917 TABLE II
SEB Detection Assay SEB Concentration # Objects Standard Deviation (ng/ml) 0.1 72 12 0.5 121 10

Claims (18)

We claim:

1. A method for solid phase, optical detection and enumeration of individual target analyte binding events comprising the steps of:
Immobilizing an analyte complex on a reflective or transmissive substrate directly from solution, said complex comprising a target analyte complexed with at least one signal generator element conjugated to at least one secondary analyte specific binding element;
Reflecting or transmitting electromagnetic radiation from or through the substrate having the analyte complex immobilized thereon;
Capturing a signal generated from said reflecting or transmitting of electromagnetic radiation; and, Analyzing the signal for the presence and/or amount of analyte present.

2. The method as defined in claim 1, wherein immobilizing the analyte complex is accomplished by adsorptive, covalent, steric, chemically mediated, liner, self assembling or force mediated binding to a solid phase or solid phase matrix.

3. The method as defined in claim 2, wherein immobilizing further.
comprises one or a plurality of intermediate layers disposed between said substrate and said analyte complex.

4. The method as defined in claim 1, wherein said target analyte is separated from a material or materials via immobilization.

5. The method as defined in claim 1, wherein said analyte is complexed in liquid phase or solid phase.

6. The method as defined in claim 1, wherein said signal generator element is selected from the group consisting of self assembling, aggregating, enzymatic, chemically active, film-forming and optically unique materials.

7. The method as defined in claim 6, wherein said signal generator element is selected from the group consisting of microparticles, colloidal metals or non-metals, polymers, glass, silial compounds, optically active materials, macromolecules and nucleic acid.

8. The method as defined in claim 6, wherein said signal generator element adds mass to said analyte complex.

9. The method as defined in claim 1, wherein a plurality of signal generator elements are complexed with said analyte complex creating a plurality of distinct signals indicative of distinct binding events.

10. The method as defined in claim 1, wherein said secondary binding element is selected from the group consisting of antibodies, antigens, macromolecules, nucleic acid and specific binding molecules.

11. The method as defined in claim 1, wherein said electromagnetic radiation source is a laser diode.

12. A system for solid phase, optical detection and enumeration of target analyte individual binding events comprising:
a means for target analyte capture, said means comprising a substrate having an analyte complex immobilized thereon, said analyte complex comprising a target analyte complexed with at least one signal generator element conjugated to at least one secondary analyte specific binding element;
a signal carrier means consisting of electromagnetic radiation, said signal carrier having a known interaction with said signal generator and generating a detectable signal evidencing analyte binding event or events;
a signal capture means having an optical resolution element or elements and configured to receive information generated from the signal carrier; and, a signal analyzing means, said means processing information generated by said signal capture means for qualitative and/or quantitative analyte detection.

13. The system as defined in claim 12, wherein said electromagnetic radiation is monochromatic wavelength within the range of 400 nm - 700 nm.

14. The system as defined in claim 12, wherein said electromagnetic radiation is multiple wavelength within the range of 400 nm - 700 nm.

15. The system as defined in claim 12, wherein said resolution element magnifies, focuses and/or controls the signal carrier means.

16. The system as defined in claim 12, wherein said resolution element parses discrete signals from an aggregate signal generated from said signal generator.

17. The system as defined in claim 12, wherein said signal carrier means is selected from the group consisting of interference, diffraction, reflection, polarization, scattering, birefringence, absorption and refraction.

18. The system as defined in claim 12, wherein said analyte complex further comprises mass enhancement means for amplifying a signal for detection related to the presence of the target analyte.