Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites

Definitions

• This invention relates to the general fields of molecular biology
• 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.
• optical ellipsometric immunoassay (OpTestTM, DDx, Inc.), a detection system for molecular and microscopic scale events, that measures interactions between biological samples and light.
• 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.
• the biosensor system Based on Imaging Ellipsometry for Visualization of 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.
• Imaging Ellipsometer Beaglehole (1988) Rev. Sci. Instrum. 59(12):2557. No type of life science or biological system application of the imaging is suggested.
• This methodology is directed to detecting labeled microparticles using microscopy, for example, an electron microscope imaging system.
• 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 systematic errors may also collectively contribute to the noise associated with measurement.
• light radiation e.g., fluorescence or chemiluminescence
• 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 imposed or induced variations. In general, efforts to augment this signal to noise (S/N) ratio have centered on
• detection limit associated with a particular analyte.
• the detection limit refers
• 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 limit.
• 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 concentration. 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
• 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
• the instant invention is based on novel methods of analyte detection as a means for detection of specific molecules using solid phase immobilization
• this invention comprises the
• this invention is directed to the solid phase, optical detection and enumeration of individual binding events mediated by specific binding interactions.
• This invention is defined by analyte solid phase immobilization, a signal
• 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 utilizing 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.
• CCD charged-coupled device
• 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: 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
• 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
• the digital 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.
• an optical signal i.e., photons.
• Stimpson et al. and Allen et al. employ the use of CCDs and pixels for detection purposes.
• the pixel regions are dimensioned such that the number of analyte complexes included 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 includes 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
• the detection techniques of the present invention can be used for detecting a wide variety of analytes.
• the assay sample medium is preferably a solid phase bound analyte complex in which detectable label not bound to an analyte may be removed through conventional washing procedures.
• 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
• 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 most part, the number of signal units per analyte complex will be more than one.
• the assay sample medium often has low concentrations of analyte, generally at picomolar or less, frequently femtomolar or less. Assay volumes are usually less than about 100 ⁇ l, frequently less than 10 ⁇ l and may be 1 ⁇ l 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 CCD pixels is irrelevant in that this is accomplished through magnifying optics.
• the elements of a specific binding pair can be referred to as "ligands” and “receptors.” Generally receptors are immobilized to the solid phase to capture, or immobilize, the
• ligand analyte of interest
• specific binding pairs may involve haptens and antigens (referred to as “ligands”) and their complementary binding elements, such as antibodies, enzymes, surface membrane protein receptors, lectins, etc. (generally known as “receptors").
• ligands haptens and antigens
• receptors complementary binding elements
• 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 binding elements comprising ligands and receptors.
• 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.
• various protocols may be employed, which may be associated with commercially available reagents or such reagents which may be modified.
• Figure 1 illustrates the determination of mass per unit volume or equivalent thereof in standard immunoassay methodology
• 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 large 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 aggregate 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 and/or 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 scanning micro-laser configurations
• Figure 14 depicts optical enhancement potential.
• Figure 15 depicts the preferred instrumentation embodiment of the instant invention.
• samples include,
• 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
• 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.
• the instant invention enables the detection of individual binding events. The principle being to narrow the size, actual or 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
• the present invention solves the problem of detection of low concentrations of specific molecules of interest (analytes) using solid phase immobilization and optical signals capable of detecting and measuring mass
• such mass changes are additively achieved or mediated by analyte complexing 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)
• mass change is subtractively achieved through specific enzymatic, chemical or other specific dissociating or lytic agents.
• assay systems utilizing specific binding or lytic interactions suitable for mass change analysis include, 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.
• such enhancement involves the alteration of one or more of the elements of the binding or lytic event generating a differentiable optical signal, or the enhancement initiates a detectable self-assembly or aggregation process.
• 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 ⁇ l) of a liquid sample or suspension.
• the immobilization area typically used for this type of assay is also at the macro- scale (> 1000 microns).
• 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 lytic 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.
• the signal generated must be differentiable against the field of
• 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, DNA probe, immuno-chromatographic or other ligand binding methods.
• 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 multi-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 ligand-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.
• 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
• immobilization examples 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 capture 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
• the solid support is reactive to analyte binding ("reactive surface").
• blocking materials include, for example, proteins such as casein and bovine serum albumin, detergents, and long-chain polymers.
• the chosen receptor is immobilized 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.
• the analyte bound to the solid phase may be additionally complexed with a mass amplifying agent through a secondary specific receptor binding to form an analyte 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.
• 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
• the Optical Signal Format of the instant invention is comprised of at least a signal carrier, a signal generator and a signal detector.
• Optical Signal Format Signal Generator
• the present invention specifically relates to a method for altering the ratio of signal to non-signal surface area, allowing for more sensitive results.
• this invention uses specific labels selected to interact with specific optical beam types to create an enhanced, differentiable or amplified signal.
• a solid phase is typically used as a separation platform to isolate an analyte from other elements of a sample and from excess reagents.
• the signal generator remains attached to the binding complex, and thus
• the mass of analyte found in the volumetric sample is converted to mass immobilized on the solid phase in a proportional manner.
• the signal generator 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 between the two.
• a signal generator element includes material which may be used to specifically label, amplify, distinguish, mark or generate a detectable signal associated with the
• 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.
• signal generators exist. 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 properties, i.e., high refractive index, chiral properties, high absorption, high levels of scatter.
• multiple signal generators may be employed to create discrete signals for different binding events.
• 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 light 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, polycarbonate or similar materials. The size of such particulate labels ranges from 10 nm to 10 ⁇ m,
• Suitable particle labels are available from Bangs Laboratories, Inc and Fishers.
• the label is attached to either a secondary
• labeled secondary receptor that binds specifically to the analyte of interest, or to an analog of the analyte (“labeled analog”), depending on the format of the assay.
• labeled analog specifically binds with the reactive surface in competition with the analyte of interest.
• labeled secondary receptor is
• analyte specific for a second epitope on the analyte. This permits the analyte to be
• the secondary receptor is also specific for a second epitope on the analyte and is labeled with a material that specifically binds an additional light scattering label.
• a biotinylated antibody may be used to sandwich the analyte, and an avidinated light scattering label is used for signal generation.
• the receptor or analog must be attached to the light scattering label to form a "labeled conjugate.”
• the light scattering labels may be covalently bonded to the receptor or analog, but this is not essential. Physical adsorption is also suitable. In such case, the attachment to
• signal generators are conjugated to binding reagents, which in turn, allow specific interaction with the target analyte
• 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 binding reagents generally include antibodies, antigens, specific binding proteins, carbohydrates, lectins, lipids, enzymes, macromolecules, nucleic acids and other specific binding molecules.
• Signal carriers useful in the instant invention are optical and near-optical pathways. These pathways interact with a signal generator 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.
• the surface e.g., a laser beam
• the surface is the production of a single result representing the mass change effects of all binding events within the assay area.
• the effective result is the same.
• optical averaging occurring over a statistically significant or an entire assay
• OTERTM single detector
• a disadvantage of this method derives from that same optical averaging effect.
• this method tends to cause results to be statistically 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.
• 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 large number of discrete "local" detection areas. Any signal generated within such a local reading zone is averaged over a much smaller area or field, and thus is
• results for any given test surface most of which report negative results.
• the local reaction zone reports a very high positive signal; the averaging over the entire area has not diluted the positive signal.
• 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 generated for local positive events.
• the enumeration methodology allows for extremely sensitive assay procedures, including the determination of individual binding events.
• An obvious application of this method is in microbiology for the detection of low numbers of microorganisms.
• the ability 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
• reaction product exists in extremely small quantities.
• individual binding event detection eliminates the need for cumbersome 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.
• the enumeration principle is illustrated in Figure 5 using a small beam diameter, to provide a local reading area. This beam provides a vastly higher
• a collimated beam of light is scanned over a test piece in a raster (X-Y) fashion.
• the beam outside diameter (OD) approximately 20 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 and/or the size of the cell or cell groups.
• a cell that is small in comparison to the beam will be difficult to detect above general noise associated with background light and detector amplification.
• Practical light sources for application of the instant invention include a beam having an OD
• laser diodes ranging approximately from 0.650-1.550 microns, i.e., laser diodes.
• Laser diodes are compact in size and utilize small diameter lenses to manipulate light, thus, facilitating variable equipment dimensions, for example, bench top, lap top and hand held equipment.
• a CCD detector could result in a significant improvement in sensitivity and shorten assay run time.
• a signal detector in general, must be receptive at the wavelength of the signal carrier and must be configured to receive the system information.
• Signal detectors may include CCD cameras, single silicon detectors and diode array
• An ellipsometer in conjunction with CCD 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
• binding events e.g., beads, cells, colony forming units.
• Figure 6 depicts topological resolution of the surface evidencing enumeration of individual binding events.
• event identification Key to practicing the enumeration method, is the ability to segment, parse or segregate discrete areas of signal 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.
• 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 8 compare the differences between the current OTER instrument configuration and one of the enumeration capable instrument configurations.
• reaction zone 2 mm in diameter
• scanning beam 20 ⁇ m in diameter
• Signal parsing may also take place at the detector.
• an aggregate signal may be divided into discrete information pathways correlating to discrete areas on the test-piece using a broad or large beam width.
• 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
• magnification, focus and carrier:detector position control are methods for keeping information commensurate throughout the system.
• the use of a monolithic or single crystal 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.
• the definition of the small read zone (local result) is not provided by the diameter
• the detector such as a photo diode array, CCD or other non- integrating signal receiver, receives the information contained in the large beam
• 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.
• 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 resolution desired.
• the examples provided herein use ellipsometry and scatterometry, see Figure 11.
• a variety of optical methods will be substantially improved by adopting the general concepts and methodologies described herein.
• effects such as absorption, refractive index change, chiral effects and diffraction may be used within essentially similar 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,
• the aspect ratio or relative height:width:breadth of various size materials that may be used as signal generators.
• organisms at the cellular scale generate very significant signal without amplification within the system.
• the thin attachment layer
• any given mass 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.
• Figure 14 specifically provides an example of this type of effect through the use of high refractive index material in an ellipsometric format.
• 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
• Detection of scattered light may occur visually or by photoelectric means.
• For visual detection the eye and brain of an observer
• situs perform the image processing steps that result in the determination of scattering or not at a particular situs.
• the terms "situs” and "site” refer, herein, to the area covered by one ligand. Scattering is observed when the situs 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 intensity at the situs.
• photodiodes charge coupled devices, photo transistors, photoresistors and photomultipliers are suitable photoelectric detection devices.
• detector arrays pixels
• pixels correspond to the array of sites on the reactive surface for signal parsing, some detectors corresponding to non-situs portions.
• 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.
• CCD charge coupled device
• a CCD camera or video camera forms an image of the entire reactive surface, including all 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 information may be displayed on a monitor, or stored in
• IPP Image Pro Plus for Windows
• IPP is also able estimate the number of objects contained within a cluster of objects.
• IPP may be programmed to perform a specific series of functions
• 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 filters.
• Enhancement techniques may include, for example, brightness: contract adjustment and spatial morphological filtering. More specifically, there are three basic categories of image enhancement: intensity index modification, spatial filtering and image frequency
• 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, gamma correction, thresholding,
• 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.
• 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
• analyte particles comprising a molecule and a labor or for rapid scanning to locate areas of interest within an image of a sample.
• 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.
• the detector is manually focused on the scattering particles.
• the image produced by the light scattering is collected and saved.
• 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.
• the detection process may be repeated for any number of test spots contained on a test piece.
• the instrument employed for the enumeration methodology disclosed herein consists of 3 defining modules: a sample stage, an optical signal format
• each module is adapted for independent translation on 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, for 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
• 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.
• a signal generator such as a light scattering label
• 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 may 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 25.
• 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 25.
• the signal carrier 40 is
• an electromagnetic radiation source and more preferably, a laser diode
• the signal detector 25, an integrally combined microscope focus tube 30 and objective 20 functionally combined with a photodetector, and preferably a CCD camera 50 are disposed, by any standard mounting means, vertically above the sample
• 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.
• 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 of 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.
• the appropriate software preparation is performed prior to engagement of the enumerator 100. For example, subfolders, default settings and macros
• 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
• a macro adapted for use in the preferred embodiment of the invention includes:
• test pieces used are:
• 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
• Polymedica Ml 020 Polyurethane Polymedica, Inc.
• DMAC N,N-dimethylacetamide
• non-reflective wafer surface using a 3.5" x 3.5" rubber stamp coated with RTV 108 silicone rubber adhesive sealant (GE Silicones, Inc.).
• RTV 108 silicone rubber adhesive sealant GE Silicones, Inc.
• the resulting circular outlines serve as a means to isolate each circular polyurethane coated test spot ( ⁇ 0.25" diameter).
• the adhesive is cured at ambient room temperature for approximately 24 hours prior to use in assay.
• each of the polyurethane coated wafer test spots are coated with 20 ⁇ l of a 1 ⁇ g/ml of biotinylated bovine serum albumin (BSA) (Sigma Chemical Co.), or alternatively a non-biotinylated BSA for use as a negative control.
• BSA biotinylated bovine serum albumin
• 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
• Streptavidin coated polystyrene microspheres 350 nm diameter (Bangs Laboratories) are serially diluted in borate buffer (0.1 M, pH 8.5 + 0.01 %
• Tween-20 for resulting dilution ranging between 1 :10 and 1 :10,000.
• 20 ⁇ l of each dilution is applied to the biotinylated and non-biotinylated test spots and the wafer incubated at 37° C for 1 hour, rinsed for 10 seconds with
• biotinylated surface and that the number of microspheres counted on the surfaces is dependent on the number applied to the surface.
• Example 2 Staphylococcal Enterotoxin B (SEB) Detection Assay
• 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 Polymedica Ml 020 Polyurethane (Polymedica, Inc.) In N,N-dimethylacetamide
• 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 «-SEB capture antibody by applying 20 ⁇ l 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.
• 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°
• SEB samples are prepared by serial dilution of a 1 mg/ml stock into sample buffer (0.1 M PBS + 1% BSA + 0.01% 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
• Biotinylated «-SEB antibody is diluted to 4 ⁇ g/ml in sample buffer. Each test spot is coated with 20 ⁇ l of this secondary antibody dilution. The wafer is incubated at 37° C for 30 minutes then rinsed 3 times with deionized water and dried with compressed air.
• Test spots are coated with 20 ⁇ l of a 1:100 dilution of streptavidin coated 350 nm diameter polystyrene microspheres in borate buffer (0.1 M, pH
• 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 Image Pro Plus.

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