JP2008529013A - Method and apparatus for quadrature phase difference interference detection of an immunoassay - Google Patents

Abstract

Phase difference orthogonal interferometry for determining the presence or absence of a target analyte in a sample. The method includes using a laser beam having a wavelength λ and a waist w ₀ to probe at least a portion of a substrate having a reflective surface exposed to the sample. The reflective surface includes at least a first region having a layer of recognition molecules specific to the target analyte and a second region not including a layer of recognition molecules specific to the target analyte. The method further explores the first region and the second region, and calculates the time-dependent intensity of substantially only the first orthogonal component at one of the pair of quadrature angles θq of the reflected diffraction signal of the probe beam. It further includes measuring using a detector. An apparatus for detecting the presence or absence of a target molecule in a planar array by the phase difference quadrature interferometer side includes a light source for generating a probe beam. The apparatus includes a platform for receiving a planar array and a first optical element array that directs the probe beam to be substantially perpendicular to the platform. The apparatus further includes an objective lens having a first surface and a second surface and having a focal length. The objective lens is separated from the platform by a first distance approximately equal to the focal length on the first surface of the lens. The apparatus further includes split light detection means for measuring the first orthogonal component and the second orthogonal component in the signal generated by reflection of the probe beam.
[Selection] Figure 1

Description

  The present invention relates generally to an apparatus for detecting the presence of a particular biological material in a sample, and more particularly to detecting the presence of biological pathogens and / or molecules of interest that bind to a target receptor on a disk. The invention relates to a laser compact disc system for performing the detection by sensing changes in the optical properties of the probe beam reflected from the disc by the pathogen and / or the analyte.

  In many chemical, biological, medical, and diagnostic applications, it is desirable to detect the presence of specific molecular structures in a sample. Many molecular structures such as cells, viruses, bacteria, toxins, peptides, DNA fragments, and antibodies are recognized by specific receptors. Biochemical techniques for detecting gene expression patterns in cancer cells, such as those involving gene chips, immunological chips, and DNA arrays, take advantage of the interaction between these molecular structures and receptors. [See, for example, the description of the paper below. Sanders, GHW and A. Manz, “Chip-based microsystems for genomic and proteomic analysis”, Trends in Anal. Chem., 2000, Vol. 19 (6), p. .364-378; Wang, J., “From DNA biosensor to gene chips”, Nucl. Acids Res., 2000, Vol. 28 (16), p.3011-3016 Hagman, M., “Doing immunology on a chip”, Science, 2000, Vol. 290, p.82-83; Marx, J., “DNA arrays Reveal cancer in its many forms ("DNA Arrays reveal cancer in its many forms"), Science, 2000, Vol. 289, p.1670-1672]. These techniques primarily employ a stationary chip prepared to contain the desired receptor (one that interacts with the target substance or the molecular structure to be tested). Since the area of the receptor can be very small, it is possible to produce a chip for testing multiple analytes. Ideally, thousands of binding receptors are provided to achieve a complete assay. When this receptor is exposed to a biological sample, only a few may bind to a particular protein or pathogen. Ideally, such receptor sites are identified as quickly as possible.

  One such technique for screening multiple molecular structures is a so-called immunological compact disc that simply contains antibody microarrays. [See, for example, the description of the paper below. Ekins, R., F. Chu, and E. Biggart, “Development of microspot multi-analyte ratiometric immunoassay using dual fluorescent-labeled antibodies. ")", Anal. Chim. Acta, 1989, Vol. 227, p.73-96; Ekins, R. and FW Chu, "Multi-analytical microspot immunoassay-" Compact Disc "for future microanalysis (" Multianalyte microspot immunoassay-Microanalytical “compact Disk” of the future ”)”, Clin. Chem., 1991, Vol. 37 (11), p.1955-1967; Ekins, R., “Ligand assay: downsizing from electrophoresis “Ligand assays: from electrophoresis to miniaturized microarrays” ”, Clin. Chem., 1998, Vol. 44 (9), p. 2015-2030]. Conventional fluorescence detection is used to sense the presence of the molecular structure under test in a microarray. Another method for addressing immunological assays uses a conventional Mach-Zehnder interferometer, including waveguides and grating couplers. [See, for example, the description of the paper below. Gao, H., et al., “Immunosensing with photo-immobilized immunoreagents on planar optical wave guides”, Biosensors and Bioelectronics, 1995 , Vol. 10, p.317-328; Maisenholder, B., et al., “A GaAs / AlGaAs-based refractometer platform for integrated optical sensing for integrated optical sensing applications. applications ”), Sensors and Actuators B, 1997, Vol. 38-39, p.324-329; Kunz, RE,“ Miniature integrated optical modules for chemical and biochemical sensing ” chemical and biochemical sensing ”)”, Sensors and Actuators B, 1997, Vol. 38-39, p.13-28; Duebendorfer, J. and RE Kunz, “Reference pads for miniature optical sensors” integrated optical sensors ")", Sensors and Actuators B, 1997, Vol. 38-39, p.116-121; Brecht, A. and G. Gauglitz, “Recent developments in optical transducers for chemical or biochemical applications”, Sensors and Actuators B, 1997, Vol. 38-39 , p.1-7]. Interferometric optical biosensors have the inherent advantage of interferometer sensitivity, but often have the characteristics of a large surface area per element, a long distance required for interaction, or a complex resonant structure. Optical biosensors with interferometers can also be sensitive to phase drift due to thermal and mechanical effects.

  Although the aforementioned techniques have proven useful for creating and reading assay information in the chemical, biological, medical, and diagnostic practice industries, existing planar arrays It is desirable to develop an improved manufacturing and reading technique for planar arrays with significantly improved performance.

In one aspect of the present invention, there is an unlabeled quadrature phase difference interferometry that detects the presence or absence of a target analyte in a biological sample. The method includes exposing the reflective surface of the substrate to a biological sample. The reflective surface has a spatial pattern consisting of a coating of receptor molecules. Each coating is specific for a particular target analyte. The method further includes using a split photodetector to measure the intensity of the far field diffraction pattern of the reflected signal. The reflected signal is obtained from an imaging probe laser beam having a wavelength λ that is incident with a waist w _{0 on} the spatial pattern of the receptor molecular coating. The reflected signal can also be obtained from scanning at least a portion of the substrate. The method further comprises measuring the intensity of at least a portion of the reflected signal in substantially orthogonal conditions by measuring the intensity of at least one of the two viewing angles with a split photodetector. Including. These two observation angles are substantially equal to a pair of orthogonal angles. The orthogonal angle θq is given by
θ _q = sin ^-1 (λ / 2w ₀ )
Defined by

  In one modification in one aspect of the invention, the intensity measurement of the far field diffraction pattern of the reflected beam is performed in the Fourier plane.

  In another modification in one aspect of the invention, the method further includes inverting the output of the split photodetector at one of the pair of orthogonal angles and splitting the inverted output at the other of the pair of orthogonal angles. Adding with the output of the photodetector.

  In another modification in one aspect of the invention, the method further includes passing the reflected signal through an objective lens prior to measuring the intensity using a split photodetector.

  In another modification in one aspect of the invention, the substrate is a disk and scanning of the substrate is performed by rotating the disk.

In another aspect of the invention, there is orthogonal interferometry for determining the presence or absence of a target analyte in a sample. The method includes using a laser beam having a wavelength λ and a waist w ₀ to probe at least a portion of the substrate. A portion of the substrate has a reflective surface that is exposed to the sample. The reflective surface includes a first region having a layer of recognition molecules specific to the target analyte and a second region having no layer of recognition molecules specific to the target analyte. The method further detects the time-dependent intensity of only the first orthogonal portion of the pair of orthogonal angles θq of the reflected diffraction signal of the probe beam while searching the first region and the second region. Including measuring in a vessel.

  In one modification in one aspect of the invention, the time dependence is caused by the relative movement of the incident laser beam relative to the substrate.

  In another modification in one aspect of the invention, the substrate is a disk, and the relative movement of the disk with respect to the incident laser beam is generated by rotating the disk.

  In another modification in one aspect of the invention, the reflected diffraction signal of the laser beam is measured by a split photodetector configuration. The method further includes inverting the first output portion of the reflected signal corresponding to one of the pair of orthogonal angles. The inverted first output is added to the second output of the reflected signal corresponding to the other of the pair of orthogonal angles.

  In another modification in one aspect of the invention, the substrate is a disk and the reflected signal is passed through the objective lens prior to intensity measurement.

  In another modification in one aspect of the invention, the method further includes passing the reflected diffraction signal of the probe beam through a π / 2 phase mask prior to intensity measurement.

In another modification in one aspect of the invention, the reflective surface is substantially flat. The orthogonal angle is calculated from the ray perpendicular to the substrate:
θ _q = sin ^-1 (λ / 2w ₀ )
Defined by

In another modification in one aspect of the invention, the substrate is a disk, and the reflective surface of the disk includes a plurality of flat lands and a plurality of terraces. The plateau has a height h. The orthogonal angle is calculated from the ray perpendicular to the substrate:
θ _q = sin ^-1 [λ / 2 -4h) / w ₀ ]
Defined by

  In another aspect of the present invention, there is a step detection method using a quadrature interferometer for determining the presence or absence of a target analysis target in a sample. The method measures the time-dependent intensity of the far-field diffraction pattern of a reflected light signal obtained from a probe laser beam incident on a disk having a spatial pattern of recognition molecules using a split photodetector configuration. Including that. The method further includes adding contributions from the first orthogonal component and the opposing second orthogonal component of the obtained optical signal. The addition of the contribution is preceded by the inversion of the contribution of the first orthogonal part.

  In another modification in one aspect of the invention, the intensity is measured on the optical signal reflected from the reflective surface of the disk.

  In another modification in one aspect of the invention, the split photodetector configuration is a split ring photodetector.

  In another modification in one aspect of the invention, the split photodetector configuration is a quadrant photodetector.

In another modification in one aspect of the invention, the split photodetector configuration includes first and second photodetectors. The probe beam incident on the disk has a wavelength λ and a waist w ₀ . First and second photodetectors, essentially measuring the intensity in a pair of quadrature angle theta _q. The orthogonal angle is calculated from the ray perpendicular to the substrate:
θ _q = sin ^-1 (λ / 2w ₀ )
Defined by

  In another modification in one aspect of the invention, the time dependent intensity is measured by rotating the disk.

  In another modification in one aspect of the invention, the disc rotates at about 80 Hz.

  In another aspect of the present invention, there is a step detection method using quadrature phase difference interference for determining the presence or absence of a target analysis target in a sample. The method includes measuring a time-dependent difference of a first portion of a reflected optical signal that substantially consists of only a first orthogonal component at a substantially first orthogonal interference angle. The reflected light signal is obtained from tracing the laser beam across alternating regions of specific and non-specific antibodies in a planar array.

  In another modification in one aspect of the invention, the method further comprises a reflected light signal consisting essentially of only the second orthogonal component obtained from tracing the laser beam across alternating repeating regions of the planar array. Measuring the time dependent difference of the second part substantially at the second orthogonal interference angle.

  In another modification in one aspect of the invention, the method further includes inverting the first output of the first portion of the reflected light signal. The method further includes adding a second output of the second portion of the reflected light signal to the inverted first output.

In another aspect of the present invention, there is a step detection method using quadrature phase difference interference without adding a label, which does not depend on a scale, for determining the presence or absence of a target analysis target in a sample. The method includes using an imaging laser beam having an incident waist w ₀ and a wavelength λ to scan the disk. The disc has a spatially patterned layer containing receptor molecules specific for the target analyte. The layer has a substantially steep layer end. The method further includes detecting intensity changes in the far-field diffraction pattern by scanning a substantially steep layer end using a split photodetector configuration. The split photodetector configuration provides a far-field diffraction pattern output at substantially at least one of a pair of right angle interference angles defined by rays perpendicular to the disk.

In another aspect of the invention, there is quadrature phase difference interferometry for determining the presence or absence of a target analyte in a sample. The method includes measuring an output of a first photodetector that is axially aligned with an array of optical elements for receiving substantially only the first orthogonal portion of the reflected light signal. Only the substantial first orthogonal component is obtained from observing the reflected light signal substantially at the first orthogonal angle. The reflected light signal is obtained from a probe laser beam having a wavelength λ and a waist w ₀ incident on the planar array. A planar array has at least one plateau defined by a layer of receptor molecules specific for the target analyte. The orthogonal angle θq is calculated from the ray perpendicular to the planar array:
θ _q = sin ^-1 (λ / 2w ₀ )
Defined by

  In another modification in one aspect of the invention, the method further includes for substantially accepting only the second opposite orthogonal component resulting from observing the reflected light signal substantially at the second orthogonal angle. Measuring the output of the second photodetector, aligned with the optical element array.

  In another modification in one aspect of the invention, the first and second photodetectors measure the far field diffraction pattern of the reflected light signal in the Fourier plane.

  In another modification in one aspect of the invention, the method further comprises inverting the output of the first photodetector and adding the inverted output of the first photodetector to the output of the second photodetector. .

  In another modification in one aspect of the invention, the optical element array includes an objective lens.

In yet another aspect of the present invention, there is a kit for interference detection of the presence or absence of a target analyte in a biological sample. The kit includes a light source of an imaging laser beam having a wavelength λ and a waist w ₀ . The light source is aligned directly or indirectly so that the laser beam is incident on the substrate. The substrate has a reflective surface with a spatially patterned biolayer. The biolayer includes a plurality of coatings of receptor molecules. Each coating is configured to bind to a specific target analyte. The kit further includes a split photodetector for measuring the intensity of the far field diffraction pattern. The split photodetector is arranged to detect intensity only at an observation angle that is substantially equal to at least one of a pair of orthogonal angles. The orthogonal angle is calculated from the ray perpendicular to the substrate:
θ _q = sin ^-1 (λ / 2w ₀ )
Defined by

  In another modification in one aspect of the invention, the split photodetector includes an aperture for blocking portions of the far-field diffraction pattern except for orthogonal angles.

In another modification in one aspect of the invention, the reflective surface comprises a 10 layer dielectric stack of TiO ₂ / SiO _{2 that} acts as a laser mirror.

  In another modification in one aspect of the invention, the split photodetector is a quadrant photodetector and the reflective surface includes a quarter wave dielectric stack.

  In yet another aspect of the invention, there is an apparatus for phase difference quadrature interference detection that detects the presence or absence of a target analyte in a planar array. The apparatus includes a laser light source for generating a probe beam. The apparatus also includes a platform for receiving the planar array. The apparatus further includes a first optical element array for directing the probe beam substantially perpendicular to the platform. The apparatus also includes an objective lens having a first side and a second side and a focal length. The objective lens is separated from the platform on a first surface of the lens by a first distance approximately equal to the focal length. The apparatus further includes split photodetector means for measuring the first and second orthogonal components obtained by reflection of the probe beam.

  In another modification in one aspect of the invention, the planar array is a disk. The apparatus further includes a rotator for rotating the disk attached to the platform.

  In another modification in one aspect of the present invention, the measurement split light detection means generates a first output for the first orthogonal component in the signal and a second output for the second orthogonal component in the signal. Arranged is a quadrature photodetector.

  In another modification of one aspect of the invention, the apparatus further includes an inverting circuit attached to one of the first output and the second output, and the inverting circuit, the first output, and the second output. And an adder circuit attached to the other.

  In another modification in one aspect of the invention, the split light detection means for measurement is a first photodetector arranged to generate a first output for the first orthogonal component in the signal, and in the signal A second orthogonal photodetector arranged to generate a second output for the second orthogonal component.

  In another modification in one aspect of the present invention, the measurement split light detection means generates a first output for the first orthogonal component in the signal and a second output for the second orthogonal component in the signal. It is a split ring photodetector arranged.

  In another modification in one aspect of the invention, the measuring means is separated from the second side of the objective lens by a second distance approximately equal to the focal length.

In yet another aspect of the invention, there is a system for quadrature phase difference detection of the presence or absence of a target analyte in a sample, the sample comprising a reflective surface comprising a plurality of spatially patterned recognition molecule coatings. And at least one of the recognition molecules is specific for the target analyte. The apparatus includes a platform for receiving a disk and a rotator for rotating the disk. The apparatus also includes a light source for the imaging laser beam of wavelength λ. The light source is aligned directly or indirectly so that the laser is incident on the disk with a waist w ₀ . The apparatus further includes means for tracing the laser beam across a plurality of coatings of spatially patterned recognition molecules. The apparatus also includes a split photodetector for measuring the intensity substantially at the orthogonal interference angle θ _q of the pair of far field diffraction patterns. A far field diffraction pattern is obtained by tracing the laser beam across a planar array. The orthogonal interference angle θq is expressed by the following equation:
θ _q = sin ^-1 (λ / 2w ₀ )
Defined by

  In one modification in one aspect of the invention, the system further includes an objective lens disposed between the disk on the platform and the split light detection means for measuring the intensity.

  In another modification in one aspect of the present invention, the split light detection means for measuring the intensity includes a first output corresponding to the intensity at one of the pair of orthogonal interference angles θq, and the pair of orthogonal interference angles. It has a second output corresponding to the intensity at the other of θq.

  In another modification in one aspect of the invention, the system further includes an inverting circuit attached to one of the first output and the second output, and the inverting circuit and the first output and the second output. And an adder circuit attached to the other.

  In another modification in one aspect of the present invention, the divided light detection means for measurement is a quadrant light detector.

[Related applications]
This application is a US patent provisional application filed on February 1, 2005, entitled “Phase-Contrast Quadrature For Spinning Disk Interferometery And Immunological Assay”. Claim priority to 60 / 649,070. This application is also filed December 30, 2005, entitled “Phase-Contrast BioCD: High-Speed Immunoassays at Sub-Picogram Detection Levels”. We also claim priority to US Provisional Patent Application No. 60 / 755,177.

  This application is related to its parent application which became US Pat. No. 6,685,885 in addition to US patent application Ser. No. 10 / 726,772 filed Dec. 3, 2003. This application is also filed with US Patent Provisional Application No. 60 / 649,071, entitled “Laser Scanning Interferometric Assays”, [and filed on the same date as this application, and priority to this provisional application. US Patent Application No.? / ???, ???] entitled “Laser Scanning Interferometric Surface Metrology”, entitled “Multiplexed Laser Scanning Interferometric Biochip” U.S. Provisional Application No. 60 / 649,043, entitled “Multiplexed Laser-Scanning Interferometric Biochips and Biodisks”, [and the name filed on the same date as this application and claims priority to this provisional application. US Patent Application No.? / ???, ???] entitled “Multiplexed Biological Analyzer Planar Array Apparatus and Methods” and the name “For Molecular Recognition” To carrier sideband light US Provisional Application No. 60 / 648,724, entitled “Method for Conducting Carrier-Wave Side-Band Optical Assays for Molecular Recognition”, [and filed on the same date as this application, US Patent Application No.? / ???, entitled "Differentially Encoded Biological Analyzer Planar Array Apparatus and Methods", claiming priority over the provisional application. Also related to ???]. The above three US patent provisional applications were all filed on February 1, 2005.

  New workarounds different from those described in the background section are currently being developed or have already been developed. One other approach utilizes a biological, optical compact disc ("Bio-Optical CD" or "Bio-CD") that includes a CD player for biological CD scanning. This system allows the use of interferometric detection techniques to sense the presence of a particular analyte in a biological sample. As will be described below, such a bio-CD device is preferably used with an interferometer detection system operating in substantially orthogonal conditions.

  Proteomics [EF Petricoin, KC Zoon, EC Kohn, JC Barrett, and LA Lotta, “Clinical proteomics: Translating benchside promise into bedside reality”, Nature Reviews Drug Discovery, vol. 1, pp. 683-695, 2002], and increasing complexity of protein-protein interaction networks [P. Bork, LJ Jensen, C. von Mering, AK Ramani, I. Lee , and EM Marmotte, “Protein interaction network from yeast to human”, Current Opinion In Structural Biology, vol. 14, pp. 292-299, 2004; S.-H Yook, ZN Oltvai, and A.-L. Barabasi, “Functional and topological characterization of protein interaction networks”, Proteomics, vol. 4, pp. 828-942, 2004], a number of analyzes It has created demand for bio-chip to enable high-speed test for recognition of elephants molecule. An important example is a protein microarray for expression testing [PF Predki, “Functional protein microarrays: ripe for discovery”, Current Opinion In Chemical Biology, vol. 8, pp .8-13, 2004; B. Schweitzer, P. Predki, and M. Snyder, “Microarrays to characterize protein interactions on a whole-proteome scale” , Proteomics, vol. 3, pp.2190-2199, 2003], and antibody chips for diagnostic medicine [SR Lal, RI Christopherson, and CG dos Remedios, “Antibody array: just emerging” "Antibody arrays: an embryonic but rapidly growing technology" ", Drug Discovery Today, vol. 7, pp. S143-S149, 2002; YP Ding, LY Chen, W. Zhang, HJ Cao, SM Ni, MF Zhou, H. Liang, ZC Ling, YY Geng, and SQ Wang, "Studies on simultaneously detecting multiple antibodies in the serum using microarray" ", Progress in Biochemistry and Biophysics, vol. 29, pp. 640-644, 2002; W. Kusnezow and JD Hoheisel, “Antibody Microarrays: Promises and Problems”, Biotechniques, vol. 33, pp. S14-S23, 2002]. Interferometry has the advantage of producing a higher photon flux than conventional fluorescence detection, thus allowing shorter detection times and / or higher signal to noise ratios.

  One or more of the inventors have introduced a biological compact disc as a highly sensitive rotating disc interferometer that operates at high speed and is self-referencing [MM Varma, HD Inerowicz, FE Regnier, and DD Nolte, See "High-speed label free detection by spinning-disk micro-interferometery", Biosensors & Bioelectronics, vol. 19, pp. 1371-1376, 2004]. The self-referencing method is preferable for performing stable interference measurement on a mechanically rotating disk. In order to be highly sensitive to the optical path length, the relative phase between the signal beam and the reference beam is locked at substantially a right angle (π / 2 phase difference), preferably regardless of mechanical vibration or motion. One or more of the inventors previously defined two classes of orthogonal interferometer detection for bio-CDs. Micro-Diffraction Class (“MD-Class” [MM Varma, DD Nolte, HD Inerowicz, and FE Regnier, “Spinning-disk self-referencing interferometry of antigen-antibody recognition” ””, Optics Letters, vol. 29, pp. 950-952, 2004, see also US Pat. No. 6,685,885 issued to Nolte et al.], And adaptive optics class (“AO-class”) [See US Patent Application No. 10 / 726,772, filed December 3, 2003, entitled “Adaptive Interferometric Multi-Analyte High-Speed Biosensor”), This patent document is hereby incorporated by reference in its entirety.

  MD-class bio-CDs are locked in quadrature using a microstructure fabricated on the disc. This microstructure diffracts the imaging laser beam into the far field with a fixed relative phase. In one embodiment, preferably 1024 gold spokes are deposited on the reflective surface with a height of λ / 8, preferably for one disc, and the biomolecules are either gold spokes or flat ground. Immobilized. Since the phase difference is set by the local microstructure elevation, it is not affected by mechanical movement or vibration. Immobilized biomolecules change the relative phase when converted to far-field amplitude modulation.

  The AO class is fixed in quadrature, preferably in a photorefractive quantum well, by self-adaptive nonlinear optical mixing [DD Nolte, “Semi-insulating semiconductor heterostructures: optoelectronic properties and applications (“ Semi-insulating semiconductor heterostructures: Optoelectronic properties and applications ”), J. Appl. Phys., vol. 85, pp. 6259, 1999; DD Nolte and MR Melloch,“ Photorefractive Quantum Wells and Thin Films , ”), Photorefractive Effects and Materials, DD Nolte, Ed. Dordrecht: Kluver Academic Publishers, 1995”. Quadrature phase adaptively tracks the phase between the signal and reference [DD Nolte, T. Cubel, LJ Pyrak-Nolte, and MR Melloch, “Adaptive beam combining and interferometry with photorefractive quantum wells (“ Adaptive Beam Combining and Interferometry using Photorefractive Quantum Wells ”)”, J. Opt. Soc. Am. B, vol. 18, pp.192-205, 2001]. In one embodiment, the patterned protein structure is sent to a photorefractive quantum well (PRQW) device and modulates the optical phase of the probe beam that is mixed with the reference local oscillator beam by two-wave mixing. This two-wave mixing self-compensates for mechanical disturbances to maintain the orthogonal condition with a compensation rate higher than kHz. The phase modulation caused by the protein structure on the rotating disk has a frequency higher than the compensation rate and is read by the photodetector.

  These bio-CD orthogonal classes balance the complexity between near and far fields. MD class bio CDs appear to require more complex microstructures on the disk, whereas AO class disks require holographic film for non-linear optical mixing. The present invention introduces a new orthogonal class that approximates the phase contrast imaging method. For this reason, various embodiments of the present invention are often referred to herein as "phase difference classes (" PC classes ").

  Before describing the various embodiments of the PC class, the intended orthogonal meaning in the interferometer detection system (s) of the present invention will be further described. In one specific application, orthogonal is an interferometer system where a common optical “mode” is divided into at least two “scattering” modes that differ by about N * π / 2 (where N is an odd number) in phase. It is possible to think in a limited way that happens. However, as used in the present invention (and previously mentioned issued patents and / or pending applications such as Nolte et al.), The interferometer system has at least one mode of “interacting” with the target molecule and others Suppose that at least one of the modes has no interaction and these modes are in quadrature when they differ in phase by approximately N * π / 2 (where N is an odd number). This quadrature definition can also be applied to interferometer systems where “other mode (s)” interact with another molecule. This interferometer system is considered to be substantially in an orthogonal condition if the phase difference is π / 2 (or N * π / 2, where N is an odd number) plus or minus about 20%.

  Furthermore, before describing the various embodiments of the PC-class, the intended meaning of “end” or “end detection” in orthogonal interferometer detection of the present invention will be further described. Various portions of the description of one or more embodiments below will refer to the end that diffracts light. For those skilled in the art, in all embodiments disclosed herein, a description of a step or end that diffracts light is actually integrated over the entire optical wavefront. It will be understood that it refers to the fact that Strictly speaking, it is not just the ends that diffract light. What is diffracted and detected in the far field is a discontinuity or step that is integrated over the entire beam. Step discontinuities with different heights impose different conditions on the left and right of the wave. It is this integrated difference that is detected as diffraction, not simply a step or end. Furthermore, in the context of this application, the term “end” or “end detection” is generally intended to cover the differential detection techniques disclosed in this application. That is, the detection of an orthogonal interferometer that detects the slope of the surface height, ie, the derivative. The signal is proportional to dh (x) / dx. Although the relatively common usage of this term appears to indicate that something is an “end-detection” process only in the special case of a discontinuous step, the term used herein is described in this paragraph. As was done, it is intended to be more broadly defined to include “tilt detection” across a step.

  The present invention relates generally to improved manufacture and reading of a rotating disk immunoassay (BioCD). In one embodiment, the quadrature phase difference interference measurement condition is preferably realized for the disc. The system preferably has a light detection efficiency of up to 100%. The system also preferably has automatic compensation for laser intensity drift.

  Various embodiments of the present invention generally relate to a method for converting spatial optical phase variation in a material to a time-dependent intensity using an orthogonal condition between a signal wave and a reference wave. In this case, the orthogonal condition is determined by diffraction from the index variation. The optical phase variation may be an intrinsic refractive index variation relative to the substrate material or may result from a material added to the substrate, such as immobilized proteins or nucleic acids. To detect phase modulation, Fourier filtering is used, consisting of a phase mask, an amplitude mask, or both a phase and amplitude mask. The mask is placed in the center of the Fourier plane or slope. This signal is detected in whole or in part by a photodetector with a detection aperture or by a split detector configuration that sums the contributions from the opposing orthogonal components. Time dependence is caused by the relative movement of the probe laser spot relative to the material, or vice versa.

  As described below, one or more embodiments of the present invention include an optical element array using one or more lenses. A special surface is defined for any optical system that uses a lens. As such, the objective surface (the surface on which the object is placed), the lens surface (the surface on which the lens is placed), the imaging surface (the surface on which imaging is placed), and the detection surface (light is detected) Surface). When there are a plurality of lenses, a plurality of imaging planes may be generated. In special optical systems where the object plane is one focal distance away from the lens surface, the Fourier plane is defined at a focal distance from the lens opposite the object plane. The aperture and mask may be placed on the Fourier plane, the imaging plane, the lens plane (immediately before or after the lens), or the detection plane. These openings and masks can adjust the amount of transmitted light (amplitude mask) or the phase of light (phase mask). The purpose of the mask in the different planes is to create the strongest constructive interference and thus a signal at the detector. For the selection of possible mask patterns and positions for adjusting the intensity detected by a single detector or detector array, a varied set of parameters is within the scope of the present invention. Is considered. In this way, the signal is optimized and maximized by proper selection of mask and location.

  In order to measure the intensity of the reflected light signal (including but not limited to far-field diffraction patterns) of the reflected light signal at two orthogonal angles, at least one, perhaps both (opposing) orthogonal conditions It should be understood that various split detector configurations and other means are possible. Such split detector configurations or means for measurement are considered to be within the scope of the present invention. In one embodiment, the split detector configuration may be a split ring photodetector. As described in further detail below, in one embodiment, a split photodetector is used in combination with an inverting and adding circuit. Alternatively, two separate photodetectors are used, each arranged to receive opposite quadrature signals. In the above and other variations described herein, the means for measuring the intensity includes apertures, various phase and / or amplitude masks, and other components known to those skilled in the art, It should be understood that it may be part of a large optical element array. Another possible modification uses a single photodetector. In doing so, the signal encounters an aperture and / or knife edge that shields at least a portion of the photodetector, so that the photodetector accepts a signal that includes substantially only a single orthogonal component. The split detector configuration may also be a quadrature photodetector. Other variants known to those skilled in the art are also considered to be within the scope of the present invention. For example, it should be noted that a split detector or quadrature detector is only an example of a detector array that has a large number of detector elements and can grow very large.

  In at least some embodiments of the invention, the role of the phase mask in the optical system is replaced by a split photodetector with a differential electronic output. The introduction of such an embodiment is believed to be at least partially advantageous in both near and far field. In the near field (compared to the MD class), it is preferable that no fine structure of the disc is required. In the far field (compared to the AO class), less complex detection is preferably used. The coping method is still preferably self-referential (providing at least some stability against mechanical motion). Furthermore, various embodiments of the present invention are locked in quadrature by step diffraction, for example, by spatially varying the immobilized protein pattern on the disk.

  With reference to FIGS. 1-4, some of the aspects of one or more embodiments of the present invention relating to step diffraction quadrature will now be described. FIG. 1 illustrates one aspect of at least some embodiments of the present invention that utilizes step diffraction and orthogonal angles.

  Referring to FIG. 1, in one embodiment of the present invention, the substrate 200 includes a plurality of flat 215 and a plateau 225, and preferably has a terminal end 205 at the boundary between the flat 215 and the plateau 225. Light ray 210 is incident on flat ground 215 and light ray 220 is incident on plateau 225. Plateau 225 includes a height h defined by plateau 225 (reference number 230 in FIG. 1) that includes the added thickness of a single layer of receptor molecule 250 in addition to the thickness between bottom surface 226 and top surface 228. have. The height derivative δh is caused by the added thickness of the target molecule 260 (target analyte / molecule includes but is not limited to protein). Other embodiments, such as embodiments in which the acceptor molecule 250 is bound to one intermediate layer rather than directly to the flat 215 of the reflective substrate 200 or the plateau 225 of the reflective substrate 200, are described in the present invention. Must be understood to be considered within the scope of

As shown in FIG. 1, the end 205 diffracts light and establishes an orthogonal condition between a light beam 210 incident on one side of the step and a light beam 220 incident on the other side of the step. For example, in a protein or other target analyte immobilized at height 230 (referred to as height h in the equations below), the orthogonal angle θq is for an incident laser beam with wavelength λ and beam width w ₀ The following formula:
0.5 * w ₀ * sinθ _q + 2h = λ / 4
Given by. Therefore, it is as the following formula.
θ _q = arcsin [(λ / 2-4h) / w _o ]

  At the orthogonal angle θq, the reflected light 222 from the plateau and the reflected light 212 from the flat ground have a relative phase difference of π / 2, that is, a quadrature phase. In the far field, the intensity at the orthogonal angle θq is equal to one half. The intensity at the orthogonal angle θq is linearly sensitive to the presence of the target molecule 260 (including but not limited to proteins or other biomolecules) on the plateau 225. One skilled in the art will understand that another equivalent description is that a discontinuous change in the optical phase will cause the reflected beam to be displaced laterally or undergo angular displacement at the detector. .

  Referring to FIG. 2, a “plateau” can have a height h (reference number 330 in FIG. 2) set by the receptor layer 350 itself (in other words, the height h is a biolayer or It should be understood that it is as small as the thickness of the receptor molecule 350). In this embodiment of the present invention, substrate 300 preferably includes a plurality of flat 315 and plateau 325, and has a terminal 305 at the boundary between flat 315 and plateau 325. The light ray 310 is incident on the flat ground 315 and the light ray 320 is incident on the plateau 325. The plateau 325 has a height h (reference number 330 in FIG. 2) defined by the thickness of the receptor molecule 350 in one layer. The height derivative δh is caused by the added thickness of the target molecule 360 that preferably binds to the receptor molecule 350 (examples of target analyte / molecule include protein, Not limited to that).

As shown in FIG. 2, end 305 diffracts light and establishes an orthogonal condition between ray 310 incident on one side of the end and ray 320 incident on the other side of the end. For example, in a protein or other target analyte that is immobilized at height 330 (referred to as height h in the equation below), the orthogonal angle θq is for an incident laser beam with wavelength λ and beam width w ₀ The following formula:
0.5 * w ₀ * sinθ _q = λ / 4
Given by. Therefore, it is as the following formula.
θ _q = arcsin [(λ / 2 * w _o )]
Again, as shown in FIG. 2, there is a linear sensitivity to the biomolecule at the orthogonal angle θq.

  If the protein is printed as a spoke pattern or as a square array element and the printed protein is scanned through a laser spot with a finite width, the orthogonality condition depends only on the size of the laser spot, It appears at a distinct angle that is substantially independent of the thickness of the protein receptor layer. Referring to FIGS. 3A and 3B, a step (or tilt) detection phase difference detection aspect that defines an orthogonal angle in the diffractive far field is depicted. Generally speaking, if the step is illuminated with a finite beam size, some of the scattered waves from the protein and bare substrate (or adjacent protein) will have a π / 2 phase difference between the partial waves. , Θq is an in-phase orthogonal component at an orthogonal angle set by θq.

FIG. 3A shows that a finite beam 440 having intensity I ₀ , wavelength λ, and width w ₀ is the end 405 of a protein 450 (preferably a printed protein pattern) printed on a reflective substrate 400. Shows where the light is illuminated. The movement of the substrate 400 (preferably a rotating disk) is indicated by arrow 475. The reflected rays 412 and 422 are traced from the midpoint of each half of the beam. At a certain angle, orthogonal angle θq, both rays have a relative π / 2 phase shift. The orthogonal angle θq is as follows.
θ _q = arcsin [λ / (2 * w ₀ )]

Referring to FIG. 3B, it can be seen that there are two angles at which the reflected rays are orthogonal. One is + θq and one is -θq. FIG. 3B shows that a finite beam 540 having intensity I ₀ , wavelength λ, and width w _{0 is applied} to the end 505 of a protein 550 (preferably part of the printed protein pattern) printed on the reflective substrate 500. Shows where it is illuminated. The base of FIG. 3B shows a (Gaussian) drop in intensity across the beam waist width w ₀ . The movement of the substrate 500 (preferably a rotating disk) is indicated by arrows 575. The reflected rays 512 and 522 are traced from the midpoint of each half of the beam and trace the + θq orthogonal angle. At this time, the reflected rays have a relative π / 2 phase transition and orthogonal interference. The reflected rays 513 and 523 are traced from the midpoint of each half of the beam and trace a -θq orthogonal angle. At this time, the reflected rays have a relative −π / 2 phase transition and orthogonal interference.

  Referring now to FIG. 4, it should be understood that similar elements are labeled with the same reference numbers as previously used. The intensity change in the far field is plotted in the upper part of FIG. 4, showing an increase in one half of the beam and a decrease in the other half. As described above, the protein diffraction step has a double orthogonal angle in the far field. One produces a positive change in intensity and the other produces a negative change. During detection, one or the other is preferably detected. Alternatively, both are detected and one phase is inverted before adding both. Therefore, when the entire field of view is detected, if one orthogonal signal is not inverted, these two orthogonal components will cancel each other. However, if half of the far field is collected, for example by a knife edge, half of the possible signal is extracted. Further, if the split detector configuration is used with an inverter and summing circuit, a complete signal is obtained as described below in connection with FIGS. 5A and 5B.

  With reference to FIGS. 5A and 5B, two embodiments of an optical layout for step or tilt detection of a printed protein or other target molecule are depicted. It should be understood that similar elements are labeled with the same reference number. The substrate 600 is preferably a rotating disk that rotates around the rotating shaft 610. The optical axis 650 of the optical element array is movable radially inward or outward from the rotational axis 610 of the substrate 600, for example, to align with some specific target area. In this optical layout, an objective lens 620 having a focal length f is preferably used, which is inserted between the substrate 600 and the photodetector 630 (FIG. 5A) or 730 (FIG. 5B). In the illustrated embodiment, the objective lens 620 is separated from the disc 600 by a distance f equal to the focal length of the lens, and further from the photodetector 630 (FIG. 5A) or 730 (FIG. 5B) by a distance f. It is done. In the embodiment of FIGS. 5A and 5B, the photodetectors 630, 730 are preferably segmented photodetectors that are segmented as indicated by 635, 735, respectively. It should be understood that various other embodiments of the split photodetector configuration are possible within the scope of the present invention. The photodetector may be, for example, an orthogonal photodetector or a split ring photodetector. Similarly, as described above, in some embodiments of the present invention, only half of the far field is sampled by a knife edge or other similar mechanism and only half of the possible signal is extracted (half of that). Is essentially only one orthogonal part).

  As shown in FIGS. 5A and 5B, the photodetectors 630, 730 are centered in the Fourier plane parallel to the long axis of the spoke to collect differential signals between the two halves of the split photodetector. Placed. In the illustrated embodiment, the signal from one of the split photodetectors 630, 730 (substantially corresponding to one orthogonal component) is summed with another portion of the photodetector (corresponding to the other orthogonal component). Before being summed through circuit 660, it is preferably sent to inverting circuit 640 to generate output signal 680. The output signal 680 is a measurement of the far field intensity and varies as the probe laser beam scans over the substrate 600 (eg, the substrate 600 rotates). Alternatively, as described above, if possible, only half of the signal corresponding to one orthogonal component may be sampled (however, a relatively small amount of overlap between the two orthogonal components can be compared with the signal-to-noise ratio). Can be tolerated without causing any noticeable adverse effects).

  With reference to FIGS. 6A and 6B, the scale independent nature of the step or tilt detection of at least some embodiments of the present invention is shown. The ends have no inherent length scale. Thus, in the Fourier plane, the end openings also have no length scale. This has the advantage that the beam size (which can vary) and the filter size need not be matched. As shown in FIGS. 6A and 6B, reducing the size of the laser spot (810a, 820a, 830a) simply increases the spot size (810b, 820b) in the split photodetector configuration 850, which is preferably placed in the Fourier plane. 830b). That is, changing the size of the laser spot does not require a change in the form of the split detector. Thus, the system is independent of the spot size or spoke width scale as long as the spoke width is greater than the beam diameter.

  With reference to FIGS. 7A and 7B, yet another aspect applicable to one or more embodiments of the present invention is depicted. As shown briefly in FIG. 7A, it is preferable to perform sideband detection without a carrier wave. Laser beam 1050 traces over the alternating spokes of specific antibody 1000 and non-specific antibody 1010 along the direction indicated by arrow 1075. The peak height at the ends of spokes 1000, 1010 is simply related to the difference between specific binding and non-specific binding. Therefore, there is no “another” measurement for non-specific binding to the substrate. Referring to FIG. 7B, a direct difference and time trace of non-specific binding is depicted. The signal height depends only on the difference between specific and non-specific binding. There is no carrier frequency and all detected intensity changes are obtained in the envelope. For details, filed February 1, 2005, entitled “Method for Conducting Carrier-Wave Side-Band Optical Assays for Molecular Recognition”. See US Provisional Patent Application No. 60 / 648,724. Furthermore, the name “Differentially Encoded Biological Analyzer Planar Array Apparatus and Methods” filed on the same date as this application and claims priority to this provisional application. U.S. Patent Application No ./?, ???

  Referring to FIG. 8, data obtained from an experimental display relating to knife edge detection of a spoke pattern on a calibration disk is shown. In one embodiment according to the present invention, experimental performance was displayed using a calibration disc that was etched to a depth of 12 nm in a glass disc. The entire field of view was detected in the top curve of FIG. 8, and the optimal signal condition was obtained at 1/2 power of the second curve from the bottom. In this transmission experiment, where the noise basis results from heterodyne mixing between the zero digit and the scattered light coming from the glass index variation, suppression of the zero digit beam further reduces the noise basis.

  Referring to FIG. 9, single spoke detection is shown. In particular, the detection of very thin spokes or spots 900 relative to beam 940 is shown. It should be understood that a spoke or spot having a size smaller than the beam width can establish orthogonal conditions in the far field. In the embodiment shown in FIG. 9, a single small spoke having a width a << w, where w is the width of the beam 940 (preferably a laser beam having a Gaussian distribution as shown). Alternatively, dots can also be detected. Small plateaus can be detected using the same split detection configuration as well as wide spokes. However, as shown in FIG. 10, when a π / 2 objective mask is used in combination with an aperture adjustment detector, a slightly higher performance is possible. As explained above, by selecting masks at various optical surfaces, it is possible to maximize structural interference at the detector. A π / 2 mask is very useful to convert the tilt and end in the disk and provide intensity modulation in the detector. In special cases, the mask can dispense with a split detector so that full intensity is detected in the far field. This method simplifies the detector but requires the insertion of a mask into the optical system.

  The biological compact disc (Bio CD) of the present invention and / or used with the present invention is a sensitive detection platform for detecting patterned biomolecules, eg, fixed on the surface of a rotating disc It is. Various embodiments of the present invention provide a rotating disk interferometer side that allows high-speed detection (10 microseconds per spot) with high reproducibility on a scale with optical path length changes of at least nanometers or less. As mentioned above, one important aspect in performing stable interference measurements on a mechanically rotating disk is the self-referencing method. That is, the phase of the signal beam and the reference beam is locked to a quadrature (π / 2 phase difference) regardless of mechanical vibration or relative motion.

  It should be understood that the phase difference class (PC class) of interferometer detection can be implemented in various ways known to those skilled in the art. In one embodiment of the invention, the protein is immobilized as a 1024 spoke pattern on the disk using photolithography (the number of spokes varies greatly with other design parameters, and is within the scope of the invention). Is considered within). The step of the printed protein diffracts the imaging laser beam, which is preferably split detector form in the Fourier plane, eg split photodetector, two separate photodetectors, quadrature photodetector And, more generally, detected by a detector, including but not limited to a photodetector array. The signal from the split detector configuration may be differentiated. This is effective in the electronic world as in the case of optical phase difference image recording.

  As detailed further below, the PC class potential in fast, label-free biosensing is demonstrated by two analyte immunoassays that adequately reject non-specific binding and low cross-reactivity between antibodies. The Immunoassays were performed on IgG immunoglobulins to detect bound analytes of 1 picogram or less. Experiments were also conducted on protein droplets to demonstrate the potential to scale to hundreds or thousands of analytes per disk.

によって与えられる角度で観察した場合、裸の表面に入射する光線と、バイオ層530を貫通する光線との間に存在する。 The reflective surface that supports the spatially patterned biolayer diffracts the imaging laser beam into an asymmetric far-field intensity pattern as shown in FIG. FIG. 4 shows an imaging Gaussian beam 540 with a waist w ₀ that is incident m on the substrate 500. As described above, the orthogonal condition is the following equation (1), that is,

When observed at an angle given by, there is between a light ray incident on the bare surface and a light ray penetrating the biolayer 530.

  The maximum change in intensity in the far field is caused by the sharp biolayer end and is observed at this orthogonal angle. There are two orthogonal interference angles with opposite signs at opposite diffraction angles. In order to obtain a protein signal, it is preferable to use a split detector including an inverting and summing circuit in the Fourier plane (examples are shown in FIGS. 5A and 5B. Other split detector configurations, eg, quadrature phase difference) Photodetectors are also considered to be within the scope of the present invention). In the case of a steep end, the signal is linearly proportional to the phase shift caused by the protein layer and thus linearly proportional to the protein height.

によって与えられる。上式において、K(θ)はフレネル係数であり、θは検出角度であり、E_inc(x)は入射ビームの照射野であり、

は、屈折率nおよび、速度νで動く変動タンパク高h(x)を持つバイオ層に対する相

を持つタンパクの位相関数である。我々の実験では、h(x)は、高さ8nmおよび屈折率1.33の、ほぼ矩形波である。式2の二乗係数から、別チャンネルにおいて分割光検出器によって検出される遠視野強度が得られる。得られた電子タンパク信号は、ビームプロフィールと、タンパク高分布dh(x)dxの第1導関数との重畳にほぼ比例する。従って、この技術は、傾斜検出技術である。タンパクパターンがさらに急峻な場合、より強力な信号が得られる。 The change induced in the far field by the patterned biolayer is the following equation (2):

Given by. In the above equation, K (θ) is the Fresnel coefficient, θ is the detection angle, E _inc (x) is the irradiation field of the incident beam,

Is the phase for a biolayer with a refractive index n and a variable protein height h (x) moving at a speed ν

It is a phase function of a protein with In our experiments, h (x) is a nearly square wave with a height of 8 nm and a refractive index of 1.33. From the square coefficient of Equation 2, the far field intensity detected by the split photodetector in another channel is obtained. The obtained electronic protein signal is approximately proportional to the superposition of the beam profile and the first derivative of the protein height distribution dh (x) dx. Therefore, this technique is a tilt detection technique. If the protein pattern is steeper, a stronger signal is obtained.

For an experimental discussion that follows the details of the apparatus and methods used to obtain the experimental data, a preferred introduction is discussed below. These detailed details are merely exemplary, and a wide range of modifications of the same are considered within the scope of the present invention. The phase difference bio CD is preferably manufactured from a 100 mm diameter, 1 mm thick borosilicate glass disk. The disc is preferably coated with a 10 layer dielectric stack consisting of Ti ₂ O ₅ / SiO ₂ , which becomes a laser mirror with a center wavelength at 633 nm. There are several different ways to immobilize proteins on the disk surface. The protein may be immobilized on the surface by physical adsorption. That is, 1) silanization of the silica surface, 2) biotin-avidin covalent bond for high affinity immobilization, or 3) covalent bond to the ATPES epoxide surface coating.

  The reflective surface of the bio-CD disc is preferably a multi-layer dielectric medium in order to enhance light reflection and maximize the surface electric field. The quarter wave stack is one of the most common dielectric structures used to achieve this condition, but an oxide layer on silicon, for example, can also be used.

  Silanization follows standard protocols using chlorooctadecyl silane treatment of silica surfaces. Proteins bind to organic end groups through hydrophobic interactions. Protein patterning is preferably achieved by gel stamping.

  In the high affinity biotin-avidin method, the surface is covered by a polysuccinimide polymer that is conjugated to biotin. Next, photographic engraving is applied. The photoresist is spun onto the polysuccinimide polymer coating, exposed, for example, through a 1024-spoke photomask and developed. The disk surface is then exposed to avidin, which adheres to biotin in the exposed areas. Next, a biotinylated antibody is added and attached to avidin that binds to the exposed areas.

  APTES epoxide coating follows standard protocols. Photo-etching is applied and the disc is treated with 1% sodium borohydride solution that etches the exposed disc surface. After removal of the photoresist, the disc has a patterned surface that covalently binds to the protein. Both physical adsorption and covalent binding to the surface yields a protein pattern with well-defined ends in the spoke pattern. The pattern is swept across the probe laser spot as the disk rotates.

  In one embodiment, the optical detection system preferably uses an objective lens with a focal length of 5 cm to image a 635 nm wavelength diode laser beam onto the disk as a diameter of about 20 microns. The reflected and scattered light is split by a beam splitter and directed to an orthogonal photodetector placed in the Fourier plane of the objective lens. The quadrature detector preferably has three output channels. That is, the overall intensity, the difference between the upper and lower halves, and the difference between the left and right. Depending on the direction of the protein spokes, one of the differential channels provides the desired phase signal. The other differential channel provides diagnostics for axial alignment and disc tremor while the sum channel provides small or negligible amplitude information related to Rayleigh and other scattering losses from the disc for uniform protein prints. give.

  The disc is preferably rotated at a steady frequency of 80 Hz by a stable rotator (Lincoln Laser, Inc.) to form a time-dependent phase and amplitude signal as the protein spoke passes through the imaging laser spot. . FIG. 11 shows the measurements of photo-engraved FITC-conjugated avidin (Sigma) and biotin-conjugated anti-rabbit IgG (Sigma) double protein layers when compared to computer simulations assuming protein spokes 8 nm high and 100 microns wide. Shows a time trace. FIG. 12 shows the power spectrum detected by the 3 kHz band. Noise from surface roughness distributed across multiple interfaces of the dielectric stack is 15 dB above the noise base of the detector and laser system. The protein signal of the protein spoke has a signal to noise ratio of 25 dB.

  To demonstrate the potential to perform PC-class bio-CD as an immunoassay, we performed specific antigen-antibody reactions in a two-component assay. Patterned polyacrylamide gel impregnated with fluorescein conjugated bovine serum albumin (FBSA) was contacted directly with a dielectric coated glass disk to print FBSA. For this experiment, the surface was activated with chlorodimethyloctadecylsilane. In the contact area between the polyacrylamide gel and the surface, the protein diffuses from the gel and is immobilized by physical adsorption. The quality of the printing technique was examined by fluorescein image recording and atomic force microscopy. BSA is relatively inert and becomes a universal carrier template on the disk surface, which can then be loaded (in the spoke region between BSA) with active or inactive proteins or other control molecules. .

  The disc is then refilled with specific antigen molecules. The disc on which BSA was printed was divided into four 90 degree quadrants. Each of the quadrants was filled with four different chemicals on the unprinted flat ground. 1) phosphate buffer, 2) rabbit IgG, 3) FBSA, and 4) horse IgG. The concentration of the refilled protein solution was 20 μg / ml. The prepared disc was then incubated in a band for specific recognition molecules. Crossing four quadrants, the three circular bands form a total of 12 virtual “wells” that serve as specific assays with multiple control assays. The inner band was incubated with anti-horse IgG and the outer band was incubated with anti-rabbit IgG. In both cases, the concentration was 20 μg / ml. Since the intermediate band was not incubated with the target sample and received the same wash as all bands, this provided a measure of assay stability and served as a negative control. Of the 12 wells, 4 are control wells for measuring drug withdrawal treatment, 4 are control wells for measuring non-specific binding of antibodies to BSA, and 2 are antibody-antigen crossings. The reactivity was examined and two were examined for specific antibody-antigen binding.

  The results of this assay are shown in FIGS. The distribution of height change in various segments after exposure to the antibody is shown in FIG. The two specific assays behave significantly different from each other, but include negative controls, nonspecific binding of antibodies to FBSA, and cross-reactivity (CR of anti-rabbit antibodies to horses and anti-rabbit antibodies to horses). ) Was not significantly different. This indicates that there is no significant cross-reactivity or non-specific binding in this experiment. The two specific two-component assays (anti-rabbit binds to rabbit and anti-horse binds to horse) gave 60% and 80% responses. Standard deviations were 20% and 30%, respectively. FIG. 14 shows the receptor operating characteristic (ROC) curve of this experiment. Non-specific binding segments are used as false positives. The curve shows a clear difference between specific and non-specific binding, with little difference between cross-reactivity and non-specific binding. The p-value was calculated to be less than 0.01 from the distribution.

  In order to test the variability of the optical detection method, before this experiment, remove the disc printed with FBSA by gel stamp printing based on physical surface adsorption and reattach it with a 90 degree total rotation When scanned again 4 times, all values were repeated within 5% standard error. If the disc is loaded again without rotation, the standard error drops to less than 2%. Thus, optical detection is stable and preferably repeatable with sufficiently small standard errors in future tests of dose-response if the fixation and incubation chemistry conditions are made more uniform.

  To demonstrate the potential to scale to hundreds or thousands of assays per disc, protein spot solution experiments were performed on ARTES epoxide coatings. After photo-etching, the disc was immersed in 1% sodium borohydride dissolved in deionized water for 12 hours. When the exposed surface was etched with a solution, it became extremely hydrophilic and no longer attracted protein. After removal of the photoresist, the coated portion of the disc retained an ARTES coating that binds strongly to the protein. Horse IgG and avian IgG regions were spotted on the disc. The area size was about 10 mm. The protein region was then incubated with a spot of anti-horse IgG of the same size. The distribution of protein height change in different droplets is shown in FIG. The difference between specific and non-specific binding can be clearly seen in this figure. The p-value was calculated to be less than 0.01. The droplet size in this experiment was 10 mm, but using an inkjet printer, the size can easily be reduced by 1 mm or even smaller, thus running thousands of or more assays per disk It is possible.

  Having described the results of some experimental data so far, further features or expected advantages of the invention, whether or not including such features, are based on the selected embodiment. explain. It will be appreciated that the present invention generally relates to a self-referencing interferometer optical biosensor that measures phase modulation obtained from proteins on a rotating disk. Optical detection of the pattern at high speed produces a noise basis that is much lower than 1 / f noise. The periodic protein pattern on the disc provides a spatial carrier that, when suitably demodulated, produces a slowly varying protein envelope that is distinguished with high accuracy.

  In one embodiment of the invention, two consecutive differential scans of the disc are distinguished without removing the disc at all. This results in a root mean surface height measurement error of only 20 pm, corresponding to 5 femtograms of protein within a 20 micron diameter focal spot. A simple area measurement relationship that predicts immunoassay performance as a function of well size is discussed below. Further discussed below is the demonstration of surface mass sensitivity of differential phase difference bio-CD, down to 0.2 pg / mm. The sensitivity of this bio-CD is comparable to that of a surface plasmon resonance sensor, but is preferably achieved without a resonant structure and is relatively simple to manufacture and operate.

  Using the above two paragraphs as an introduction to the use of differential phase difference detection in combination with a spatially patterned protein that provides a spatial carrier that produces a slowly varying protein envelope when frequency demodulated. Details will be discussed below. Detecting the pattern optically at high speed produces a noise basis that is much lower than 1 / f noise. A distinction is made between two consecutive differential scans of the disc, which results in only a mean square surface height measurement error of only 20pm, which corresponds to 5 femtograms of protein within a 20 micron diameter focal spot. Absent. A surface sensitivity of 0.2 pg / mm is suitably achieved if the scales are scaled appropriately.

  With respect to the above, the optical scanning system is preferably a differential phase difference system as described above. Such a system may include a stable motor (Lincoln Laser), a 635 nm laser source (Coherent), and a split quadrature detector where the output signal is distinguished between channels perpendicular to the disk surface motion. preferable. Thus, the net signal is proportional to the spatial first derivative of the height of the disk surface, or in the case of a disk bearing a patterned protein, assuming that the phase modulation is proportional to the surface density, the protein surface mass density derivative. Proportional to function.

In one embodiment of the present invention, the bio CD disc is a multilayer dielectric mirror having a center wavelength of 635 nm. The SiO ₂ top surface of the disk is patterned with avidin on a coating of biotinylated polysuccinimide polymer attached with silane to the silica surface. The avidin pattern includes a series of terraces with a spatial periodicity that varies linearly as a function of the radius of the disk. A typical period is about 150 microns. The disc is rotated at 5000 rpm and the laser is imaged into a 20 micron diameter spot. The detection frequency is typically 50 kHz, which is far from the 1 / f noise of lasers and electronic amplifiers. The optical performance is within 1.5 orders of magnitude of the shot noise limit. It should be understood that various operating parameters are contemplated as being within the scope of the present invention. The aforementioned spatial period, disk rotation speed, imaging laser spot diameter, and detection frequency are merely examples.

  Referring to FIG. 16, the surface relationship of protein spokes in selected portions of the disc is shown. The differential signal looks to the eye like a shaded 3D topology. The bright signal is from the leading protein step, while the dark negative signal is from the corresponding subsequent step. The spatial carrier frequency is demodulated to obtain the protein envelope function shown in FIG. The envelope varies slowly depending on the size of the probe laser beam. As mentioned above, in various embodiments of the present invention, it is preferred that the difference between two successive scans be quantified.

  A histogram of the data distinguished between two consecutive scans is shown in FIG. Without demodulation, the width of the height distribution is 75pm. After demodulation, the distribution is only 20pm. This is the average error in height measurement per spot area spotted. This is governed by the mechanical performance of the system (relocation error between scans) and is not limited by the stability of the laser, nor is it a fundamental limitation. This surface height uncertainty translates to surface mass sensitivity in differential phase difference bio-CDs.

The assumption of the basic measure provided in the following inference is the assumption of an uncorrelated random distribution of measurement errors in two consecutive scans for the bio-CD surface height distribution. Let me briefly touch on deviations from this condition, but this assumption is related to the normal conditions found in bio-CDs. Deviations from the uncorrelated and random surface roughness assumption lead to different scaling and therefore different mass per area values. For example, if the surface differential measurement error is spatially correlated, Smm will increase compared to the value derived below (current systems have a limit of about 10 pg / mm ^2). However, this number will be smaller on systems with improved stability.)

上式において、W_measは、測定された高さの差△h_measに関連するスポット直径である。△h_meas = 20 pmで、w₀ = 20ミクロンの場合、△h_mm = 0.4 pmが得られる。この表面高さの平均感度が、プロトンの半径よりも小さいことを注記するのは興味あることである。このタンパク高に関連する質量は、下記の式(4)によって与えられる。すなわち、

上式から、△h_mm = 0.4 pmの場合、△m_mm = 0.4 pgが得られる。 From FIG. 18, the surface height uncertainty between two repeated scans was determined to be 20 pm per focal spot. This corresponds to 5 femtograms of protein per focal spot with a diameter of 20 microns. The surface mass sensitivity associated with this measurement is 0.004 pg / (0.02 mm) ² = 10 pg / mm ² . However, to compare with other surface mass detection techniques, such as surface plasmon resonance, this number needs to be properly scaled to the corresponding size of 1 mm under uncorrelated and random surface roughness conditions. Yes, it reduces the standard error of the measurement by the square root of the sensor area. The equivalent scale surface sensitivity at a scale of 1 mm is given by the following equation (3). That is,

_Where W _meas is the spot diameter associated with the measured height difference _{Δh meas} . When _{Δh meas} = 20 pm and w ₀ = 20 microns, Δh _mm = 0.4 pm is obtained. It is interesting to note that the average sensitivity of this surface height is smaller than the proton radius. The mass associated with this protein height is given by equation (4) below. That is,

From the above equation, Δm _mm = 0.4 pg is obtained when Δh _mm = 0.4 pm.

上式から、感度が下記の式(6)のように求められる。すなわち、

これは、質量／長さの単位を持つ。 Combining the above equations (3) and (4) to obtain a general measure for surface mass sensitivity yields the following equation (5). That is,

From the above equation, the sensitivity is obtained as in the following equation (6). That is,

It has a mass / length unit.

In a single assay that distributes the sensor over area A, the minimum capture mass that can be detected in that assay is given by equation (7) below. That is,

この面積依存性感度は、SPRによって定められる最善値に匹敵する。この感度は、共鳴を要することなく獲得することが可能であり、従って、高感度を実現するのに共鳴に依存する他の干渉法または共鳴法よりも、はるかに安定で、製造も容易である。 As an example, if the assay area is 1 mm ² , the detected mass is 0.2 pg. From this we conclude that, when properly scaled, the surface mass sensitivity in 1 square millimeter is given by equation (8) below. That is,

This area-dependent sensitivity is comparable to the best value defined by SPR. This sensitivity can be obtained without the need for resonance and is therefore much more stable and easier to manufacture than other interferometry or resonance methods that rely on resonance to achieve high sensitivity. .

The square root scale setting in Equation (7) above is the result of averaging the signal over a relatively large area. This provides a powerful advantage over measurements in bio CD. The sensitivity per focal spot obtained by interferometer sensitivity is increased by averaging the signal over an area several times that of a single focal spot. For example, when a 20 micron diameter spot enters an area of 1 mm ² , a factor of 50 is obtained. This square root is a factor of 7, or an improvement of almost an order of magnitude.

  The performance of the PC class bio CD comes between the aforementioned MD class and AO class performance by presenting a useful compromise. The phase difference signal is significantly stronger than the MD class and the immobilization is more uniform on the silica surface. On the other hand, the PC-class detects the derivative h '(x) of the protein pattern on the disc, whereas the AO-class reacts directly to the protein profile h (x). The AO class method is also relatively stable because of its active adaptability, but is relatively difficult to implement. As an example, photodetectors 444A and 444B are depicted in connection with the AO class related disclosure found in US Patent Application Publication No. 2004 / 0166593A1. However, in the AO class, the two beams originate from different spatial modes and are separated separately. The two beams also carry very different information. The “direct” beam is mostly responsible for amplitude information and the “diffracted” beam is responsible for phase information. Accordingly, the photodetectors 444A and 444B of FIG. 21 of US Patent Application Publication No. 2004 / 0166593A1 combine the phase and information rather than making them separate. On the other hand, in the PC class, it is preferable that there is only a single generated spatial mode that is asymmetrically scattered. When this scattered light is detected and distinguished in the split light detection mode of the present invention, only the phase information is collected. Amplitude information is obtained simply by detecting all the light.

  In summary, this novel bio-CD orthogonal class occupies a promising position in the promotion of diverse, multi-analyte assays, thanks to the advantages of the PC class, which is simple to manufacture and simple to detect. The PC class is preferably a class having a self-referential interferometer that is perpendicular to the free surface of the rotating disk and does not have a resonance structure. Preferably, the interferometer element has a surface area as small as the focal spot of the laser, the length of interaction is slightly the same as the thickness of the biolayer, and makes the structure difficult to manufacture. It does not depend on optical resonators at all.

  The foregoing detection system has been described and illustrated as detection system (s) for use with a bio-CD configured to utilize a reflected signal. It should be understood that the present invention also includes a detection system configured to be used with a bio-CD configured to generate a transmitted signal beam, which is considered within the scope of the present invention. Don't be.

  Although bio-CDs and related detection systems have been described for use in detecting the presence of blood proteins in biological samples, bio-CDs and related detection systems are also useful in other applications, such as water or other fluids. It should be understood that it may be used to analyze environmental samples, including samples.

  The system is receptive to various modifications and alternative forms, exemplary embodiments of which are illustrated through example drawings and have been described in detail herein. However, there is no intention to limit the system to the particular forms disclosed, but on the contrary, all modifications, equivalents, which are included within the spirit and scope of the system as defined by the appended claims. And it should be understood that it is intended to handle alternatives.

FIG. 1 shows one aspect of the present invention relating to an orthogonal condition of a light beam incident on one side of a step with respect to a light beam incident on the other side of the step in a substrate having a plateau and a flat surface. FIG. 2 is similar to the embodiment of FIG. 1 but shows an embodiment where the height of the “plateau” is as small as the thickness of the biolayer. FIG. 3A shows a quadrature that realizes step detection in the diffractive far field. FIG. 3B is a schematic diagram of the intensity distribution of the laser beam imaged on the protein level difference and the incident laser beam waist. At two orthogonal angles, the phase transition (+ π / 2 or −π / 2) caused by the protein layer defines a quadrature phase in the diffracted far-field light intensity. FIG. 4 illustrates one aspect of the present invention where the diffractive protein step has a double quadrature in the far field. FIGS. 5A and 5B show an embodiment of the optical layout in a split photodetector configuration to perform printed protein step detection with slight variations. 6A and 6B illustrate the scale-independent nature of step detection in one or more embodiments of the present invention. Figures 7A and 7B show the direct difference of non-specific binding and the resulting time trace. FIG. 8 shows data obtained from an experimental display for the knife edge detection of a spoke pattern on a calibration disk. FIG. 9 shows single spoke detection. FIG. 10 shows the detection of very thin spokes or spots versus beam size in the case of a split detector (dotted curve) and a π / 2 mask (solid line). FIG. 11 shows a measured time trace of protein spokes when compared to a computer simulation of 8 nm protein spokes. FIG. 12 shows the measured power spectrum of the protein signal. FIG. 13 shows the distribution of changes in protein height in different segments of the disc after incubation. FIG. 14 shows the receptor operating characteristics of the two component assay. FIG. 15 shows the distribution of protein height after incubation in different protein droplets. FIG. 16 shows the spatial topology of the printed avidin plateau on the bio CD. FIG. 17 shows a spatial frequency demodulated image of the disk of FIG. FIG. 18 shows a histogram of avidin differential height in bio CD obtained without removing the disc.

Claims (45)

A label-free quadrature interferometry method for detecting the presence or absence of a target analyte in a biological sample,
Exposing a reflective surface of a substrate to a biological sample, the reflective surface having a spatial pattern of coatings of receptor molecules, and each coating is specific to a particular label analyte A process,
While scanning at least a portion of the substrate, the intensity of the far-field diffraction pattern of the reflected signal obtained from the imaging probe laser beam with wavelength λ, incident at a waist w ₀ against the spatial pattern of the receptor molecule coating. Using a split photodetector to measure,
Including
The intensity is a portion of the reflected signal in substantially orthogonal conditions by measuring its intensity with a split photodetector for at least one of two viewing angles that is substantially equal to a pair of orthogonal angles. The orthogonal angle θq is determined by the following equation for a ray perpendicular to the substrate:
θ _q = sin ^-1 (λ / 2w ₀ )
Said method, characterized in that it is defined by   Further comprising the steps of inverting the output of the split photodetector at one of the pair of orthogonal angles, and adding the inverted output to the output of the split photodetector at the other of the pair of orthogonal angles. The method of claim 1.   The method of claim 2, further comprising the step of passing the reflected signal through an objective lens before measuring the intensity using a split photodetector.   Method according to claim 3, characterized in that the intensity measurement of the far-field diffraction pattern of the reflected beam is performed in the Fourier plane.   The method of claim 1, wherein the substrate is a disk and scanning of the substrate is performed by rotating the disk. An orthogonal interferometer side method for determining the presence or absence of a target analyte in a sample,
Using a laser beam having a wavelength λ and a waist w ₀ to probe at least a portion of a substrate having a reflective surface exposed to a sample, the reflective surface being at least specific to a target analyte Including a first region having a layer of a specific recognition molecule and a second region having no layer of a recognition molecule specific for a target analyte;
A step of measuring the time-dependent intensity at the photodetector for substantially only the first orthogonal portion of the pair of orthogonal angles θq of the reflected diffraction signal of the probe beam while searching the first region and the second region. ,
Including said method.   The method of claim 6, wherein the time dependence is caused by the relative movement of the incident laser beam relative to the substrate.   The method of claim 7, wherein the substrate is a disk and the relative movement of the disk relative to the incident laser beam is generated by rotating the disk.   The reflected diffraction signal of the laser beam is measured by a split photodetector configuration, inverting the first output portion of the reflected signal corresponding to one of the pair of orthogonal angles, and the inverted first output 9. The method of claim 8, further comprising adding to the second output of the reflected signal corresponding to the other of the orthogonal angles.   Method according to claim 9, characterized in that the substrate is a disc and the reflected diffraction signal is passed through the objective lens before the intensity measurement.   The method of claim 7, further comprising the step of passing the reflected diffraction signal of the probe beam through a π / 2 phase mask prior to intensity measurement. The reflection surface is substantially flat and the orthogonal angle is calculated from the ray perpendicular to the substrate:
θ _q = sin ^-1 (λ / 2w ₀ )
The method of claim 6, characterized by: The substrate is a disk, the reflective surface of the disk includes a plurality of flat surfaces and a plurality of plateaus, the plateau has a height h, and the orthogonal angle is calculated from the light ray perpendicular to the substrate as follows:
θ _q = sin ^-1 [λ / 2 -4h] / w ₀ ]
The method of claim 6, characterized by: A step detection method using phase difference orthogonal interference to determine the presence or absence of a target analysis target in a sample,
Measuring a time-dependent intensity of a far-field diffraction pattern of a reflected light signal obtained from a probe laser beam incident on a disk having a spatial pattern of recognition molecules using a split photodetector configuration; and
The step of adding the contribution from the first orthogonal component and the opposing second orthogonal component of the obtained optical signal, the addition of the contribution precedes the inversion of the contribution of the first orthogonal component,
Including said method.   15. The method of claim 14, wherein the intensity is measured for an optical signal reflected from the reflective surface of the disk.   The method of claim 15, wherein the split photodetector configuration is a split ring photodetector.   The method of claim 15, wherein the split photodetector configuration is a quadrant photodetector. The split photodetector configuration includes first and second photodetectors, the probe beam incident on the disk has a wavelength λ and a waist w ₀ , and the first and second photodetectors are substantially paired. Intensity is measured at an orthogonal angle θ _q of
θ _q = sin ^-1 (λ / 2w ₀ )
The method of claim 15, characterized by:   The method of claim 15, wherein the time-dependent intensity is measured by rotating the disk.   The method of claim 19, wherein the disk rotates at about 80 Hz. A step detection method using phase difference orthogonal interference for determining the presence or absence of a target analysis target in a sample,
Measuring a time-dependent difference of a first portion of the reflected light signal substantially consisting only of the first orthogonal component at a substantially first orthogonal interference angle, wherein the reflected light signal is specific to the planar array. A step resulting from tracing the laser beam across alternating regions of antibody and non-specific antibody;
Including said method.   The time-dependent difference of the second part of the reflected optical signal, which consists essentially of only the second orthogonal component, obtained by tracing the laser beam across the alternating repeating area of the planar array, is substantially the second orthogonal interference. 23. The method of claim 22, further comprising measuring at a corner. Inverting the first output of the first portion of the reflected light signal;
Adding the second output of the second portion of the reflected light signal to the inverted first output;
24. The method of claim 22, further comprising: A scale-independent, label-free, step detection method using quadrature phase difference interference to determine the presence or absence of a target analyte in a sample,
Using an imaging laser beam having an incident waist w ₀ and a wavelength λ to scan the disk, the disk containing spatially patterned containing receptor molecules specific for the target analyte A layer having a substantially steep layer end,
Detecting an intensity change in a far-field diffraction pattern caused by scanning a substantially sharp layer end using a split photodetector configuration, wherein the split photodetector configuration is perpendicular to the disk Providing a far-field diffraction pattern output at substantially at least one of a pair of right-angle interference angles defined by the rays;
Including said method. Orthogonal interferometry for determining the presence or absence of a target analyte in a sample,
Measuring the output of the first photodetector, axially aligned with the optical element array for receiving substantially only the first orthogonal component of the reflected light signal, wherein the first orthogonal component is the reflected light signal The reflected light signal is obtained from a probe laser beam having a wavelength λ and a waist w ₀ incident on the planar array, the planar array being directed to the target analyte. Having at least one plateau defined by a single layer of receptor molecules specific to
The orthogonal angle θq is calculated from the ray perpendicular to the planar array:
θ _q = sin ^-1 (λ / 2w ₀ )
Said method, characterized in that it is defined by   Measuring the output of the second photodetector, aligned to an optical element array for substantially receiving only the second opposite orthogonal component, wherein the second opposite orthogonal component substantially reflects the reflected light signal. 26. The method of claim 25, further comprising the step of obtaining from observation at a second orthogonal angle. Inverting the output of the first photodetector;
Adding the inverted output of the first photodetector with the output of the second photodetector;
27. The method of claim 26, further comprising:   27. The method of claim 26, wherein the optical element array includes an objective lens.   30. The method of claim 28, wherein the first and second photodetectors measure a far field diffraction pattern of the reflected light signal in a Fourier plane. A kit for interference detection of the presence or absence of a target analyte in a biological sample,
A light source of an imaging laser beam having a wavelength λ and a waist w _0, which is directly or indirectly aligned so that the laser beam is incident on the substrate;
A substrate having a reflective surface having a spatially patterned biolayer, wherein the biolayer includes a plurality of coatings of receptor molecules, each coating configured to bind to a specific target analyte. A substrate,
A split photodetector for measuring the intensity of a far-field diffraction pattern, arranged to detect intensity only at an observation angle substantially equal to at least one of a pair of orthogonal angles, From the light beam perpendicular to the substrate:
θ _q = sin ^-1 (λ / 2w ₀ )
A split photodetector as defined by said kit.   31. The kit of claim 30, wherein the split photodetector includes an aperture for blocking portions of the far-field diffraction pattern except for orthogonal angles. Reflective surface acts as a laser mirror, consisting of TiO ₂ / SiO _2, characterized in that it comprises a dielectric stack of 10 layers The apparatus of claim 30.   32. The apparatus of claim 30, wherein the split photodetector is a quadrant photodetector and the reflective surface comprises a quarter wave dielectric stack. An apparatus for phase difference quadrature interference detection that detects the presence or absence of a target analyte in a planar array,
A laser light source for generating a probe beam;
A platform for receiving a planar array;
A first optical element array for directing the exploration beam substantially perpendicular to the platform;
An objective lens having a first side and a second side, and a focal length, the objective lens being separated from the platform by a first distance approximately equal to the focal length on the first side of the lens;
A split photodetector means for measuring the first and second orthogonal components in the signal obtained from the reflection of the probe beam;
Including said device.   35. The apparatus of claim 34, wherein the planar array is a disk and the apparatus further comprises a rotator for rotating the disk attached to the platform.   The split light detection means for measurement is an orthogonal photodetector arranged to generate a first output for the first orthogonal component in the signal and a second output for the second orthogonal component in the signal. 35. The device of claim 34, characterized.   An inverter circuit attached to one of the first output and the second output; an inverter circuit; and an adder circuit attached to the other of the first output and the second output. The apparatus of claim 36.   A split light detection means for measurement generates a first output for generating a first output for the first orthogonal component in the signal, and generates a second output for the second orthogonal component in the signal 35. The apparatus of claim 34, wherein the apparatus is a second orthogonal photodetector arranged in the manner described above.   The split light detector for measurement is a split ring photodetector arranged to generate a first output for the first quadrature component in the signal and a second output for the second quadrature component in the signal. 36. The device of claim 35.   35. The apparatus of claim 34, wherein the measuring means is separated from the second side of the objective lens by a second distance substantially equal to the focal length. A system for phase difference orthogonal interference detection of the presence or absence of a target analyte in a sample, wherein the sample is exposed to a disk having a reflective surface comprising a plurality of coatings of spatially patterned recognition molecules, At least one of the recognition molecules is specific for the target analyte;
A platform for receiving discs,
A rotator for rotating the disk;
A light source for an imaging laser beam of wavelength λ, wherein the laser is directly or indirectly axially aligned to enter the disc with a waist w ₀ ;
Means for tracing the laser beam across a plurality of coatings of spatially patterned recognition molecules;
In effect, a split photodetector for measuring intensity at a quadrature interference angle θ _q of a pair of far-field diffraction patterns, the far-field diffraction pattern tracing a laser beam across a planar array And the orthogonal interference angle θq is given by:
θ _q = sin ^-1 (λ / 2w ₀ )
Split photodetector, defined by
Including the system.   42. The system of claim 41, further comprising an objective lens disposed between a disk on the platform and a split light detection means for measuring intensity.   The split light detection means for measuring the intensity has a first output corresponding to the intensity at one of the pair of orthogonal interference angles θq, and a second output corresponding to the intensity at the other of the pair of orthogonal interference angles θq, 42. The system of claim 41, comprising:   An inverter circuit attached to one of the first output and the second output; and an adder circuit attached to the inverter circuit and the other of the first output and the second output. 44. The system of claim 43.   44. The system of claim 43, wherein the measuring split light detection means is a quadrant light detector.

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