Abstract:
A planar array having plurality of biological recognition molecules including at least two types of biological recognition molecules distributed about a substrate is disclosed. A first type of biological recognition molecules is distributed according to a first frequency and a second type of biological recognition molecules is distributed according to a second frequency. Another planar array having a plurality of biological recognition molecules including at least two kinds of biological recognition molecules is disclosed. The recognition molecules are distributed about a substrate with first kind of biological recognition molecules distributed at a first height or depth relative to a surface of the substrate and a second kind of biological recognition molecules distributed at a second height or depth relative to the surface. An apparatus including a surface normal interferometry platform including a scanning pathway and a plurality of analyzer molecules adapted to detect the presence or absence of a plurality of target analytes is also disclosed. The plurality of analyzer molecules are distributed about the scanning pathway according to a multiplexing scheme. A method including multiplexing a plurality of kinds of capture molecules about a detection pathway is further disclosed. The method also includes contacting a biological sample to the array, detecting the presence or absence of binding of the plurality of kinds of capture molecules and a plurality of target analytes using interferometry.

Description:
CROSS REFERENCE 
     This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/649,043, entitled “MULTI-PLEXED LASER SCANNING INTERFEROMETRIC BIOCHIPS AND BIODISKS,” filed on Feb. 1, 2005 and the same is expressly incorporated herein by reference. 
    
    
     TECHNICAL FIELD 
     The present invention generally relates to a device for detecting the presence of specific biological material in a sample, and more particularly to a laser compact disc system for detecting the presence of biological pathogens and/or analyte molecules bound to target receptors on the disc by sensing changes in the optical characteristics of a probe beam reflected from the disc caused by the pathogens and/or analytes. 
     BACKGROUND 
     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 particular receptors. Biochemical technologies including gene chips, immunological chips, and DNA arrays for detecting gene expression patterns in cancer cells, exploit the interaction between these molecular structures and the receptors. [For examples see the descriptions in the following articles: Sanders, G. H. W. 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 biosensors 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 . Science, 2000, Vol. 289, p. 1670-1672]. These technologies generally employ a stationary chip prepared to include the desired receptors (those which interact with the target analyte or molecular structure under test). Since the receptor areas can be quite small, chips may be produced which test for a plurality of analytes. Ideally, many thousand binding receptors are provided to provide a complete assay. When the receptors are exposed to a biological sample, only a few may bind a specific protein or pathogen. Ideally, these receptor sites are identified in as short a time as possible. 
     One such technology for screening for a plurality of molecular structures is the so-called immunological compact disk, which simply includes an antibody microarray. [For examples see the descriptions in the following articles: Ekins, R., F. Chu, and E. Biggart,  Development of microspot multi - analyte ratiometric immunoassay using dual flourescent - labelled antibodies . Anal. Chim. Acta, 1989, Vol. 227, p. 73-96; Ekins, R. and F. W. Chu,  Multianalyte microspot immunoassay—Microanalytical “compact Disk” of the future . Clin. Chem., 1991, Vol. 37(11), p. 1955-1967; Ekins, R.,  Ligand assays: from electrophoresis to miniaturized microarrays . Clin. Chem., 1998, Vol. 44(9), p. 2015-2030]. Conventional fluorescence detection is employed to sense the presence in the microarray of the molecular structures under test. Other approaches to immunological assays employ traditional Mach-Zender interferometers that include waveguides and grating couplers. [For examples see the descriptions in the following articles: 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 applications . Sensors and Actuators B, 1997, Vol. 38-39, p. 324-329; Kunz, R. E.,  Miniature integrated optical modules for chemical and biochemical sensing . Sensors and Actuators B, 1997, Vol. 38-39, p. 13-28; Dübendorfer, J. and R. E. Kunz,  Reference pads for miniature 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]. 
     While the abovementioned techniques have proven useful for producing and reading assay information within the chemical, biological, medical and diagnostic application industries, developing improved fabrication and reading techniques for planar arrays with significant improvement in performance over existing planar array technology is desirable. 
     SUMMARY 
     One embodiment according to the present invention includes a planar array having plurality of biological recognition molecules including at least two types of biological recognition molecules distributed about a substrate. A first type of biological recognition molecules is distributed according to a first frequency and a second type of biological recognition molecules is distributed according to a second frequency. 
     Another embodiment according to the present invention includes a planar array having plurality of biological recognition molecules including at least two kinds of biological recognition molecules distributed about a substrate. A first kind of biological recognition molecules is distributed at a first height or depth relative to a surface of the substrate and a second kind of biological recognition molecules is distributed at a second height or depth relative to the surface. 
     A further embodiment according to the present invention includes an apparatus including a surface normal interferometry platform including a scanning pathway and a plurality of analyzer molecules adapted to detect the presence or absence of a plurality of target analytes. The plurality of analyzer molecules are distributed about the scanning pathway according to a multiplexing scheme. 
     Another embodiment of the present invention includes a method including multiplexing a plurality of kinds of capture molecules about a detection pathway, contacting a biological sample to the array, detecting the presence or absence of binding of the plurality of kinds of capture molecules and a plurality of target analytes using interferometry. 
     Additional embodiments, aspects, and advantages of the present invention will be apparent from the following description. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a top schematic view of a distribution of elements including multiple analyzer molecules according to one embodiment of the present invention. 
         FIG. 2  is a graph illustrating harmonic signals indicating the detection of analytes by the analyzer molecules of  FIG. 1 . 
         FIG. 3  is a top schematic view of a bio-CD according to one embodiment of the present invention. 
         FIG. 4  is a side sectional schematic view of a biosensor platform according to one embodiment of the present invention. 
         FIG. 5  is a side sectional schematic view of a biosensor platform according to one embodiment of the present invention. 
         FIG. 6  is a top schematic view of a distribution of elements including multiple analyzer molecules according to one embodiment of the present invention. 
         FIG. 7  is a view of orthogonal bit sequences relating to the distribution of elements of  FIG. 6 . 
     
    
    
     DETAILED DESCRIPTION 
     For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated device, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates. 
     With reference to  FIG. 1  there is shown a distribution of elements  100  according to one embodiment of the present invention. The distribution of elements  100  includes at least three types of analyzer molecules  110 ,  120  and  130  distributed about scanning pathway  104  at three frequencies. Analyzer molecules  110  are distributed at a frequency of every other element of distribution  100 . Analyzer molecules  120  are distributed at a frequency of every fourth element of distribution  100 . Analyzer molecules  130  are distributed at a frequency of every eighth element of distribution  100 . Distribution  100  also includes elements  150  which do not include analyzer molecules  110 ,  120 , or  130 . Additional types of analyzer molecules could be present at elements  150  and could, for example, be distributed at different frequencies such as every 16 elements, every 32 elements, or at other frequencies. Elements  150  could also not include analyzer molecules. As indicated by ellipses  106  and  108 , elements  100  and reading pathway  104  can extend beyond the segment illustrated in  FIG. 1  with the distribution of various analyzer molecules occurring at various frequencies as described above. 
     Scanning footprint  102  travels over the distribution of elements  100  along scanning pathway  104  in the direction indicated by arrow R. In a preferred embodiment of the present invention, the distribution of elements  100  can be about a bio-CD which is scanned with a laser beam to detect the presence or absence of analytes bound to the analyzer molecules. In one embodiment the bio-CD is preferably scanned using surface normal self referencing phase quadrature interferometry techniques. In this embodiment elements  100  might, for example, be microstructures such as radial spokes formed on the surface of the bio-CD, and analyzer molecules  110 ,  120 , and  130  may be immobilized, for example, as monolayers, fractional monolayers, partial monolayers, or near monolayers on surfaces of the microstructures. Furthermore, scanning pathway  104  can be one of multiple substantially concentric tracks which may be scanned with a laser, for example, using interferometry techniques such as phase quadrature interferometric detection techniques. Examples of phase quadrature interferometric techniques include the micro-diffraction quadrature class (“MD-class”) and adaptive optic quadrature class (“AO-class”) as described in U.S. application Ser. No. 10/726,772 filed on Dec. 3, 2003 entitled “Adaptive Interferometric Multi-Analyte High-Speed Biosensor” (published on Aug. 26, 2004 as U.S. Pub. No. 2004/0166593), the contents of which are incorporated herein by reference. Other examples of phase quadrature interferometric techniques include the phase-contrast quadrature class (“PC-class”) as described in U.S. Provisional Patent Application No. 60/649,070, filed Feb. 1, 2005, entitled “Phase-Contrast Quadrature For Spinning Disk Interferometry And Immunological Assay”, U.S. Provisional Patent Application No. 60/755,177, filed Dec. 30, 2005, entitled “Phase-Contrast BioCD: High-Speed Immunoassays at Sub-Picogram Detection Levels”, and U.S. application Ser. No. 11/345,462 being filed the same day as the present application that claims priority to these two provisional applications and entitled “Method And Apparatus For Phase Contrast Quadrature Interferometric Detection Of An Immunoassay”. The disclosures of the utility application entitled “Method And Apparatus For Phase Contrast Quadrature Interferometric Detection Of An Immunoassay” is incorporated herein by reference. In these embodiments, the scanning pathway is preferably a ring shaped track of a bio-CD. During scanning the bio-CD is rotated at a scanning rate, for example, 223 revolutions per minute, to successively pass elements through the footprint of the laser beam. Under these conditions, the spatial frequency of the distribution of elements  100  corresponds to a temporal frequency. 
     Distribution of elements  100  is one exemplary embodiment of frequency-domain multiplexing in which different analyzer molecules are distributed at different spatial frequencies. Frequency-domain multiplexing can be used to test for many different analytes along a scanning pathway without requiring spatial separation of analyzer molecules used to detect particular analytes into sectors. In embodiments utilizing a rotating disk as a detection platform, systematics that depend on theta, the angular position about the disk, are substantially averaged out using this technique. 
     In addition to the embodiments described above, a variety of additional exemplary embodiments are also contemplated. For example, elements and analytes might be distributed in a variety of shapes such as substantially circular, oval, ellipsoid, square, polygonal, or other shapes. A variety of scanning pathway configurations might be utilized, including substantially linear or circular pathways, open pathways, closed pathways, curvilinear pathways, or spiral pathways, for example. A variety of frequency distributions of analyzer molecules can be used in addition to or instead of those described above. A variety of spacing of elements or analyzer molecules may also be used, for example, successive elements or analyzer molecules may be adjacent or adjoining, or may be spaced at intervals differing from those shown in connection with  FIG. 1 , or may overlap or occupy the same position. A variety of substrates and platforms may also be used including disks or chips supporting planar arrays including, for example, those described herein. Additionally, the variations and additional embodiments described elsewhere herein may apply to the present embodiment. 
     With reference to  FIG. 2  there is shown graph  200  illustrating an example of detection of target analytes using the distribution of elements  100  illustrated and described in connection with  FIG. 1 . The x axis of graph  200  is frequency which increases from origin  206  in the direction indicated by x axis arrow  204 . The y axis of graph  200  is harmonic signal intensity which increases from origin  206  in the direction indicated by y axis arrow  202 . 
     As shown in  FIG. 2 , the results of a scanning of the distribution of elements  100  can be represented in the frequency domain. Harmonic signals  210 ,  220  and  230  indicate detection of binding of target analytes to analyzer molecules  110 ,  120 , and  130 , respectively. Signal  230  has center frequency  231 , signal  220  has center frequency  221 , and signal  210  has center frequency  211 . Center frequencies  231 ,  221 , and  211  correspond to the spatial frequencies of analyzer molecules  130 ,  120 , and  110 , respectively, and the rate of scanning. Since the spatial frequency of analyzer molecules  110  is 16 times that of analyzer molecules  130  and 4 times that of analyzer molecules  120 , the harmonic signal  210  has a center frequency  211  sixteen times that of the center frequency  231  of harmonic signal  231  and four times that of center frequency  221  of harmonic signal  220 . Detection of signals at a variety of different frequencies corresponding to spatial frequencies of analyzer molecules is possible. Furthermore, time domain detection techniques can be utilized. 
     With reference to  FIG. 3  there is shown a bio-CD  300  according to another embodiment of the present invention. Bio-CD  300  includes sectors  310 ,  320 ,  330 ,  340 ,  350 ,  360 ,  370 ,  380  and  390 . A substantially circular scanning pathway  302  is defined about the read surface of Bio-CD  300  and scanning footprint  304  follows pathway  302  when bio-CD  300  is rotated. Bio-CD  300  may also include multiple other scanning pathways substantially concentric with pathway  302  located inside the outer perimeter  306  of bio-CD  300 . Additionally, bio-CD  300  may include an aperture located at its center for receiving a device used to rotate bio-CD  300 . 
     Each of sectors  310 ,  320 ,  330 ,  340 ,  350 ,  360 ,  370 ,  380  and  390  could include a different type of analyzer molecule to test for a different analyte in a single sample, or could include the same type of analyzer molecule and test different samples for the same analyte. Combinations of these two examples are also possible, for example, the eight sectors shown in  FIG. 3  could be used to test two samples for four different analytes, four samples for two different analytes, two samples for one analyte and two samples for three analytes, one sample for one analyte another sample for two analytes and another sample for five analytes, or various other combinations using all or some of the sectors for various other combinations of analyzer molecule(s) and test sample(s). 
     In a preferred embodiment according to the present invention, a bio-CD including a plurality of tracks as reading pathways provides a platform that can be divided into sectors as described above and used in a surface normal self referencing phase quadrature interferometric detection system. One such embodiment preferably includes 1024 interferometric elements per track and is divided into sixteen sectors for receiving sixteen samples. The total number of assays in this case is sixteen times the number of tracks. At 1000 tracks this allows 16,000 assays. Another such preferred embodiment includes 8192 interferometric elements per track and is divided into 128 sectors for receiving 128 samples. The total number of assays in this case is 128 times the number of tracks. For 1000 tracks, this is 128,000 assays. Thus, very high numbers of assays may be conducted in the time required to scan a bio-CD, which can be as little as about twenty minutes or less. 
     With reference to  FIG. 4  there is shown a multi-layer biosensor platform  400  according to one embodiment of the present invention. Platform may be a disk or chip including, for example, those described herein. Platform  400  includes an upper surface  404  and scanning levels  410 ,  412 , and  414  which are positioned at different distances from surface  404 . Scanning levels  410 ,  412 , and  414  include interferometric elements  420 A,  420 B, and  420 C, respectively. For simplicity, only a few interferometric elements are labeled, but additional interferometric elements are present as indicated by ellipses  421 A,  421 B, and  421 C. Analyzer molecules may be provided on scanning surfaces of interferometric elements  420 A,  420 B, and  420 C, and may be exposed to test samples via microfluidic channels internal to platform  400 . Confocal microscope optics can be used to direct a laser beam  402  to scan interferometric elements  420 A,  420 B, and  420 C. As shown in  FIG. 4  laser beam  402  is scanning elements  420 B of level  412 . Thus, it is possible to isolate a signal from a specific scanning level of platform  400 . 
     In a preferred embodiment of the present invention platform  400  is a multi-level bio-CD. In this embodiment, the interferometric elements can be transparent or partially transparent elements, placed in a stack at different levels in a fashion similar to that of a high-density DVD. To focus at a different level, the disk is moved to or away from the objective, or else the optical train is moved toward or away from the disk or chip. 
     The embodiments described above in connection with  FIGS. 3 and 4  are examples of space domain multiplexing in which spatially separate segments of a biosensor platform carry different analytes. Additionally, the variations and additional embodiments described elsewhere herein may apply to the present embodiment. 
     With reference to  FIG. 5  there is shown a biosensor platform  500  according to one embodiment of the present invention. Platform  500  includes substrate  504  having upper surface  505 . Microstructures  510 ,  520  and  530  are disposed on surface  505  and have a variety of heights. Microstructures  510 ,  520  and  530  and are scanned by laser beam  502 . Laser beam  502  preferably includes a plurality of wavelengths of laser light represented by arrows LA, LB, and LC. The height of microstructure  510  shown by arrows HA is ⅛ wavelength LA, the height of microstructure  520  shown by arrows HB is ⅛ wavelength LB, and the height of microstructure  530  shown by arrows HC is ⅛ wavelength LC. Thus, microstructures  510 ,  520 , and  530  are individually tuned to produce a phase quadrature condition for the different wavelengths LA, LB, and LC, respectively. As described above, each of microstructures  510 ,  520 , and  530  could include a different analyzer molecule immobilized to its surface for detecting a different analyte. It is also contemplated that additional or fewer microstructure heights and/or light wavelengths could be used. 
     The embodiments shown and described above in connection with  FIG. 5  is one example of wavelength-domain multiplexing which includes having multiple types of microstructures on a single track that are individually tuned to different wavelengths. A variety of additional embodiments and variations are also contemplated. In a preferred embodiment according to the present invention biosensor platform  500  is a bio-CD and the scanning of elements is accomplished by rotating the bio-CD to pass the interferometric microstructures through the footprint of the laser beam. In this embodiment microstructures can be radial spokes formed at different heights. The heights can be selected so that a microstructure is in quadrature for one wavelength, but null for a different wavelength. Furthermore, the distribution of analyzer molecules about the microstructures can be according to frequency-domain multiplexing, space-domain multiplexing or code-domain multiplexing schemes including those described above and below. 
     In other embodiments according to the present invention different height microstructures are not required, rather a laser probe contains a continuum of wavelengths, and readout in the far field can use an imaging spectrometer to separate theta and wavelength, for example, or readout can use interferometry techniques. In one preferred embodiment according to the present invention a phase contrast bio-CD provides an interferometry platform including a multiplexed distribution of analyzer molecules without different height microstructures. This phase contrast platform can be scanned with a plurality of wavelengths to define quadrature conditions and quadrature angles. Different wavelengths interact differently with this phase contrast platform and can be separated during detection to extract independent information from the wavelengths, for example, using interferometry. Multiple discrete wavelengths can be utilized or one or more wavelength continuums can be utilized. Other exemplary platforms not including different height microstructures include surface plasmon resonance platforms including a multiplexed distribution of analyzer molecules. Scanning of such platforms with multiple wavelengths can be read out as angular shifts or as frequency shifts. 
     Wavelength-domain multiplexing techniques allow probing at many different wavelengths, bringing spectroscopic molecular specificity to bear on the laser-scanning interferometry. There can be significant information contained in the spectral absorption of biomolecules. This information can be used in detecting target analytes in a sample. Additionally, the variations and additional embodiments described elsewhere herein may apply to the present embodiment. 
     With reference to  FIG. 6  there is shown a distribution of elements  600  according to another embodiment of the present invention. The distribution of elements  600  includes three types of analyzer molecules  610 ,  620  and  630  distributed about scanning pathway  604  according to a pseudorandom sequence or a random sequence. As indicated by ellipses  606  and  608 , elements  600  and reading pathway  604  can extend beyond the segment illustrated in  FIG. 6  with the distribution of various analyzer molecules occurring pseudorandomly or randomly as described above. 
     Scanning footprint  602  travels over the distribution of elements  600  along scanning pathway  604  in the direction indicated by arrow RR. In a preferred embodiment of the present invention, the distribution of elements  600  can be on a bio-CD which is scanned with a laser beam to detect the presence or absence of analytes bound to the analyzer molecules preferably using surface normal self referencing phase quadrature interferometry techniques. In this embodiment elements  600  may be microstructures such as radial spokes formed on the surface of the bio-CD and analyzer molecules  610 ,  620 , and  630  may be immobilized as monolayers, fractional monolayers, partial monolayers, or near monolayers on surfaces of the microstructures. Alternatively, in this and other embodiments, the elements may be defined regions on a substrate without microstructures. Furthermore, scanning pathway  604  can be one of multiple substantially concentric tracks which may be scanned with a laser, for example, as described above. In this embodiment, the scanning pathway is preferably a substantially ring shaped track of a bio-CD. 
     Scanning of distribution of elements  600  can yield a multiplexed bit sequence which can be processed using known orthogonal bit sequence, for example with matched gating techniques, to produce bit sequences corresponding to each analyzer molecule.  FIG. 7  shows a group of mutually orthogonal bit sequences  702  corresponding to the occurrences of analyzer molecules  610 ,  620 , and  630  shown and described above in connection with  FIG. 6 . Orthogonal bit sequence  710  corresponds to the occurrences of molecules  610 , orthogonal bit sequence  720  corresponds to the occurrences of molecules  620 , and orthogonal bit sequence  730  corresponds to the occurrences of molecules  630 . 
     Distribution of elements  600  is one exemplary embodiment of code-division multiplexing in which different analyzer molecules are distributed according to a known pseudorandom or random sequence. A variety of additional exemplary embodiments are also contemplated. For example, greater numbers of analyzer molecules could be used. Additionally, the variations and additional embodiments described above and below may also apply to the present embodiment. 
     Various embodiments according to the present invention can include a variety of biosensor platforms including those described above. For example, these platforms include bio-CDs such as micro-diffraction bio-CDs, adaptive-optical bio-CDs, phase-contrast bio-CDs, and others. Details relating to these various classes of bio-CDs can be found, for example, in the aforementioned patent applications incorporated herein by reference. These platforms further include bio-chips, immunological chips, gene chips, DNA arrays, platforms used in connection with fluorescence assays and other platforms and substrates supporting planar arrays including analyzer molecules. 
     Various embodiments according to the present invention can include a variety of analyzer molecules useful in detecting the presence or absence of a variety of target analytes in a solution to be tested. For example, these analyzer molecules can include antibodies or immunoglobulins, antigens, DNA fragments, cDNA fragments, aptameres, peptides, proteins, and other molecules. Various embodiments according to the present invention can include combinations of one or more the foregoing and other types of analyzer molecules known to those of ordinary skill in the art arranged, for example, in a planar array. 
     Various embodiments according to the present invention can be used in connection with a variety of scanning and detection techniques. For example, such techniques include interferometry, including surface normal interferometry techniques, and preferably phase quadrature interferometry techniques where one detected optical mode differs in phase from another by about π/2 or an odd integer multiple thereof, and/or self referencing interferometry techniques where a reference wave is generated locally with respect to a signal wave so that the reference and signal waves experience common aberrations and path length changes and thus maintain a constant relative phase without the need for active stabilization of different light paths, florescence techniques and platforms, resonance techniques and platforms, and other techniques and platforms. 
     As used herein terms relating to properties such as geometries, shapes, sizes, physical configurations, speeds, rates, frequencies, periods, amplitudes, include properties that are substantially or about the same or equal to the properties described unless explicitly indicated to the contrary. 
     While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only preferred embodiments have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected.