Patent Publication Number: US-2009231980-A1

Title: Optical disc assemblies for performing assays

Description:
This application claims priority from U.S. Provisional Application Ser. No. 60/254,394, filed Dec. 8, 2000; U.S. Provisional Application Ser. No. 60/255,233, filed Dec. 12, 2000; U.S. Provisional Application Ser. No. 60/293,917, filed May 24, 2001; U.S. Provisional Application Ser. No. 60/294,051, filed May 29, 2001; U.S. Provisional Application Ser. No. 60/294,052, filed May 29, 2001; U.S. Provisional Application Ser. No. 60/303,437, filed Jul. 6, 2001; U.S. Provisional Application Ser. No. 60/306,226, filed Jul. 18, 2001; and U.S. Provisional Application Ser. No. 60/323,405, filed Sep. 19, 2001. 
    
    
     FIELD OF INVENTION 
     The present invention relates to optical discs and their use to perform assays. More particularly, this invention relates to optical discs and methods for using the optical discs to discriminably obtain signals generated by investigational features, including analyte-specific signal elements, that are disposed upon surfaces of optical disc inserts. 
     BACKGROUND OF THE INVENTION 
     Optical discs have been used to detect microscopic samples. The advantages of using optical discs for detection and characterization of microscopic structures are discussed in WO 96/09548 (Gordon) and commonly assigned U.S. application Ser. No. 09/421,870 (Worthington), the disclosures of which are incorporated herein by reference in their entirety. However, consumers may be reluctant to use optical discs because microscope slides, rather than optical discs, are the convertional choice for performing assays. 
     Therefore, there exists a need to make an optical disc assembly that is adapted to accommodate microscope slides or the like. Nonoperational features or investigational features and structures can be disposed on the microscope slides. A standard optical disc reader may be used to both track the optical disc assembly and acquire investigational signals generated by the investigational features. 
     SUMMARY OF THE INVENTION 
     Therefore, it is an object of the present invention to provide an optical disc assembly capable of receiving an insert upon which an analyte of interest may be disposed. The optical disc assembly can be read by an optical disc reader, and the analyte disposed on the insert can be detected by the optical disc reader. 
     In accordance with one aspect of the present invention, the optical disc assembly includes (1) a first layer containing optically readable structures which are trackable by an optical disc reader and which have encoded speed information enabling the optical disc reader to rotate the optical disc assembly at a speed that is determinable from the speed information; and (2) at least one insert having a surface capable of receiving an analyte which can be detected by the optical disc reader. The optical disc reader may be a CD reader or a DVD reader. 
     In one embodiment one surface of the first layer is impressed with the optically readable structures which are coated with a reflective layer. The insert may be laser-proximal to the first layer and capable of focusing the reading beam of the optical disc reader on the reflective layer. The analyte-bearing surface of the insert may face the first layer. The insert may also be laser-distal to the first layer, and the reflective layer is a semi-reflective layer which may have a reflectivity of between 18 and 30%. 
     In another embodiment, the optical disc assembly includes a second layer, and the insert and the reflective layer are located between the first layer and the second layer. The second layer may be located laser-proximal to the first layer and the insert and function like a lens to focus the reading beam of the optical disc reader on the reflective layer. The analyte-bearing surface of the insert may face either the first layer or the second layer. 
     In yet another embodiment the insert is replaceable, and contains glass or plastic. 
     In a preferred embodiment, the optical disc assembly includes at least one counterweight insert to balance the disc assembly against the analyte-bearing insert. 
     In another preferred embodiment, the optical disc assembly is a dual data layer disc, which includes two data layers. Each of the two data layers has a surface impressed with optically readable structures which may have encoded operational information or assay information and which may be coated with a reflective or semi-reflective layer, The analyte-bearing surface of the insert may face either of the two data layers. One of the two data layers may function as a lens to focus the reading beam of the optical disc reader on the optically readable structures contained in either of the two layers. 
     In accordance with another aspect of this invention, the insert is at least partially embedded in the operational layer of the disc assembly. The disc assembly may include another layer, functioning as a lens, to focus the reading beam on the operational structures. 
     In accordance with yet another aspect of this invention, the optical disc assembly includes a hologram containing optically readable structures which have encoded tracking information and speed information enabling the optical disc reader to rotate the optical disc assembly at a speed that is determinable from the speed information. This embodiment of the present invention further includes at least one insert having a surface capable of receiving an analyte which can be detected by the optical disc reader. The optical disc reader preferably is a CD reader or a DVD reader. The optical disc assembly may include a lens layer capable of focusing the reading beam of the optical disc reader on an image plane of the hologram. The analyte-bearing surface of the insert may be within or adjacent to the image plane of the hologram. The insert may be located between the hologram and a second layer in the optical disc assembly. One surface of the second layer may be impressed with optically readable structures which may have encoded operational information or assay information and which may be coated with a reflective or semi-reflective layer. The hologram may also have encoded assay information. 
     In one embodiment, the optical disc assembly includes the analyte which is disposed on the insert. The analyte may be a biological, chemical, or biochemical analyte. The analyte may be adjacent to the operational structures or other optically readable structures in the optical disc assembly. When a hologram is used, the analyte may be adjacent to the image plane of the hologram. Preferably, at least part of the analyte is within the image plane of the hologram. 
     In accordance with another aspect of this invention, the analyte held by the insert in the optical disc assembly is detected using a CD or DVD reader. The detection includes the steps of (1) introducing the optical disc assembly into a CD or DVD reader; (2) reading the optically disc assembly; and (3) obtaining a signal which is indicative of the presence of the analyte. 
     In accordance with yet another aspect of this invention, a kit is provided for detecting an analyte. The kit includes a disc including a first layer that contains optically readable structures which have encoded tracking information and speed information enabling an optical disc reader to rotate the disc at a speed that is determinable from the speed information. The kit according to this aspect of the present invention further includes an insert having a surface capable of receiving the analyte. This insert is capable of being coupled to the disc to enable the optical disc reader to detect the analyte held by the insert. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS  
       All the drawings used herein are given by way of illustration, not limitation. 
         FIG. 1  shows a typical optical pickup used by an optical disc reader. 
         FIG. 2  shows a side cross-sectional view of a CD-type disc. 
         FIG. 3  illustrates a perspective view of the operational surface of a CD-R disc, 
         FIG. 4  depicts the position of three reading beams, as used by a typical three-beam pickup, relative to the tracks on the operational surface of an optical disc. 
         FIG. 5  is an exemplary optical disc detector used by a three-beam optical pickup. 
         FIG. 6  shows an illustrative block diagram of a chip set used by a CD-type optical disc reader. 
         FIG. 7  shows an illustrative block diagram of a chip set used by a DVD-type optical disc reader. 
         FIG. 8  depicts investigational structures applied to the air-incident, laser-proximal surface of a typical single layer CD-type optical disc. 
         FIG. 9  demonstrates an inverted CD-type single layer optical disc, with laser incident from below. 
         FIG. 10  illustrates an inverted optical disc impressed with reverse image wobble grooves. 
         FIG. 11  depicts an optical disc assembly including an insert associated with analytes. 
         FIG. 12  is a side cross sectional view of an optical disc assembly including an insert and a nonintegral laser-proximal, laser-refractive cover. 
         FIG. 13  shows another optical disc assembly including a reflective hologram which has encoded operational features, wherein the encoded operational features appear to be located in the image plane of the hologram, and wherein analytes may be positioned within or adjacent to the image plane. 
         FIG. 14  is a top perspective view of a polycarbonate laser-refracting cover. 
         FIG. 15  shows a side cross-sectional view of a dual layer DVD-type disc. 
         FIG. 16  is an exploded side perspective view of a dual data layer optical disc assembly. 
         FIG. 17  depicts a side cross-sectional view of another dual data layer optical disc assembly including internal channels. 
         FIG. 18  shows a side cross-sectional view of yet another dual data layer optical disc assembly including assay-facilitating features. 
         FIG. 19  illustrates a side cross-sectional view of another dual data layer optical disc assembly according to one embodiment of the present invention. 
         FIG. 20  shows a dual data layer optical disc assembly, wherein one of the data layers is a hologram. 
         FIG. 21  presents illustrative shapes of suitable inserts in accordance with the present invention. 
         FIG. 22  depicts multiple ring-shaped inserts according to the present invention which can provide multiple sessions on a single disc. 
         FIG. 23  shows an arrangement of differently shaped inserts on an optical disc assembly implemented in accordance with the present invention. 
         FIG. 24  is an illustrative example for using a counterweight insert according to this invention. 
         FIG. 25  demonstrates an annular counterweight channel according to one embodiment of the present invention. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     As used herein, “operational structures” or “operational features” in an optical disc assembly refer to optically readable structures which are impressed or encoded in the optical disc assembly and which enable an optical disc reader to track, synchronize, or perform other customized operational functions. Operational structures may act as phase components or provide interference patterns. Operational structures may have encoded speed information that enables the optical disc reader to rotate the disc assembly at a speed determinable from the speed information. For instance, frame synchronization words or wobble grooves may be used to provide speed information. Light returned from or passed over operational structures can be acquired by the optical disc reader to generate operational signals. These operational signals are used by the optical disc reader to track, focus, synchronize, or perform other operational functions. 
     Operational structures may be imprinted or impressed in a surface of a layer in the disc assembly. Such a layer is referred to as an “operational layer,” and such a surface is referred to as an “operational surface.” In a typical CD disc, the operational layer is the polycarbonate disc, and the operational surface is the laser-distal surface of the polycarbonate disc. An optical disc assembly may have more than one operational layer or operational surface. Preferred operational structures include, but are not limited to, wobble grooves, pits and lands, dye marks, or any combination thereof. 
     According to the present invention, operational structures may be advantageously encoded in a hologram. Light returned from or transmitted through the hologram can create an image plane within which the encoded operational structures appear to be positioned. The encoded operational structures, as appeared in the image plane of the hologram, preferably are in the form of wobble grooves, tracks of pits and lands, or any other type of operational structures that may be physically impressed in an optical disc&#39;s operational layer. 
     Operational structures, impressed or encoded, may be in a variety of formats. Suitable formats for this invention include, but are not limited to, CD formats, DVD formats, any combination thereof; or other optical disc formats. CD formats include, but are not limited to, CD-ROM, CD-R, and CD-RW formats. DVD formats include, but are not limited to, DVD-R, DVD-RW, and DVD-RAM formats. As appreciated by those of skill in the art, other CD or DVD formats or other optical disc formats, including those that have been or will be developed in the future, may be used in the present invention. 
     As used herein, an “investigational feature” or an “investigational structure” refers to any feature or structure which is placed or disposed on or within an optical disc assembly to be examined. When read by an optical disc reader, an investigational feature or structure may be capable of producing a signal which is not required for the optical disc reader to operate the disc assembly. Investigational features or structures include analytes or analyte-specific signal elements. An “analyte-specific signal element” refers to any structure that may be used to signal the presence of a specific analyte that is disposed in an optical disc assembly. Analyte-specific signal elements include both those structures that are alone detectable by an optical disc reader and those that require additional components before becoming detectable. 
     In accordance with one aspect of this invention, the optical disc assembly is designed based on modification of standard optical discs, The optical disc assembly is read by an optical disc reader, which may use the optical pickup of a conventional optical disc reader, or an optical pickup modified therefrom. The optical disc reader may be a CD reader or a DVD reader. As used herein, CD readers include, but are not limited to, CD-ROM readers, CD Recordable (CD-R) readers, CD-Rewriteable (CD-RW) readers, or any reader capable of reading CD-format discs. Industry standard CD readers may be used in the present invention. A preferred CD reader for this invention is a CD-RW reader or a modification thereof. As used herein, DVD readers include, but are not limited to, DVD-R readers, DVD-RAM readers, DVD-RW readers, or any reader that can read DVD-format discs. Industry standard DVD readers may be used. As appreciated by one of skill in the art other CD readers, DVD readers or optical disc readers, including those that have been or will be developed in the future, may be used in the present invention. 
     Features of a conventional optical disc reader and an optical disc are described in  FIGS. 1 ,  2 , and  3 .  FIG. 1  depicts a conventional reader&#39;s optical pickup  810  which directs at least one reading beam to a CD-type optical disc  811 . The path of the reading beam is indicated by the dashed lines.  FIG. 2  provides an enlarged side cross-sectional view of disc  811 . With reference to  FIGS. 1 and 2 , the optical pickup  810  includes laser source  819 , lenses  812 ,  813 , and  814 , beam splitter  815 , quarter wave plate  820 , and detector  818 . Light source  819 , typically a laser diode, emits a light beam which passes through beam splitter  815  and then is collimated by lens  812 . Objective lens  813  focuses the laser beam onto a reflective layer in optical disc  811 . 
     The laser beam can be reflected from the reflective layer and returned through objective lens  813  and quarter wave plate  820  to beam splitter  815 . Quarter wave plate  820  changes the polarization of the laser beam so that beam splitter  815  can direct the reflected laser beam through lens  814 , which may focus the reflected laser beam onto detector  818 . Astigmatic element  816 , which may be a cylindrical lens, may be included between beam splitter  815  and detector  818  to introduce astigmatism in the reflected laser beam. 
     As shown in greater detail in  FIG. 2 , the CD-type disc  811  includes three layers. From laser-proximal to laser-distal, these three layers are transparent substrate  112 , reflective layer  114 , and protective layer  116 . As used herein, a layer in an optical disc assembly refers to a thickness of material. For instance, a layer may be a substrate disc or a coating of reflective material. A layer may also be an insert which can be assembled into another layer in the disc assembly. A layer may be flat or not flat. A layer may be homogeneous or non-homogenous. The depth of a layer may be uniform or not uniform. A layer may be an assembly of several parts. 
     The total depth of disc  811  is about 1.2 mm. The senior standard for compact discs, republished as IEC (International Electrotechnical Commission)  908 , colloquially the “Red Book,” permits a CD-type disc to have a physical thickness of between 1.1 and 1.5 mm. Discs with other thickness can also be used in this invention. 
     Although the disc shown in  FIG. 2  includes three physical layers, there is only a single data layer. Such a disc is thus referred to as a single data-layer (or “single layer”) disc. Such a disc may also be described as a “CD-type” disc. CD discs, including CD-ROM, CD-R, and CD-RW discs, are examples of CD-type discs. In comparison, a DVD disc includes two data layers, and therefore is referred to as a dual data layer, “multiple layer”, or “dual layer” disc. DVD discs include DVD-R, DVD-RW, and DVD-RAM disc. 
     Transparent substrate  112  makes up most of the 1.2 mm thickness of a typical CD-type disc, as measured along the optical axis. Transparent substrate  112  also provides both optical and structural features necessary for disc operation. 
     With respect to the optical features, the refractive properties of transparent substrate  112  serve to focus the incident laser light on reflective layer  114 . On the most laser-proximal surface of a CD-type disc, the laser spot has a diameter of approximately 800 μm. Transparent substrate  112  further focuses the beam, achieving a diameter of approximately 1.7 μm at reflective layer  114 . In design and manufacture of optical discs, the thickness and index of refraction of transparent substrate  112  are selected to achieve this focusing function 
     Transparent substrate  112  also provides the principal structural integrity of the disc. Reflective layer  114  is approximately 0.05 to 0.10 microns in thickness, and protective layer  116  typically includes a lacquer material that hardens when exposed to ultraviolet light, and has a thickness between 10 and 30 microns. Thus, transparent substrate  112  makes up the major layer, and is the only layer capable of imparting sufficient rigidity to the disc. 
     Substrate layer  112  is typically impressed with a spiral track that starts at the innermost readable portion of the disc and then spirals out to the outermost readable portion of the disc. In a non-recordable disc, this track is made up of a series of embossed pits, each having a depth of approximately ¼ of the wavelength of the light used to read the disc. The pits have varying lengths. The length and spacing of the pits encode data. As further discussed below, the spiral wobble groove of a recordable disc contains a dye rather than pits. As used herein, a spiral wobble groove can be considered as a series of wobble grooves connected consecutively to form a spiral track Two portions of such a spiral wobble groove  118  are shown in a perspective view in  FIG. 3 . Reference numeral  110  denotes the land of the wobble groove. 
     Transparent substrate  112  is typically manufactured by an injection molding process, in which molten polycarbonate is injected into a mold cavity having a “stamper.” The stamper has a reverse image of spiral groove  118 , and is made from a master by electroforming. The injection molding process typically takes 5 to 10 seconds per disc. 
     Reflective layer  114  is approximately 0.05 to 0.10 microns in thickness, and typically contains a reflective metallic material, such as aluminum, silver, gold, or copper. For the CD-R format, a reflection coefficient of approximately 65 percent is recommended in the official format specification, but few discs actually meet this level. Most drives have gain control circuitry, and are capable of reading discs having a much lower reflection coefficient. When the disc is being read, reflective layer  114  reflects the laser beam back to the sensors in the disc reader. 
     Reflective layer  114  may be prepared using a magnetron sputtering process, in which a solid target is bombarded with ions, releasing metal molecules that are used to form reflective layer  114 . The vapor deposition process is slow, and is generally only used for mastering discs. A chemical wet “silvering” process (using silver, nickel, or other metal) may also be used to form reflective layer  114  on transparent substrate  112 . 
     Protective layer  116  typically contains a lacquer material that hardens when exposed to ultraviolet light, a process called “curing.” Protective layer  116  has a thickness between 10 and 30 microns, Protective layer  116  serves to protect reflective layer  114  from scratches and oxidation, and provides a convenient surface on which a label may be printed. Protective layer  116  is typically applied to transparent substrate  112  and reflective layer  114  through a spin-coating process, whereby a small amount of a material that hardens when exposed to ultraviolet light is sprayed on the disc near the inner diameter of reflective layer  114 , and the disc then is spun at high speed, causing a thin layer of the material to cover the surface of the disc. The disc is subsequently exposed to ultraviolet light causing the material to harden. 
     The various CD and DVD standards contemplate discs having a nominal depth (in the dimension defined by the optical axis) of 1.2 mm and a nominal diameter in the radial dimension of 120 mm. The senior standard for compact disc technology (colloquially, the “Red Book”) permits physical thickness of between 1.1 and 1.5 mm (for all layers combined). Disc readers are capable of accommodating some additional variance, and discs suitable for CD and DVD drives may have a depth maximally of about 2.4 mm and minimally of about 0.3 mm, preferably from 1.0 to 1.4 mm, more preferably from 1.1 to 1.3 mm, and most preferably 1.2 mm. With respect to the nominal 120 mm diameter, disc readers may accommodate discs of radial diameter from about 100 to 140 mm, preferably from about 110 to 130 mm, more preferably from 115 to 125 mm, and most preferably 120 mm. 
     In addition, the standard allows discs with radial diameter of 8 cm (80 mm). The dimensions of the mounting and clamping rings remain the same as those for 120 mm discs, and only the outer diameter is reduced, therefore reducing the data area of the disc. Commercially available CD and DVD readers and reader/writers accommodate discs of this diameter in their disc trays. A disc of this diameter presents certain advantages in the practice of the present invention, among which are a commensurate reduction in assay sample volume required to effect contact with the entire disc surface, as well as the ability to package such a disc in a sleeve dimensioned identically to the sleeve of a 3½ inches magnetic floppy disc. 
     Furthermore, various additional standards, such as those defining a magneto-optical “minidisc” or analog laser disc have been, or will be, developed. Thus, a disc of the present invention may have a radial diameter as small as 50 mm and as large as that for a standard laser disc, and may be adapted to such size standards as will be developed in the future. One skilled in the art will appreciate that the term “disc” contemplates any suitably rotatable media, whether or not perfectly circular, whether or not flat. 
     Referring now to  FIG. 5 , exemplary detector  81  and its associated electronics are described. Detector  818  typically includes a central quad detector flanked by two additional detector elements. The central quad detector is split into four sensor elements a, b, c, and d. Each of sensor elements a, b, c, d, e, and f provides an electrical signal indicative of the intensity of the reflected laser beam striking that element. 
     A CD reader can use a three-beam pickup, wherein the light beam is split into three beams consisting of a main beam and two tracking beams. The main beam is focused onto the operational surface of an optical disc and is centered on a track. The tracking beams fall on either side of the track. For example, as shown in  FIG. 4 , main beam  821  is centered on track  824  as defined by pits  822 . The two tracking beams  823  fill on either side of track  824 . By design, the three beams are reflected from the optical disc and directed to detector  818  such that main beam  821  falls on the central quad detector, and the two tracking beams  823  fall on sensor elements e and f. The sum of the electrical signals generated by the central quad detector (a+b+c+d), is a radio frequency (RF) signal, also referred to as a high frequency (HF) or quad-sum signal. The circuitry in  FIG. 5  is just one example of circuitry that is suitable for providing focusing and tracking error signals to an optical disc player. 
     The RF signal, obtained from summing the signals from all of sensor elements a, b, c, and d, may be used to extract data recorded in the optical disc. Typically, the process is performed by analog circuitry in combination with one or more integrated circuit chips. Often, the circuitry may take the form of a special chip set. 
       FIGS. 6 and 7  are illustrative block diagrams of exemplary chip sets for a typical CD drive and DVD drive respectively. 
     Numerous designs and configurations of optical pickups and associated electronics that may be suitable for the present invention are described in  Compact Disc Technology,  by Nakajima and Ogawa, IOS Press, Inc. (1992);  The Compact Disc Handbook,  by Pohlmann, A-R Editions, Inc. (1992);  Digital Audio and Compact Disc Technology,  by Baert et al. (eds.), Books Britain (1995); and  CD - Rom Professional&#39;s CD - Recordable Handbook: The Complete Guide to Practical Desktop CD,  Starrett et al. (eds.), ISBN:0910965188 (1996), all of which are incorporated herein in their entirety by reference. 
       FIG. 6  includes buffer amplifiers  826 ,  827 , and  828 . These amplifiers enable external circuitry, such as oscilloscopes and analog to digital converters, to be connected to the various signals generated by the optical disc drive without interfering with normal drive operation. For more information about reading and processing signals, such as those produced by investigational features or structures, see U.S. Patent Application Ser. No. 90/xxx,xxx, entitled “Disc Drive System and Methods for Use with Blo-disc” filed Nov. 9, 2001; U.S. Provisional Application Ser. No. 60/270,095, entitled “Signal Processing Apparatus and Methods for Obtaining Signal Signatures of Investigational Features Detected on a Surface of an Optical Disc Assembly” filed Feb. 20, 2001; and U.S. Provisional Application Ser. No. 60/292,108, entitled “Signal Processing Apparatus and Methods for Obtaining Signal Signatures of Investigational Features Detected on a Surface of an Optical Disc Assembly” filed May 18, 2001, all of which are incorporated herein by reference. 
     As a general principle, in order for an optical disc reader to properly operate an optical disc, the optical disc reader is typically required to be able to (1) accurately focus above the operational surface of the optical disc, (2) accurately follow the spiral track or utilize some form of uniform radial movement across the optical disc surface, and (3) recover enough information to facilitate a form of speed control. 
     As discussed above in conjunction with  FIG. 2 , transparent substrate layer  112  of the optical disc can focus the reader&#39;s laser upon the reflective layer  114 . Failure to maintain correct thickness, transparency, and refractive index of transparent substrate layer  112  may render the optical disc unreadable. 
     As shown in  FIG. 2 , operational structures are impressed in the laser-distal surface of the substrate layer  112  and coated with reflective layer  114 , Operational structures must be read in order to maintain correct tracking. In a standard pressed CD, the disc reader tracks a pitted spiral track that is impressed in a surface of the substrate layer. In a recordable CD, the disc reader tracks a spiral groove that is also impressed in the substrate layer. The spiral groove may be filled with dye. 
       FIG. 8  demonstrates a typical CD-type disc  811 ′ which is associated with analytes  40  and  40 ′. Analytes  40  and  40 ′ are applied to the air-incident, the most laser-proximal surface of the disc  811 ′, and are laser-proximal to the focal point of the incident laser. Analytes so disposed would be undetectable by standard means. For instance, the analytes would have to be in the range of the beam size at the incident surface, which may be about 800 μm, in order to be detectable by the laser source. If so large, the analytes may, by virtue of their interposition between the laser and the reflective surface  114 ′, interfere with the reading of the operational structures embossed in substrate layer  112 ′. 
     Thus, in order to adapt standard optical disc technology for detection of analyte-specific structures or signal elements, there is a need to design an optical disc having geometries and tracking schemes that overcome the above problems. Illustrative suitable disc geometries and tracking schemes are described, for example, in WO 00/26677, entitled “Trackable Optical Discs with Concurrently Readable Analyte Material” which is incorporated herein by reference herein in its entirety. The optical disc assembly of the present invention may be used in accordance with such geometries and schemes. 
     Disc inserts of the present invention may be used in conjunction with a number of different types of discs, such as single data layer discs. The physical orientation of a standard, single data-layer disc, such as a CD or a CD-type disc, may effectively be inverted, presenting what would otherwise be the most laser-distal surface as the most laser-proximal surface of the disc. To compensate for the inverted physical orientation, an inverted image of the disc&#39;s operational structures may be used. 
       FIG. 9  shows physical inversion of the disc  811  of  FIG. 2 , Light is incident from below. Protective layer  116   a  now becomes the most laser-proximal layer in the disc  811   a.  Reflective layer  114   a  is laser-distal to protective layer  116   a,  and transparent substrate  112   a  becomes the most laser-distal layer in the disc  811   a.    
     From the perspective of the optical pickup of the reader, physical inversion of an optical disc which has an operational surface impressed with a spiral wobble groove effectively converts each original land on the operational surface to a groove, and each original groove to a land. Inversion may also effect changes in the non-symmetric features in the operational surface, such as the direction of the spiral of the wobble groove. To restore the proper orientation of data after physical inversion of the disc, and in particular to restore the proper orientation of operational structures, a reverse image wobble groove may be used to provide operational structures in an inverted disc.  FIG. 10  shows an example of an inverted disc with a reverse image wobble groove. The reverse image wobble groove may be impressed into substrate  112   b  using a conventional mother stamper, as opposed to a son stamper that is typically used to make a conventional wobble groove disc. More details about making an optical disc impressed with a reverse image wobble groove can be found in WO 00/26677, entitled “Trackable Optical Discs with Concurrently Readable Analyte Material” which is incorporated herein by reference. Physical inversion of an optical disc including a track of pits and lands may also be used in the present invention. In such a case, the original pits become bumps on the operational surface, 
       FIG. 11  shows a single data layer disc that has its protective layer removed. insert  500  has been attached, such as glued, to the reflective layer of disc  130 . Analytes or analyte-specific signal elements  136  have been disposed upon insert  500 . Like other figures,  FIG. 11  is not drawn to scale. If the insert is thick enough, analytes  136  preferably are positioned between insert  500  and substrate  132 . The operational structures in the disc may be a wobble groove or a track of pits and lands. 
     In this type of assay disc, the analytes or analyte-specific signal elements may be located substantially confocal with or adjacent to the tracking or other operational features embossed in the laser-proximal surface of substrate  132 . This geometry greatly simplifies the problem of achieving and maintaining focus concurrently on the disc&#39;s operational features and the analytes or analyte-specific signal elements. 
     It will be understood that the analyte-specific signal elements and the operational structures, particularly those having encoded tracking and synchronization information, need not be in the identical focal plane. It suffices that the signal elements and operational structures are sufficiently confocal or adjacent to each other. As used herein, an object is adjacent to a structure if the object and the structure are close enough so as to permit a disc reader&#39;s optical head to detect them both. An object is adjacent to a trackable surface or layer if the object is closed enough to the trackable surface or layer such that an optical disc reader can both detect the object and track the surface or layer. Likewise, an analyte-bearing surface is adjacent to a trackable surface or layer if an optical disc reader can both detect the analytes disposed on the analyte-bearing surface and track the trackable surface or layer. An object is substantially confocal with a structure or surface if the object and the structure or surface are within a focal depth of the reading beam of the optical disc reader. The focal depth of the reading beam may be about 2 micrometers. 
     As shown in  FIG. 11 , the reflective layer is now laser-distal to insert  500 . There is no transparent substrate layer present to assist the laser to focus on the reflective layer, as shown in  FIG. 2 . One solution is to add an extra focus correction lens to the disc reader&#39;s optical pickup. 
     Alternatively, or in addition thereto, the distance between the optical pickup and the disc&#39;s most laser-proximal surface can be adjusted so that the laser focuses on the reflective layer of the disc. If desired, however, insert  500  may be formed of sufficient thickness and from materials whose refractive properties are well known in order to focus the light beam correctly on the disc&#39;s operational structures. In one embodiment, the analyte-bearing surface of insert  500  faces the reflective layer, while insert  500  serves as a lens to focus the reading laser beam. In another embodiment the disc uses one or more inserts of sufficient size and geometry to cover all or substantially all of the reflective layer of disc  130 . 
     In yet another embodiment a laser-refracting member is used as the most laser-proximal layer in the disc assembly. The cover serves to refract, and thus is capable of focusing the incident reading beam on the insert or the disc&#39;s reflective layer. Suitably designed, the cover alone or in combination with inserts may obviate alteration in the reader&#39;s optics. Inserts can be at least partially inserted into or otherwise attached to the cover if desired. As used herein, a layer in an optical disc is capable of focusing a reading beam of an optical disc reader upon an object if the reading beam, which is directed to the object while the optical disc reader is operating the optical disc, can pass through the layer and become focused on the object. Preferably, the refractive index of the layer is significantly greater than the refractive index of air, as appreciated by those of skill in the art. For instance, in a typical CD-recordable (CD-R) disc, the transparent substrate is capable of focusing the reading beam on the operational structures. The substrate is composed principally of polycarbonate, and has a refractive index of between 1.55 and 1.58. Other layer or layers may also exist in the optical path of the reading beam to help the reading beam become focused on the object. 
     In a preferred embodiment, the disc assembly in  FIG. 11  can be read by a disc reader in such a manner that substrate  132  is laser-proximal to insert  500 . A second reflective layer may exist, and is positioned laser-distal to insert  500 . In this configuration, substrate  132  functions as a lens and may focus the reading beam upon analytes  136 . Preferably, the operational structures of the disc are impressed in the laser-distal surface of substrate  132  and are coated with a semi-reflective layer. Because the low reflectivity (for instance, about 18 to 30%) of the semi-reflective layer, a CD-R or DVD) reader may preferably be used in this embodiment. 
       FIG. 12  shows a disc  630  associated with a non-integral cover  640 . Disc  630  includes disc substrate  632  and reflective layer  634 . Wobble groove  638  (or a track of pits and lands) is impressed in substrate  632  and coated by reflective layer  634 . Insert  502  can be partially inserted into the cover  640 . Analyte-specific signal elements  636  are disposed on insert  502 . Another example of a suitable cover is cover  640 ′, as shown in a top perspective view in  FIG. 14 . In one embodiment, the disc is configured such that the analyte can be disposed between reflective layer  634  and insert  502 . 
     Preferably, the disc  630  may be read in such a manner that substrate  632  is laser-proximal to insert  502  and the signal elements  636 . A second reflective layer may exist, and is positioned laser-distal to insert  502  and the signal elements  636 . The reflective layer  634  preferably is semi-reflective, and has a low reflectivity such as from about 18 to 30%. The disc reader may be, but is not limited to, a CD-R reader or a DVD reader. 
     In a preferred embodiment, the disc assembly (which includes disc  630 , the insets, and the cover) is dimensioned to approximate the standard size of a unitary optical disc, that is, 1.2 mm in depth and either 80 mm or 120 mm in diameter. However, it is also contemplated that the size of the disc assembly may vary substantially therefrom. The disc assembly may still be capable of meeting the necessary optical and mechanical requirements in order for a standard disc reader to read the disc assembly. For instance, the light beam of the standard disc reader may be able to correctly focus on the disc&#39;s operational plane. The disc assembly may clamp properly onto the spindle of the disc reader. The weight of the disc assembly may not vary substantially from the standard weight because otherwise the reader&#39;s motor might not be able to maintain a proper rotational speed. 
     The laser refracting disc cover can be in the form of a nonintegral or an integral cover, hingeably or otherwise moveably or modifiably attached. In the nonintegral embodiment, the disc cover and substrate may be assembled and disassembled by use of machine-type threads, detents, or other mechanical means and provided in a kit including analyte and counterweight inserts. Nonintegral covers may be reversibly (e.g. removably) or irreversibly attachable to the other part of the disc assembly. 
     Cover  640  and insert  502  may include polycarbonate to take advantage of its well known optical qualities and the ready availability of devices adapted to its molding. In addition, other types of plastics may be used, such as polymethyl acrylic, polyethylene, polypropylene, polyacrylate, polymethyl methacrylate, polyvinylchloride, polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal, polysulfone, celluloseacetate, cellulosenitrate, nitrocellulose, or mixtures thereof. Glass can be used as well. 
     As noted above, analyte-specific signal elements are preferably disposed substantially confocally with the operational structures in a single data layer disc assembly. This permits the reading beam to focus substantially confocally on the analyte-specific signal elements and the operational structures. When signal elements fall into the operational structures, for instance into a wobble groove, the confocal effect is optimized. 
     Analyte-specific signal elements preferably are placed on insert  500 , rather than on an external surface of the optical disc assembly. When the analyte-specific signal elements are disposed on a plastic insert, for example, the number of potential chemistries that can be used to affix the signal elements to the insert increases. For example, although gold-sulfur bonds prove widely adaptable, plastic presents a far wider selection of available attachment chemistries. Also, although a gold surface can be patterned to present discrete sites for signal element attachment, a plastic surface can be more readily derivatized to present chemically reactive groups in spatially defined patterns, which facilitate the attachment of analyte-specific signal elements in spatially addressable patterns. Some of these patterns, and their advantages, are described in WO 00/05582, entitled “Optical Disc-Based Assay Devices and Methods” and commonly assigned U.S. patent application Ser. No. 09/120,049, filed Jul. 21, 1998, entitled “Optical Disc-Based Assay Devices and Methods” both of which are incorporated herein by reference. 
     Another advantage of disposing analyte-specific signal elements on an insert is that the disc assembly or the cover may be reused. 
     Polystyrene is useful in the construction of inserts or covers because many current clinical assays are conducted on polystyrene surfaces. Standard microtiter plates or dishes, used in enzyme-linked immunosorbent assays (ELISA) and radioimmunoassay (RIA), are made of polystyrene. Also, a wealth of experience attends the conduct of clinical assays on polystyrene surfaces, and such assays may thus readily be adapted to the present invention. Additionally, precision molding of polystyrene is well known. 
     The thickness of the cover, the insert, or both, can be adjusted to account for differences in the refractive index of the chosen plastic in order to focus the light beam correctly. 
     In accordance with another aspect of this invention, a hologram is used to provide operational signals.  FIG. 13  shows an illustrative single data layer disc  190 , in which operational structures are encoded in a reflective hologram  194 , rather than physically unpressed in disc substrate  192 . 
     Disc  190  includes disc substrate  192 , hologram  194 , and transparent protective coating  198 . Hologram  194  is a reflective hologram having encoded operational structures required by the disc reader to operate the disc. In operation, when a laser is reflected from hologram  194 , it will appear as though the operational structures, such as a wobble groove with the correct orientation or a track of pits and lands, are present at hologram image plane  195 . 
     Hologram image plane  195  may be laser-proximal to the hologram physical plane  194  and substantially confocal with analyte-specific signal elements  646  disposed on insert  504 . In operation, the laser is focused on the plane  195  which is shared by both the analyte-specific signal elements and the encoded operational structures, such as the image of a wobble groove encoded in the hologram. This configuration permits, if desired, concurrent and discriminable acquisition of operational data (e.g., tracking data) and analyte-specific data. 
     The light beam of the disc reader may be focused on the image plane of the hologram. This necessitates that the light beam would be less tightly focused on the hologram&#39;s physical plane. Yet the very nature of holographic imaging not only tolerates such “error” but benefits therefrom. For example, each portion of a hologram&#39;s physical surface may generate the entirety of the image that is interferometrically encoded thereon. As the illuminated portion decreases in size, however, the resolution degrades. Conversely, the larger the portion of the hologram illuminated, the better the image. Thus, the larger the illuminating laser spot on the hologram&#39;s physical plane, the better the image of the disc&#39;s operational structures (e.g. a wobble groove). 
     Furthermore, although shown as integral to disc  190 , hologram  194  may be removable, This permits hologram  194  to be mass-produced using existing high-speed holographic printing processes. Furthermore, depending on the application, hologram  194  may be reversibly attachable to disc substrate  192 , potentially permitting reuse of substrate  192 . These features permit low cost mass-production of disc assemblies readable by a wide range of optical readers. 
     As in the embodiments in which the operational structures are embossed and reflectively coated, a holographic optical disc assembly may include a laser-proximal, nonintegral cover that assists the laser to focus. The focal plane of the reading beam can be made substantially confocal with the hologram image plane. 
     Disc substrate  192  in  FIG. 13  and disc substrate  632  in  FIG. 12  need not meet the optical requirements of standard transparent disc substrate, such as substrate  112  in  FIG. 2 . This is because disc substrates  192  and  632  are located laser distal to the embossed operational structures or the hologram. 
     In accordance with yet another aspect of this invention, a multiple data layer disc may be used. The multiple layer disc may have the features as specified in the Digital Versatile Disc (DVD) format. 
       FIG. 15  shows a side cross-sectional view of a typical dual layer DVD format disc. Disc  280  includes laser-proximal substrate  282 , semi-reflective layer  284 , spacer layer  286 , reflective layer  288 , and laser-distal substrate  290 . 
     Proximal substrate  282 , also referred to as layer  0 , contains a transparent optical material, such as polycarbonate, having an index of refraction chosen to assist in focusing the reading beam onto either one of the two reflective layers. Proximal substrate  282  may be manufactured by an injection molding process similar to the process described above for manufacturing CD-Recordable format discs. Proximal substrate  282  is typically embossed with data at its laser-distal surface. 
     The data-bearing surface of proximal substrate  282  is coated with the semi-reflective layer  284 . The semi-reflective layer  284  includes a very thin coating of a material such as silicon, gold, aluminum, silver, or copper that reflects some light and transmits some light. The semi-reflective layer  284  typically has a reflectivity of approximately 18%, although a range of reflectivities can be accommodated. For instance, the semi-reflective layer  284  may have a reflectivity from approximately 18 to 30%. When a dual data layer disc is used, a DVD reader or CD-RW reader preferably is used. DVD or CD-RW reader has suitable gain control circuitry for detecting and processing signals reflected from a semi-reflective layer having a low reflectivity. 
     Distal substrate  290 , also referred to as layer  1 , includes a material, such as polycarbonate, that can be molded with a spiral data track at its laser-proximal surface. Since the reading beam usually will not pass through distal substrate  290 , its optical characteristics are unimportant. Distal substrate  290  can be manufactured by an injection molding process. 
     Distal substrate  290  is embossed with data in a spiral data track that may run parallel with the spiral data track in layer  0 , for example, from the inner portion of the disc to the outer portion, or run in the opposite direction of the spiral data track in layer  0 , for example, from the outer portion of the disc to the inner portion. 
     The data-bearing surface of distal substrate  290  is coated with reflective layer  288 , which may include a thin layer of a reflective material, such as gold, aluminum, silver, or copper. Reflective layer  288  typically has a reflectivity that is designed to obviate readjustment of gain control when the disc reader switches its reading from one layer to the other. A readjustment in gain control may adversely affect tracking. Layer  1  in a dual layer disc often has a reflectivity lower than 70%. 
     Spacer layer  286  provides 40 to 70 microns of space between layer  0  and layer  1  of the two layer disc, and serves to bind proximal substrate  282  to distal substrate  290 . Spacer layer  286  can be, for example, an optical adhesive having an index of refraction that is close to the index of refraction of the material from which proximal substrate  282  is manufactured. 
     In use, a DVD reader can focus the reading beam either on the semi-reflective layer  284  to read data encoded in layer  0 , or on the reflective layer  288  to read data encoded in layer  1 . The multiple layer nature of DVD discs and the concomitant dual-focus of DVD readers make DVD well-suited for use in the present invention, For instance, the plane occupied by the operational features of the disc may be segregated physically from the plane occupied by analyte-specific elements, facilitating discriminable acquisition of both types of data. 
     Investigational structures, such as analyte-specific signal elements, may be placed on an insert. The insert may be constructed so that the investigational structures can be substantially confocal with or adjacent to the laser-distal surface of data layer  0 , while the disc&#39;s tracking and other operational structures are positioned in data layer  1 . In another embodiment, the sample-bearing surface of the insert is substantially confocal with or adjacent to the laser-proximal surface of layer  1 , whereas the disc&#39;s tracking and other operational structures are located in layer  0 . 
       FIG. 16  presents an exploded side perspective view of a multi-insert DVD-type dual data layer disc assembly. Disc assembly  300  includes two portions: main portion  302  and cover portion  303 . These portions may be permanently affixed to one another, or may be separate but assemblable. Preferably, the main portion  302  and cover portion  303  are reversibly assemblable. Inserts  506 ,  507 ,  508 , and  509  are inserted into or otherwise attached to cover portion  303 . Cover portion  303  is assembled over outer assay area  306  of main portion  302 . Opening  308  and area  304  can be dimensioned so as to permit a snug and reliable coupling between the main portion and the cover portion. 
     Analogously to the single data layer embodiments described above, outer assay area  306  can be embossed with a wobble groove, or other substantially radial plane tracking features, to provide tracking information to an optical disc reader. Pursuant to DVD standards, the wobble groove can be either a forward image groove or a reverse image groove. A zoned constant linear velocity (ZCLV) format can be used. 
     Main portion  302  includes inner data area  304 . Inner data area  304  is formatted in a manner similar to a normal dual layer DVD disc. Programs and data can be stored in layer  0  and/or layer  1  of this area of the disc. In particular, inner data area  304  preferably encodes instructions that direct the optical disc reader to adjust its focus to the correct data layer in order to read the analyte-specific signals disposed on inserts  506 ,  507 ,  508 , and  509 . Furthermore, inner data area  304  can store data used to adjust the firmware or “flash” components of the drive chipset, as may be needed by the drive to correctly read and interpret analyte-specific signals. 
     Cover portion  303  and inserts  506 ,  507 ,  508 , and  509  can include a translucent optical material, such as polycarbonate, polymethyl acrylic, or glass. The material can be selected to optimize the detection of the signal elements. Inserts  506 ,  507 ,  508 , and  509 , and cover portion  303  can be configured such that the analyte-specific signal elements are physically segregated from operational structures. 
     There are many variations that could be made based on the embodiment shown in  FIG. 16 . For example, if large amounts of data and/or programming are needed to interpret the results of an assay, inner data area  304  could have data written both on layer  0  and on layer  1 , without altering the data structure in the outer assay area  306 . 
       FIG. 17  shows a side cross-sectional view of another multi-layer embodiment of an assay disc assembly built in accordance with the present invention. Disc assembly  320  includes one or more spaces or chambers  322 . Assays may be performed by introducing into spaces  322 , inserts  512  or  514 . Investigational structures or analytes are placed on the inserts. Layer  1  of disc assembly  320  is embossed with a wobble groove in its laser-proximal surface, and provides at least the minimal operational structures required by an optical disc reader to operate the disc assembly. Layer  0  of disc  320  may contain data necessary for the optical disc reader to detect the investigational structures or interpret the detection results. 
     Multiple assays can be performed using a disc assembly having multiple separate assay spaces, such as spaces  322 . In addition the location of spaces  322  within the disc assembly can be varied. For example, spaces  322  can be closer to either layer  0  or layer  1 , or roughly at the hallway point between layer  0  and layer  1 . In either case, the analyte disposed within spaces  322  may be detected using a light beam which can be focused on either the laser-proximal surface of layer  1  or the laser-distal surface of layer  0 . 
       FIG. 18  shows a side cross-sectional view of yet another multiple layer assay disc assembly. The assay disc assembly  330  is similar to disc assembly  320  in  FIG. 17 . In the case of disc  330 , spaces  332  are located toward the outer portion of the assembly resulting in a central portion  334  as a “standard” two layer disc. Inserts  516  and  518  can be sized to fit into spaces  332 . Layer  0  of disc  330  is divided into two sections. Section  334  of Layer  0  can be used to store data and/or programs. Section  336  of layer  0  may include a translucent material having optical properties different from those of section  334 . In a preferred embodiment, the optical properties of section  336  are optimized for focusing a light beam onto the operational structures of the disc, or for detecting the analyte disposed on inserts  516  and  518  that are disposed in spaces  332 . Disc  330  can be read by a DVD reader, but can also be read by a CD reader such as a CD-R reader or preferably a CD-RW reader. 
       FIG. 19  shows a disc assembly  340 . Data encoded in layer  0  can be arranged according to a DVD format and may be read by a standard DVD player. Data encoded in layer  1 , which includes layer  344  and layer  342 , can be in a CD-Recordable format. Layer  344  may be embossed with operational structures. Spaces  346 , which are adjacent to layer  344 , are capable of receiving inserts to perform assays. 
       FIG. 20  shows a two-layer disc  350 , Data and operational structures in layer  1  are provided by hologram  352 . The operational structures and data encoded in hologram  352  appear to reside in the hologram&#39;s image plane  354 . The image plane may be either laser distal or laser proximal relative to hologram  352 . In  FIG. 20 , the image plane  354  is laser proximal relative to the hologram  352 . Inserts can be inserted into spaces  356 . Analytes disposed on inserts  500  may be detectable using a light beam that is focused on the image plane  354 . 
     A hologram similar to hologram  352  can be used to provide layer  1  in nearly any of the multiple layer discs described above, Analytes may be disposed directly on the surface of the hologram, or within or adjacent to the image plane of the hologram. 
     The foregoing embodiments for dual data layer discs can be easily extended to use in discs which have move than two data or operational layers. Spaces or channels that are used to receive inserts can be located between any two data layers. Each of the layers may provide required operational structures or encode data for conducting analyte assays. In one embodiment, the most laser-proximal data layer does not encode operational information. 
     An insert in a disc assembly of this invention may have any shape, such as those shown in  FIG. 21 , including a substantially rectangular shape  526  which may conform to the shape of a standard microscope slide. This shape can be used in both the disc assemblies of the present invention and conventional microscopy. Other shapes may also be used. For example, the insert may have a substantially triangular shape  528  (including pie-shaped), a semi-circular shape  530 , a ring shape  532 , or any other suitable shapes. Substantially triangular inserts may minimize interference with the operational characteristics of the present system, and may maximize the orthogonality of insert edges and tracks. 
     Multiple ring-shaped inserts may be used, for example, to perform multiple assays on a single disc, as shown in  FIG. 22 . As shown in  FIG. 23 , a single disc assembly  550  can be configured to accommodate different shaped inserts  534 ,  535 ,  536 ,  538 , and  540 . 
     The weight of the insert Nay cause the disc assembly to spin in an unbalanced fashion. Thus, counterweights can be used to balance the weight of the disc. Covers or the optical discs themselves can be manufactured, for example, with one or more section or attachment sites.  FIG. 24  shows sites  556  and  558  can be diametrically positioned on the opposite sides in the disc assembly. Counterweight  554 , which approximates the weight of the sample and insert  552 , may be placed into site  558  which is opposite to site  556  in which insert  552  is positioned. The counterweights may be any suitable solid or fluid. In one embodiment, the disc can be manufactured with an irregular portion that is positioned opposite to the insertion or attachment site and weighted to compensate for the weight of the insert. If desired, a circumferential channel  562  can be used, as shown in  FIG. 25 . Annular channel  562  can be filled with materials capable of traveling inside the channel in order to balance the disc assembly against insert  564 . 
     A method for using an optical disc assembly of the present invention is described as follows. A technician can, for example, apply a sample of fluid or tissue containing an analyte of interest to an insert. After applying the sample, the insert is inserted or otherwise attached to a disc assembly at an appropriate site. Preferably, the disc assembly has a cover. The disc assembly thus prepared is inserted into a CD or DVD reader, and the sample is assayed and analyzed, as described, for example in U.S. patent application Ser. No. 09/378,878, entitled “Methods and Apparatus for Analyzing Operational and Nonoperational Data Acquired from Optical Discs”; U.S. Provisional Application Ser. No. 60/270,095, filed Feb. 20, 2001, entitled “Signal Processing Apparatus and Methods for Obtaining Signal Signatures of Investigational Features Detected on a Surface of an Optical disc Assembly”; and U.S. Provisional Application Ser. No. 60/292,108, filed May 18, 2001, entitled “Signal Processing Apparatus and Methods For Obtaining Signal Signatures of Investigational Features Detected on a Surface of an Optical disc Assembly” all of which are incorporated herein by reference. Signals that are indicative of the presence of the analyte can be obtained from the CD or DVD reader, and further processed using circuitry connected to the CD or DVD reader. 
     While preferred illustrative embodiments of the present invention are described, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the invention. The present invention contemplates all such changes and modifications which fall within the true spirit and scope of the invention. In addition, the reader&#39;s attention is directed to the provisional applications from which the present application claims priority. The contents of all these provisional applications are incorporated herein by reference.