Abstract:
A wide variety of current diagnostic and other biochemical tests employ a substance, such as a chromagen, that undergoes a detectable color development or change of fluorescent emission in the presence of the analyte of interest. The intensity of the color or fluorescence developed may be time dependent and proportional to the concentration of the analyte of interest. Systems, methods, and components usable for quantifying the concentration of an analyte of interest in a biological sample on optical biodiscs are disclosed herein. Analytes may include, for example, glucose, cholesterol, and triglycerides. In one embodiment, reagents are immobilized on the optical disc prior to the assay.

Description:
RELATED APPLICATIONS  
       [0001]     This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Ser. No. 60/452,313, filed Mar. 5, 2003, the disclosure of which is incorporated by reference herein in its entirety. 
     
    
     BACKGROUND OF THE INVENTION  
       [0002]     1. Field of the Invention  
         [0003]     This invention relates in general to assays and, in particular, colorimetric and fluorescent assays. More specifically, but without restriction to the particular embodiments hereinafter described in accordance with the best mode of practice, this invention relates to sample preparation for colorimetric and fluorescent assays as performed on optical analysis discs.  
         [0004]     2. Description of the Related Art  
         [0005]     Detection and quantification of analytes in body fluids, such as blood, may be important for diagnosis of diseases, elucidation of the pathogenesis, and monitoring the response to drug treatment. Traditionally, diagnostic assays are performed in laboratories by trained technicians using complex apparatus. Performing these assays is usually time-consuming and costly. Thus, there is a significant need to make diagnostic assays and forensic assays faster and more local to the end-user. Ideally, clinicians, patients, investigators, the military, other health care personnel, and consumers should be able to test themselves for the presence of certain risk factors or disease indicators in their systems, and to test for the presence of certain biological material at a crime scene or on a battlefield. At present, there are a number of medical diagnostic, silicon-based, devices with nucleic acids and/or proteins attached thereto that are commercially available or under development. These chips are not for use by the end-user, or for use by persons or entities lacking very specialized expertise and expensive equipment.  
         [0006]     Commonly assigned U.S. Pat. No. 6,030,581 entitled “Laboratory in a Disk” issued Feb. 29, 2000 (the &#39;581 patent) is hereby incorporated by reference in its entirety. The &#39;581 patent discloses an apparatus that includes an optical disc, adapted to be read by an optical reader, which has a sector having a substantially self-contained assay system useful for localizing and detecting an analyte suspected of being in a sample. U.S. Pat. No. 5,993,665, issued Nov. 30, 1999 (the &#39;665 patent) entitled “Quantitative Cell Analysis Methods Employing Magnetic Separation” discloses analysis of biological specimens in a fluid medium where the specimens are rendered magnetically responsive by immuno-specific binding with ferromagnetic colloid. The &#39;665 patent is hereby incorporated by reference in its entirety.  
       SUMMARY OF THE INVENTION  
       [0007]     The present invention relates to performing colorimetric and fluorescent assays on an optical analysis disc. The invention includes methods for preparing assays, methods for depositing the reagents for the assays, discs for performing assays, and detection systems.  
         [0008]     A wide variety of current diagnostic and other biochemical tests employ a substance (chromagen) that undergoes a detectable color development or change of fluorescent emission in the presence of the analyte of interest. The intensity of the color or fluorescence developed is time dependent and proportional to the concentration of the analyte of interest. For colorimetric assays, the intensity of the color is measured by optical density measurement at specific wavelengths using a spectrophotometer.  
         [0009]     The present invention includes methods for quantifying the concentration of an analyte of interest in a biological sample on optical biodiscs using colorimetric assays. Analytes may include, for example, glucose, cholesterol, and triglycerides. In one embodiment, reagents are immobilized on the optical disc prior to the assay. To perform the assay, the sample (preferably serum, but other types of body fluids could also be used) is loaded into the channel via the injection port. After injection, the ports may be sealed, such as with tape or other suitable means. Depending on the assay protocol, the bio-disc is incubated at room temperature, or other desired temperature, for an appropriate time, e.g., 3 to 7 minutes. The optical disc reader then quantifies the intensity of the color developed. After data collection and processing, the results of the assay are displayed on a computer monitor. It should be noted that some diagnostic colorimetric assays in clinical laboratories are carried out at 37 degrees Celsius to facilitate and accelerate color development. For ease of operation, colorimetric assays performed on optical discs may advantageously be optimized to run at ambient temperature. The optimization may include selection of enzyme sources, enzymes concentrations, and sample preparation.  
         [0010]     In one embodiment, Chromagen selection is important in optimizing colorimetric assays for optical density measurements on bio-discs since chromagens are detected at specific wavelengths. CD-R type disc readers, for example, are capable of detecting chromagens in the infrared region (750 nm to 800 nm). Other types of optical disc systems may be used in the present invention including DVD, DVD-R, fluorescent, phosphorescent, and any other similar optical disc reader. The amplitude of optical density measurements depends on the optical pathlength, the molar extinction coefficient of the chromagen and the concentration of the analyte of interest (Beer&#39;s law). To optimize the sensitivity of colorimetric assays on optical discs, several chromagens with high molar extinction coefficients at the wavelengths of interest have been identified and evaluated.  
         [0011]     Chromagens suitable for colorimetric assays on CD-R type optical discs include, but are not limited to, N,N′-Bis(2-hydroxy-3-sulfopropyl)tolidine, disodium salt (SAT-3), N-(Carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)-diphenylamine sodium salt (DA-64), 2,2′-azino-dimethylthiozoline-6-sulfonate (ABTS), Trinder&#39;s reagents N-Ethyl-N-(2-hydroxy-3-sulfopropyl)3-methylaniline, sodium salt, dihydrate (TOOS) with the coupling reagent 3-(N-Methyl-N-phenylamino)-6-aminobenzenesulfonic acid, and sodium salt (NCP-11). 
     
    
     BRIEF DESCRIPTION OF THE DRAWING FIGURES  
       [0012]     Further objects of the present invention together with additional features contributing thereto and advantages accruing therefrom will be apparent from the following description of the preferred embodiments of the invention which are shown in the accompanying drawing figures with like reference numerals indicating like components throughout, wherein:  
         [0013]      FIG. 1  is a pictorial representation of a bio-disc system;  
         [0014]      FIG. 2  is an exploded perspective view of a reflective bio-disc;  
         [0015]      FIG. 3  is a top plan view of the disc shown in  FIG. 2 ;  
         [0016]      FIG. 4  is a perspective view of the disc illustrated in  FIG. 2  with cut-away sections showing the different layers of the disc;  
         [0017]      FIG. 5  is an exploded perspective view of a transmissive bio-disc;  
         [0018]      FIG. 6  is a perspective view representing the disc shown in  FIG. 5  with a cut-away section illustrating the functional aspects of a semi-reflective layer of the disc;  
         [0019]      FIG. 7  is a graphical representation showing the relationship between thickness and transmission of a thin gold film;  
         [0020]      FIG. 8  is a top plan view of the disc shown in  FIG. 5 ;  
         [0021]      FIG. 9  is a perspective view of the disc illustrated in  FIG. 5  with cut-away sections showing the different layers of the disc including the type of semi-reflective layer shown in  FIG. 6 ;  
         [0022]      FIG. 10  is a perspective and block diagram representation illustrating the system of  FIG. 1  in more detail;  
         [0023]      FIG. 11  is a partial cross sectional view taken perpendicular to a radius of the reflective optical bio-disc illustrated in  FIGS. 2, 3 , and  4  showing a flow channel formed therein;  
         [0024]      FIG. 12  is a partial cross sectional view taken perpendicular to a radius of the transmissive optical bio-disc illustrated in  FIGS. 5, 8 , and  9  showing a flow channel formed therein and a top detector;  
         [0025]      FIG. 13  is a partial longitudinal cross sectional view of the reflective optical bio-disc shown in  FIGS. 2, 3 , and  4  illustrating a wobble groove formed therein;  
         [0026]      FIG. 14  is a partial longitudinal cross sectional view of the transmissive optical bio-disc illustrated in  FIGS. 5, 8 , and  9  showing a wobble groove formed therein and a top detector;  
         [0027]      FIG. 15  is a view similar to  FIG. 11  showing the entire thickness of the reflective disc and the initial refractive property thereof;  
         [0028]      FIG. 16  is a view similar to  FIG. 12  showing the entire thickness of the transmissive disc and the initial refractive property thereof;  
         [0029]      FIG. 17A  is an exploded perspective view of a reflective bio-disc incorporating equi-radial channels of the present invention;  
         [0030]      FIG. 17B  is a top plan view of the disc shown in  FIG. 17A ;  
         [0031]      FIG. 17C  is a perspective view of the disc illustrated in  FIG. 17A  with cut-away sections showing the different layers of the equi-radial reflective disc;  
         [0032]      FIG. 18A  is an exploded perspective view of a transmissive bio-disc utilizing the e-radial channels of the present invention;  
         [0033]      FIG. 18B  is a top plan view of the disc shown in  FIG. 18A ;  
         [0034]      FIG. 18C  is a perspective view of the disc illustrated in  FIG. 18A  with cut-away sections showing the different layers of this embodiment of the equi-radial transmissive bio-disc;  
         [0035]      FIG. 19  is a graphical representation of the generation of a calibration curve for a glucose assay; and  
         [0036]      FIG. 20  is a graphical representation of the generation of a calibration curve for a cholesterol assay. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0037]     The present invention relates in general to preparation of biomedical samples and analysis of same using an optical bio-disc system. More specifically, this invention is directed to colorimetric and fluorescent assays. The invention includes methods for preparing assays, methods for depositing the reagents for the assays, discs for performing assays, and detection systems. Each of the aspects of the present invention is discussed below in further detail.  
         [heading-0038]     Drive System and Related Discs  
         [0039]      FIG. 1  is a perspective view of an optical bio-disc  110  according to the present invention as implemented to conduct the cell counts and differential cell counts disclosed herein. The present optical bio-disc  110  is shown in conjunction with an optical disc drive  112  and a display monitor  114 . Further details relating to this type of disc drive and disc analysis system are disclosed in commonly assigned and co-pending U.S. patent application Ser. No. 10/008,156 entitled “Disc Drive System and Methods for Use with Bio-discs” filed Nov. 9, 2001 and U.S. patent application Ser. No. 10/043,688 entitled “Optical Disc Analysis System Including Related Methods For Biological and Medical Imaging” filed Jan. 10, 2002, both of which are herein incorporated by reference.  
         [0040]      FIG. 2  is an exploded perspective view of the principal structural elements of one embodiment of the optical bio-disc  110 .  FIG. 2  is an example of a reflective zone optical bio-disc  110  (hereinafter “reflective disc”) that may be used in the present invention. The principal structural elements include a cap portion  116 , an adhesive member or channel layer  118 , and a substrate  120 . The cap portion  116  includes one or more inlet ports  122  and one or more vent ports  124 . The cap portion  116  may be formed from polycarbonate and is preferably coated with a reflective surface  146  ( FIG. 4 ) on the bottom thereof as viewed from the perspective of  FIG. 2 . In the preferred embodiment, trigger marks or markings  126  are included on the surface of the reflective layer  142  ( FIG. 4 ). Trigger markings  126  may include a clear window in multiple, or all, layers of the bio-disc, an opaque area, or a reflective or semi-reflective area encoded with information that sends data to a processor  166 , as shown  FIG. 10 , that in turn interacts with the operative functions of the interrogation or incident beam  152 ,  FIGS. 6 and 10 .  
         [0041]     The second element shown in  FIG. 2  is an adhesive member or channel layer  118  having fluidic circuits  128  or U-channels formed therein. The fluidic circuits  128  are formed by stamping or cutting the membrane to remove plastic film and form the shapes as indicated. Each of the fluidic circuits  128  includes a flow channel  130  and a return channel  132 . Some of the fluidic circuits  128  illustrated in  FIG. 2  include a mixing chamber  134 . Two different types of mixing chambers  134  are illustrated. The first is a symmetric mixing chamber  136  that is symmetrically formed relative to the flow channel  130 . The second is an off-set mixing chamber  138 . The off-set mixing chamber  138  is formed to one side of the flow channel  130  as indicated.  
         [0042]     The third element illustrated in  FIG. 2  is a substrate  120  including target or capture zones  140 . The substrate  120  is preferably made of polycarbonate and has a reflective layer  142  deposited on the top thereof,  FIG. 4 . The target zones  140  are formed by removing the reflective layer  142  in the indicated shape or alternatively in any desired shape. Alternatively, the target zone  140  may be formed by a masking technique that includes masking the target zone  140  area before applying the reflective layer  142 . The reflective layer  142  may be formed from a metal such as aluminum or gold.  
         [0043]      FIG. 3  is a top plan view of the optical bio-disc  110  illustrated in  FIG. 2  with the reflective layer  142  on the cap portion  116  shown as transparent to reveal the fluidic circuits  128 , the target zones  140 , and trigger markings  126  situated within the disc.  
         [0044]      FIG. 4  is an enlarged perspective view of the reflective zone type optical bio-disc  110  according to one embodiment of the present invention. This view includes a portion of the various layers thereof, cut away to illustrate a partial sectional view of each principal layer, substrate, coating, or membrane.  FIG. 4  shows the substrate  120  that is coated with the reflective layer  142 . An active layer  144  is applied over the reflective layer  142 . In the preferred embodiment, the active layer  144  may be formed from polystyrene. Alternatively, polycarbonate, gold, activated glass, modified glass, or modified polystyrene, for example, polystyrene-co-maleic anhydride, may be used. In addition, hydrogels can be used. Alternatively as illustrated in this embodiment, the plastic adhesive member  118  is applied over the active layer  144 . The exposed section of the plastic adhesive member  118  illustrates the cut out or stamped U-shaped form that creates the fluidic circuits  128 . The final principal structural layer in this reflective zone embodiment of the present bio-disc is the cap portion  116 . The cap portion  116  includes the reflective surface  146  on the bottom thereof. The reflective surface  146  may be made from a metal such as aluminum or gold.  
         [0045]     Referring now to  FIG. 5 , there is shown an exploded perspective view of the principal structural elements of a transmissive type of optical bio-disc  110  according to the present invention. The principal structural elements of the transmissive type of optical bio-disc  110  similarly include the cap portion  116 , the adhesive or channel member  118 , and the substrate  120  layer. The cap portion  116  includes one or more inlet ports  122  and one or more vent ports  124 . The cap portion  116  may be formed from a polycarbonate layer. Optional trigger markings  126  may be included on the surface of a thin semi-reflective layer  143 , as illustrated in  FIGS. 6 and 9 . Trigger markings  126  may include a clear window in all three layers of the bio-disc, an opaque area, or a reflective or semi-reflective area encoded with information that sends data to the processor  166 ,  FIG. 10 , which in turn interacts with the operative functions of the interrogation beam  152 ,  FIGS. 6 and 10 .  
         [0046]     The second element shown in  FIG. 5  is the adhesive member or channel layer  118  having fluidic circuits  128  or U-channels formed therein. The fluidic circuits  128  are formed by stamping or cutting the membrane to remove plastic film and form the shapes as indicated. Each of the fluidic circuits  128  includes the flow channel  130  and the return channel  132 . Some of the fluidic circuits  128  illustrated in  FIG. 5  include the mixing chamber  134 . Two different types of mixing chambers  134  are illustrated. The first is the symmetric mixing chamber  136  that is symmetrically formed relative to the flow channel  130 . The second is the off-set mixing chamber  138 . The off-set mixing chamber  138  is formed to one side of the flow channel  130  as indicated.  
         [0047]     The third element illustrated in  FIG. 5  is the substrate  120 , which may include the target or capture zones  140 . The substrate  120  is preferably made of polycarbonate and has the thin semi-reflective layer  143  deposited on the top thereof,  FIG. 6 . The semi-reflective layer  143  associated with the substrate  120  of the disc  110  illustrated in  FIGS. 5 and 6  may be significantly thinner than the reflective layer  142  on the substrate  120  of the reflective disc  110  illustrated in  FIGS. 2, 3  and  4 . The thinner semi-reflective layer  143  allows for some transmission of the interrogation beam  152  through the structural layers of the transmissive disc as shown in  FIGS. 6 and 12 . The thin semi-reflective layer  143  may be formed from a metal such as aluminum or gold.  
         [0048]      FIG. 6  is an enlarged perspective view of the substrate  120  and semi-reflective layer  143  of the transmissive embodiment of the optical bio-disc  110  illustrated in  FIG. 5 . The thin semi-reflective layer  143  may be made from a metal such as aluminum or gold. In the preferred embodiment, the thin semi-reflective layer  143  of the transmissive disc illustrated in  FIGS. 5 and 6  is approximately 100-300 Å thick and does not exceed 400 Å. This thinner semi-reflective layer  143  allows a portion of the incident or interrogation beam  152  to penetrate and pass through the semi-reflective layer  143  to be detected by a top detector  158 ,  FIGS. 10 and 12 , while some of the light is reflected or returned back along the incident path. As indicated below, Table 1 presents the reflective and transmissive characteristics of a gold film relative to the thickness of the film. The gold film layer is fully reflective at a thickness greater than 800 Å, while the threshold density for transmission of light through the gold film is approximately 400 Å.  
                                                   TABLE 1                           Au Film Reflection and Transmission (Absolute Values)            Thickness (Angstroms)   Thickness (nm)   Reflectance   Transmittance                    0   0   0.0505   0.9495       50   5   0.1683   0.7709       100   10   0.3981   0.5169       150   15   0.5873   0.3264       200   20   0.7142   0.2057       250   25   0.7959   0.1314       300   30   0.8488   0.0851       350   35   0.8836   0.0557       400   40   0.9067   0.0368       450   45   0.9222   0.0244       500   50   0.9328   0.0163       550   55   0.9399   0.0109       600   60   0.9448   0.0073       650   65   0.9482   0.0049       700   70   0.9505   0.0033       750   75   0.9520   0.0022       800   80   0.9531   0.0015                    
         [0049]     With reference next to  FIG. 8 , there is shown a top plan view of the transmissive type optical bio-disc  110  illustrated in  FIGS. 5 and 6  with the transparent cap portion  116  revealing the fluidic channels, the trigger markings  126 , and the target zones  140  as situated within the disc.  
         [0050]      FIG. 9  is an enlarged perspective view of the optical bio-disc  110  according to the transmissive disc embodiment of the present invention. The disc  110  is illustrated with a portion of the various layers thereof cut away to show a partial sectional view of each principal layer, substrate, coating, or membrane.  FIG. 9  illustrates a transmissive disc format with the clear cap portion  116 , the thin semi-reflective layer  143  on the substrate  120 , and trigger markings  126 . In this embodiment, trigger markings  126  include opaque material placed on the top portion of the cap. Alternatively the trigger marking  126  may be formed by clear, non-reflective windows etched on the thin reflective layer  143  of the disc, or any mark that absorbs or does not reflect the signal coming from the trigger detector  160 ,  FIG. 10 .  FIG. 9  also shows the target zones  140  formed by marking the designated area in the indicated shape or alternatively in any desired shape. Markings to indicate target zone  140  may be made on the thin semi-reflective layer  143  on the substrate  120  or on the bottom portion of the substrate  120  (under the disc). Alternatively, the target zones  140  may be formed by a masking technique that includes masking all, or a portion, of the thin semi-reflective layer  143  except the target zones  140 . In this embodiment, target zones  140  may be created by silk screening ink onto the thin semi-reflective layer  143 . In the transmissive disc format illustrated in  FIGS. 5, 8 , and  9 , the target zones  140  may alternatively be defined by address information encoded on the disc. In this embodiment, target zones  140  do not include a physically discernable edge boundary.  
         [0051]     With continuing reference to  FIG. 9 , an active layer  144  is illustrated as applied over the thin semi-reflective layer  143 . In the preferred embodiment, the active layer  144  is a 10 to 200 μm thick layer of 2% polystyrene. Alternatively, polycarbonate, gold, activated glass, modified glass, or modified polystyrene, for example, polystyrene-co-maleic anhydride, may be used. In addition, hydrogels can be used. As illustrated in this embodiment, the plastic adhesive member  118  is applied over the active layer  144 . The exposed section of the plastic adhesive member  118  illustrates the cut out or stamped U-shaped form that creates the fluidic circuits  128 .  
         [0052]     The final principal structural layer in this transmissive embodiment of the present bio-disc  110  is the clear, non-reflective cap portion  116  that includes inlet ports  122  and vent ports  124 .  
         [0053]     Referring now to  FIG. 10 , there is a representation in perspective and block diagram illustrating optical components  148 , a light source  150  that produces the incident or interrogation beam  152 , a return beam  154 , and a transmitted beam  156 . In the case of the reflective bio-disc illustrated in  FIG. 4 , the return beam  154  is reflected from the reflective surface  146  of the cap portion  116  of the optical bio-disc  110 . In this reflective embodiment of the present optical bio-disc  110 , the return beam  154  is detected and analyzed for the presence of signal elements by a bottom detector  157 . In the transmissive bio-disc format, on the other hand, the transmitted beam  156  is detected, by a top detector  158 , and is also analyzed for the presence of signal elements. In the transmissive embodiment, a photo detector may be used as a top detector  158 .  
         [0054]      FIG. 10  also shows a hardware trigger mechanism that includes the trigger markings  126  on the disc and a trigger detector  160 . The hardware triggering mechanism is used in both reflective bio-discs ( FIG. 4 ) and transmissive bio-discs ( FIG. 9 ). The triggering mechanism allows the processor  166  to collect data when the interrogation beam  152  is on a respective target zone  140 . Furthermore, in the transmissive bio-disc system, a software trigger may also be used. The software trigger uses the bottom detector to signal the processor  166  to collect data as soon as the interrogation beam  152  hits the edge of a respective target zone  140 .  FIG. 10  further illustrates a drive motor  162  and a controller  164  for controlling the rotation of the optical bio-disc  110 .  FIG. 10  also shows the processor  166  and analyzer  168  implemented in the alternative for processing the return beam  154  and transmitted beam  156  associated the transmissive optical bio-disc.  
         [0055]     As shown in  FIG. 11 , there is presented a partial cross sectional view of the reflective disc embodiment of the optical bio-disc  110  according to the present invention.  FIG. 11  illustrates the substrate  120  and the reflective layer  142 . As indicated above, the reflective layer  142  may be made from a material such as aluminum, gold or other suitable reflective material. In this embodiment, the top surface of the substrate  120  is smooth.  FIG. 11  also shows the active layer  144  applied over the reflective layer  142 . As also shown in  FIG. 11 , the target zone  140  is formed by removing an area or portion of the reflective layer  142  at a desired location or, alternatively, by masking the desired area prior to applying the reflective layer  142 . As further illustrated in  FIG. 11 , the plastic adhesive member  118  is applied over the active layer  144 .  FIG. 11  also shows the cap portion  116  and the reflective surface  146  associated therewith. Thus when the cap portion  116  is applied to the plastic adhesive member  118  including the desired cutout shapes, flow channel  130  is thereby formed. As indicated by the arrowheads shown in  FIG. 11 , the path of the incident beam  152  is initially directed toward the substrate  120  from below the disc  110 . The incident beam then focuses at a point proximate the reflective layer  142 . Since this focusing takes place in the target zone  140  where a portion of the reflective layer  142  is absent, the incident light continues along a path through the active layer  144  and into the flow channel  130 . The incident beam  152  then continues upwardly traversing through the flow channel to eventually fall incident onto the reflective surface  146 . At this point, the incident beam  152  is returned or reflected back along the incident path and thereby forms the return beam  154 .  
         [0056]      FIG. 12  is a partial cross sectional view of the transmissive embodiment of the bio-disc  110  according to the present invention.  FIG. 12  illustrates a transmissive disc format with the clear cap portion  116  and the thin semi-reflective layer  143  on the substrate  120 .  FIG. 12  also shows the active layer  144  applied over the thin semi-reflective layer  143 . In the preferred embodiment, the transmissive disc has the thin semi-reflective layer  143  made from a metal such as aluminum or gold approximately 100-300 Angstroms thick and does not exceed 400 Angstroms. This thin semi-reflective layer  143  allows a portion of the incident or interrogation beam  152 , from the light source  150 ,  FIG. 10 , to penetrate and pass upwardly through the disc to be detected by a top detector  158 , while some of the light is reflected back along the same path as the incident beam but in the opposite direction. In this arrangement, the return or reflected beam  154  is reflected from the semi-reflective layer  143 . Thus in this manner, the return beam  154  does not enter into the flow channel  130 . The reflected light or return beam  154  may be used for tracking the incident beam  152  on pre-recorded information tracks formed in or on the semi-reflective layer  143  as described in more detail in conjunction with  FIGS. 13 and 14 . In the disc embodiment illustrated in  FIG. 12 , a physically defined target zone  140  may or may not be present. Target zone  140  may be created by direct markings made on the thin semi-reflective layer  143  on the substrate  120 . These marking may be formed using silk screening or any equivalent method. In the alternative embodiment where no physical indicia are employed to define a target zone (such as, for example, when encoded software addressing is utilized) the flow channel  130  in effect may be employed as a confined target area in which inspection of an investigational feature is conducted.  
         [0057]      FIG. 13  is a cross sectional view taken across the tracks of the reflective disc embodiment of the bio-disc  110  according to the present invention. This view is taken longitudinally along a radius and flow channel of the disc.  FIG. 13  includes the substrate  120  and the reflective layer  142 . In this embodiment, the substrate  120  includes a series of grooves  170 . The grooves  170  are in the form of a spiral extending from near the center of the disc toward the outer edge. The grooves  170  are implemented so that the interrogation beam  152  may track along the spiral grooves  170  on the disc. This type of groove  170  is known as a “wobble groove”. A bottom portion having undulating or wavy sidewalls forms the groove  170 , while a raised or elevated portion separates adjacent grooves  170  in the spiral. The reflective layer  142  applied over the grooves  170  in this embodiment is, as illustrated, conformal in nature.  FIG. 13  also shows the active layer  144  applied over the reflective layer  142 . As shown in  FIG. 13 , the target zone  140  is formed by removing an area or portion of the reflective layer  142  at a desired location or, alternatively, by masking the desired area prior to applying the reflective layer  142 . As further illustrated in  FIG. 13 , the plastic adhesive member  118  is applied over the active layer  144 .  FIG. 13  also shows the cap portion  116  and the reflective surface  146  associated therewith. Thus, when the cap portion  116  is applied to the plastic adhesive member  118  including the desired cutout shapes, the flow channel  130  is thereby formed.  
         [0058]      FIG. 14  is a cross sectional view taken across the tracks of the transmissive disc embodiment of the bio-disc  110  according to the present invention as described in  FIG. 12 , for example. This view is taken longitudinally along a radius and flow channel of the disc.  FIG. 14  illustrates the substrate  120  and the thin semi-reflective layer  143 . This thin semi-reflective layer  143  allows the incident or interrogation beam  152 , from the light source  150 , to penetrate and pass through the disc to be detected by the top detector  158 , while some of the light is reflected back in the form of the return beam  154 . The thickness of the thin semi-reflective layer  143  is determined by the minimum amount of reflected light needed by the disc reader to maintain its tracking ability. The substrate  120  in this embodiment, like that discussed in  FIG. 13 , includes the series of grooves  170 . The grooves  170  in this embodiment are also preferably in the form of a spiral extending from near the center of the disc toward the outer edge. The grooves  170  are implemented so that the interrogation beam  152  may track along the spiral.  FIG. 14  also shows the active layer  144  applied over the thin semi-reflective layer  143 . As further illustrated in  FIG. 14 , the plastic adhesive member or channel layer  118  is applied over the active layer  144 .  FIG. 14  also shows the cap portion  116  without a reflective surface  146 . Thus, when the cap is applied to the plastic adhesive member  118  including the desired cutout shapes, the flow channel  130  is thereby formed and a part of the incident beam  152  is allowed to pass therethrough substantially unreflected.  
         [0059]      FIG. 15  is a view similar to  FIG. 11  showing the entire thickness of the reflective disc and the initial refractive property thereof.  FIG. 16  is a view similar to  FIG. 12  showing the entire thickness of the transmissive disc and the initial refractive property thereof. Grooves  170  are not seen in  FIGS. 15 and 16  since the sections are cut along the grooves  170 .  FIGS. 15 and 16  show the presence of the narrow flow channel  130  that is situated perpendicular to the grooves  170  in these embodiments.  FIGS. 13, 14 ,  15 , and  16  show the entire thickness of the respective reflective and transmissive discs. In these figures, the incident beam  152  is illustrated initially interacting with the substrate  120  which has refractive properties that change the path of the incident beam as illustrated to provide focusing of the beam  152  on the reflective layer  142  or the thin semi-reflective layer  143 .  
         [0060]     Alternative embodiments of the bio-disc according to the present invention will now be described with reference to  FIGS. 17A, 17B ,  17 C,  18 A,  18 B, and  18 C. Various features of the discs of these latter embodiments have been already illustrated with reference to FIGS.  1  to  16 , and therefore such common features will not be described again in the following. Accordingly, and for the sake of simplicity, as a general rule in  FIGS. 17 and 18 , the features differentiating the bio-disc  110  from those of FIGS.  1  to  21  are represented.  
         [0061]     Furthermore, the following description of the bio-disc of the invention can be readily applied to a transmissive-type as well as to a reflective-type optical bio-disc described above in conjunction with FIGS.  2  to  9 .  
         [0062]      FIG. 17A  is an exploded perspective view of a reflective bio-disc incorporating equi-radial channels  200  of the present invention. This general construction corresponds to the radial-channel disc shown in  FIG. 2 . The e-rad or eRad implementation of the bio-disc  110  shown in  FIG. 17A  similarly includes the cap  116 , the channel layer  118 , and the substrate  120 . The channel layer  118  includes the equi-radial fluid channels  200 , while the substrate  120  includes the corresponding arrays of target zones  140 .  
         [0063]      FIG. 17B  is a top plan view of the disc shown in  FIG. 17A .  FIG. 17B  further shows a top plan view of an embodiment of eRad disc with a transparent cap portion, which disc has two tiers of circumferential fluid channels with ABO chemistry and two blood types (A+ and AB+). As shown in  FIG. 17B , it is also possible to provide a priori, at the manufacturing stage of the disc of the invention, a plurality of entry ports, eventually at different radial coordinate, so that a range of equi-radial, spiralling, or radial reaction sites and/or channels are possible on one disc. These channels can be used for different test suites, or for multiple samples of single test suites.  
         [0064]      FIG. 17C  is a perspective view of the disc illustrated in  FIG. 17A  with cut-away sections showing the different layers of the e-radial reflective disc. This view is similar to the reflective disc shown in  FIG. 4 . The e-rad implementation of the reflective bio-disc shown in  FIG. 17C  similarly includes the reflective layer  142 , active layer  144  as applied over the reflective layer  142 , and the reflective layer  146  on the cap portion  116 .  
         [0065]      FIG. 18A  is an exploded perspective view of a transmissive bio-disc utilizing the e-radial channels of the present invention. This general construction corresponds to the radial-channel disc shown in  FIG. 5 . The transmissive e-rad implementation of the bio-disc  110  shown in  FIG. 18A  similarly includes the cap  116 , the channel layer  118 , and the substrate  120 . The channel layer  118  includes the equi-radial fluid channels  200 , while the substrate  120  includes the corresponding arrays of target zones  140 .  
         [0066]      FIG. 18B  is a top plan view of the transmissive e-rad disc shown in  FIG. 18A .  FIG. 18B  further shows two tiers of circumferential fluid channels with ABO chemistry and two blood types (A+ and AB+). As previously discussed, the assays are performed in the target, capture, or analysis zones  140 .  
         [0067]      FIG. 18C  is a perspective view of the disc illustrated in  FIG. 18A  with cut-away sections showing the different layers of this embodiment of the e-rad transmissive bio-disc. This view is similar to the transmissive disc shown in  FIG. 9 . The e-rad implementation of the transmissive bio-disc shown in  FIG. 18C  similarly includes the thin semi-reflective layer  143  and the active layer  144  as applied over the thin semi-reflective layer  143 .  
         [heading-0068]     Quantification of Glucose and Cholesterol Using the Optical Bio-Disc  
         [0069]     A criterion that defines a good diagnostic assay is the ease by which one performs the assay. For colorimetric assays on optical bio-discs, the reagents used for the assay may advantageously be immobilized on the disc prior to the assay. There are several methods that can be used for reagent deposition. They include air or vacuum evaporation, enzyme immobilization by chemical linkage, lyophilization, or reagent printing on a suitable medium (i.e. filter paper or membrane strips). The above methods have been tested on bio-discs. In an advantageous embodiment, a reagent printing process is used to apply the reagents on the membrane strips because reagent stability for several weeks or months is preserved. In one embodiment, the printing process may be performed using a printing device, such as an ink jet printer.  
         [0070]     For each assay, the reagents are printed on 3×5×0.3 mm strips. The printing can be done manually with a pipettor, or by automatic applicators. The volume of reagents deposited on the strips varies from 2 to 5 ul. The strips are deposited on the bio-disc at the time of assembly. The thickness of the reagent strips is such that they will fit securely within the channels of the bio-disc.  
         [0071]     The selection of membrane strips for reagent deposition affects the success of the assay. Membrane strips are traditionally used in dipstick or lateral flow assays, where the chemistry typically occurs on a solid phase. However, for colorimetric assays on optical analysis discs, the chemistry between the sample and the reagents occurs in solution. For this reason, the use of membrane strips in colorimetric assays on bio-discs is rather unique. Further, instead of using nitrocellulose membranes that are normally used in lateral flow assays, the membrane strips chosen for reagent deposition in colorimetric assays should have a good absorbing capacity to accommodate the volume of reagent deposited, while retaining good release efficiency. A membrane strip with good release efficiency allows the reagents to be released from the storage medium (membrane strip) into solution as soon as the sample is injected into the reaction chamber, where they effectively catalyze the desired reactions. This allows for the color development from the reaction to be homogenous throughout the reaction chamber. The membrane strips for reagent deposition can be prepared independently of the discs and easily deposited within the disc during disc assembly. Numerous membrane strips have been tested for this particular function. In one embodiment, a membrane strip for reagent deposition is a hydrophilic polyethersulfone membrane of pore size 0.2 um or above (Pall, Port Washington, New York). In another embodiment, a membrane strip for reagent deposition is a bibulous hydrophilic material. Those of skill in the art will also recognized that other materials that have the above discussed properties may readily be used for membrane strips.  
         [0072]     On optical bio-discs, calibrators that are normally used in colorimetric assays may be replaced by calibration bars, which express the concentrations of the calibrators in terms of the relative amount of light transmitted or reflected. The calibration bars could be created either in the software or directly on the disc. The creation of calibration bars reduces the assay time significantly and makes the assay much more user friendly.  
         [0073]     According to one aspect of the present invention, there are provided detection methods for quantifying the concentration of an analyte of interest in a biological sample on the bio-discs. The detection includes directing a beam of electromagnetic energy from a disc drive toward the capture field and analyzing electromagnetic energy returned from or transmitted through the capture field.  
         [0074]     The optical density change in colorimetric assays can be quantified by the optical disc reader by two related ways. These include measuring the change in light either reflected or transmitted. The disc may be referred to as reflective, transmissive, or some combination of reflective and transmissive. In a reflective disc, an incident light beam is focused onto the disc (typically at a reflective surface where information is encoded), reflected, and returned through optical elements to a detector on the same side of the disc as the light source. In a transmissive disc, light passes through the disc (or portions thereof) to a detector on the other side of the disc from the light source. In a transmissive portion of a disc, some light may also be reflected and detected as reflected light. Different detection systems are used for different types of bio-discs (top versus bottom detector).  
         [0075]     The conversion of data captured by the CD reader into meaningful concentration units is mediated via data processing software specific for the assay of interest. In one embodiment, the data captured by the CD reader may be used to determine additional characteristics of, or related to, the assay, such as an amount of a target substance present.  
         [0076]     The apparatus and methods in embodiments of the present invention can be designed for use by an end-user, inexpensively, without specialized expertise and expensive equipment. The system can be made portable, and thus usable in remote locations where traditional diagnostic equipment may not generally be available.  
         [0077]     Alternatively, fluorescent assays can be carried out to quantify the concentration of an analyte of interest in a biological sample on the optical discs. In this case, the energy source in the disc drive preferably has a wavelength controllable light source and a detector that is or can be made specific to a particular wavelength. Alternatively, a disc drive can be made with a specific light source and detector to produce a dedicated device, in which case the source may need fine-tuning.  
         [0078]     More specifically, the present invention is directed to sample preparation and generation of calibration bars for colorimetric and fluorescent assays as implemented on optical analysis discs.  
         [0079]     A criterion that defines a good diagnostic assay is the ease by which one performs the assay. For colorimetric assays on optical bio-discs, the reagents used for the assay may be immobilized on the disc prior to the assay. At the time of the assay, the end-user just needs to dilute the sample with water then injects the sample into the channel. Alternatively, undiluted samples may be used directly.  
         [0080]     Colorimetric assays on bio-disc can use either serum or blood as sample sources. Serum can be a direct substrate for the assays. Blood can also be used as sample source by selective filtration of red blood cells using membranes such as HemaSep or CytoSep (Pall, Port Washington, New York).  
         [0081]     In lab-based colorimetric assays, the concentrations of unknown samples were normally derived from calibrators or solutions with known concentrations. The use of calibrators necessitated additional preparation steps, which were more time-consuming and error prone. On optical bio-discs, calibrators in colorimetric assays may be replaced by calibration bars. The creation of calibrator bars is achieved by measuring the amount of light transmitted or reflected by known concentrations of analytes. The amount of light transmitted or reflected may then be expressed relative to the minimum and maximum amount of light transmitted or reflected. The maximum amount of light transmitted or reflected may be obtained in the absence of any solution in the reaction zone. The minimum amount of light transmitted or reflected may be the amount of light transmitted or reflected from a blocked reaction zone. The blocking can be mediated with any available light blocking structure, such as a piece of black tape, for example. The calibration bars could be created either in the software or directly on the disc.  
         [0082]      FIGS. 19 and 20  illustrate the generation of calibration curves for the glucose and cholesterol assays, respectively. The first step in the generation of the calibration curves was filling the fluidic channel or analysis chambers with calibrators of known concentrations. One analysis chamber was left empty to measure the maximum of light that can be transmitted. Another analysis chamber was blocked with a black tape; the voltage measured in that channel represents the minimum of light that can be transmitted or reflected. The table illustrated in the figures expresses the percentage of light transmitted by the calibrators with respect to the references. The calibration curves shown expresses the inverse relationship between the calibrator concentrations and the amount of light transmitted or reflected.  
         [heading-0083]     Other Implementations of the Current Invention  
         [0084]     This invention or different aspects thereof may be readily implemented in or adapted to many of the discs, assays, and systems disclosed in the following commonly assigned and co-pending patent applications: U.S. patent application Ser. No. 09/378,878 entitled “Methods and Apparatus for Analyzing Operational and Non-operational Data Acquired from Optical Discs” filed Aug. 23, 1999; U.S. Provisional Patent Application Ser. No. 60/150,288 entitled “Methods and Apparatus for Optical Disc Data Acquisition Using Physical Synchronization Markers” filed Aug. 23, 1999; U.S. patent application Ser. No. 09/421,870 entitled “Trackable Optical Discs with Concurrently Readable Analyte Material” filed Oct. 26, 1999; U.S. patent application Ser. No. 09/643,106 entitled “Methods and Apparatus for Optical Disc Data Acquisition Using Physical Synchronization Markers” filed Aug. 21, 2000; U.S. patent application Ser. No. 09/999,274 entitled “Optical Biodiscs with Reflective Layers” filed Nov. 15, 2001; U.S. patent application Ser. No. 09/988,728 entitled “Methods and Apparatus for Detecting and Quantifying Lymphocytes with Optical Biodiscs” filed Nov. 16, 2001; U.S. patent application Ser. No. 09/988,850 entitled “Methods and Apparatus for Blood Typing with Optical Bio-discs” filed Nov. 19, 2001; U.S. patent application Ser. No. 09/989,684 entitled “Apparatus and Methods for Separating Agglutinants and Disperse Particles” filed Nov. 20, 2001; U.S. patent application Ser. No. 09/997,741 entitled “Dual Bead Assays Including Optical Biodiscs and Methods Relating Thereto” filed Nov. 27, 2001; U.S. patent application Ser. No. 09/997,895 entitled “Apparatus and Methods for Separating Components of Particulate Suspension” filed Nov. 30, 2001; U.S. patent application Ser. No. 10/005,313 entitled “Optical Discs for Measuring Analytes” filed Dec. 7, 2001; U.S. patent application Ser. No. 10/006,371 entitled “Methods for Detecting Analytes Using Optical Discs and Optical Disc Readers” filed Dec. 10, 2001; U.S. patent application Ser. No. 10/006,620 entitled “Multiple Data Layer Optical Discs for Detecting Analytes” filed Dec. 10, 2001; U.S. patent application Ser. No. 10/006,619 entitled “Optical Disc Assemblies for Performing Assays” filed Dec. 10, 2001; U.S. patent application Ser. No. 10/020,140 entitled “Detection System For Disk-Based Laboratory and Improved Optical Bio-Disc Including Same” filed Dec. 14, 2001; U.S. patent application Ser. No. 10/035,836 entitled “Surface Assembly for Immobilizing DNA Capture Probes and Bead-Based Assay Including Optical Bio-Discs and Methods Relating Thereto” filed Dec. 21, 2001; U.S. patent application Ser. No. 10/038,297 entitled “Dual Bead Assays Including Covalent Linkages for Improved Specificity and Related Optical Analysis Discs” filed Jan. 4, 2002; U.S. patent application Ser. No. 10/043,688 entitled “Optical Disc Analysis System Including Related Methods for Biological and Medical Imaging” filed Jan. 10, 2002; U.S. Provisional Application Ser. No. 60/348,767 entitled “Optical Disc Analysis System Including Related Signal Processing Methods and Software” filed Jan. 14, 2002 U.S. patent application Ser. No. 10/086,941 entitled “Methods for DNA Conjugation Onto Solid Phase Including Related Optical Biodiscs and Disc Drive Systems” filed Feb. 26, 2002; U.S. patent application Ser. No. 10/087,549 entitled “Methods for Decreasing Non-Specific Binding of Beads in Dual Bead Assays Including Related Optical Biodiscs and Disc Drive Systems” filed Feb. 28, 2002; U.S. patent application Ser. No. 10/099,256 entitled “Dual Bead Assays Using Cleavable Spacers and/or Ligation to Improve Specificity and Sensitivity Including Related Methods and Apparatus” filed Mar. 14, 2002; U.S. patent application Ser. No. 10/099,266 entitled “Use of Restriction Enzymes and Other Chemical Methods to Decrease Non-Specific Binding in Dual Bead Assays and Related Bio-Discs, Methods, and System Apparatus for Detecting Medical Targets” also filed Mar. 14, 2002; U.S. patent application Ser. No. 10/121,281 entitled “Multi-Parameter Assays Including Analysis Discs and Methods Relating Thereto” filed Apr. 11, 2002; U.S. patent application Ser. No. 10/150,575 entitled “Variable Sampling Control for Rendering Pixelization of Analysis Results in a Bio-Disc Assembly and Apparatus Relating Thereto” filed May 16, 2002; U.S. patent application Ser. No. 10/150,702 entitled “Surface Assembly For Immobilizing DNA Capture Probes in Genetic Assays Using Enzymatic Reactions to Generate Signals in Optical Bio-Discs and Methods Relating Thereto” filed May 16, 2002; U.S. patent application Ser. No. 10/194,418 entitled “Optical Disc System and Related Detecting and Decoding Methods for Analysis of Microscopic Structures” filed Jul. 12, 2002; U.S. patent application Ser. No. 10/194,396 entitled “Multi-Purpose Optical Analysis Disc for Conducting Assays and Various Reporting Agents for Use Therewith” also filed Jul. 12, 2002; U.S. patent application Ser. No. 10/199,973 entitled “Transmissive Optical Disc Assemblies for Performing Physical Measurements and Methods Relating Thereto” filed Jul. 19, 2002; U.S. patent application Ser. No. 10/201,591 entitled “Optical Analysis Disc and Related Drive Assembly for Performing Interactive Centrifugation” filed Jul. 22, 2002; U.S. patent application Ser. No. 10/205,011 entitled “Method and Apparatus for Bonded Fluidic Circuit for Optical Bio-Disc” filed Jul. 24, 2002; U.S. patent application Ser. No. 10/205,005 entitled “Magnetic Assisted Detection of Magnetic Beads Using Optical Disc Drives” also filed Jul. 24, 2002; U.S. patent application Ser. No. 10/230,959 entitled “Methods for Qualitative and Quantitative Analysis of Cells and Related Optical Bio-Disc Systems” filed Aug. 29, 2002; U.S. patent application Ser. No. 10/233,322 entitled “Capture Layer Assemblies for Cellular Assays Including Related Optical Analysis Discs and Methods” filed Aug. 30, 2002; U.S. patent application Ser. No. 10/236,857 entitled “Nuclear Morphology Based Identification and Quantification of White Blood Cell Types Using Optical Bio-Disc Systems” filed Sep. 6, 2002; U.S. patent application Ser. No. 10/241,512 entitled “Methods for Differential Cell Counts Including Related Apparatus and Software for Performing Same” filed Sep. 11, 2002; U.S. patent application Ser. No. 10/279,677 entitled “Segmented Area Detector for Biodrive and Methods Relating Thereto” filed Oct. 24, 2002; U.S. patent application Ser. No. 10/293,214 entitled “Optical Bio-Discs and Fluidic Circuits for Analysis of Cells and Methods Relating Thereto” filed on Nov. 13, 2002; U.S. patent application Ser. No. 10/298,263 entitled “Methods and Apparatus for Blood Typing with Optical Bio-Discs” filed on Nov. 15, 2002; U.S. patent application Ser. No. 10/307,263 entitled “Magneto-Optical Bio-Discs and Systems Including Related Methods” filed Nov. 27, 2002; U.S. patent application Ser. No. 10/341,326 entitled “Method and Apparatus for Visualizing Data” filed Jan. 13, 2003; U.S. patent application Ser. No. 10/345,122 entitled “Methods and Apparatus for Extracting Data From an Optical Analysis Disc” filed on Jan. 14, 2003; U.S. patent application Ser. No. 10/347,155 entitled “Optical Discs Including Equi-Radial and/or Spiral Analysis Zones and Related Disc Drive Systems and Methods” filed on Jan. 15, 2003; U.S. patent application Ser. No. 10/347,119 entitled “Bio-Safe Dispenser and Optical Analysis Disc Assembly” filed Jan. 17, 2003; U.S. patent application Ser. No. 10/348,049 entitled “Multi-Purpose Optical Analysis Disc for Conducting Assays and Related Methods for Attaching Capture Agents” filed on Jan. 21, 2003; U.S. patent application Ser. No. 10/348,196 entitled “Processes for Manufacturing Optical Analysis Discs with Molded Microfluidic Structures and Discs Made According Thereto” filed on Jan. 21, 2003; U.S. patent application Ser. No. 10/351,604 entitled “Methods for Triggering Through Disc Grooves and Related Optical Analysis Discs and System” filed on Jan. 23, 2003; U.S. patent application Ser. No. 10/351,280 entitled “Bio-Safety Features for Optical Analysis Discs and Disc System Including Same” filed on Jan. 23, 2003; U.S. patent application Ser. No. 10/351,244 entitled “Manufacturing Processes for Making Optical Analysis Discs Including Successive Patterning Operations and Optical Discs Thereby Manufactured” filed on Jan. 24, 2003; U.S. patent application Ser. No. 10/353,777 entitled “Processes for Manufacturing Optical Analysis Discs with Molded Microfluidic Structures and Discs Made According Thereto” filed on Jan. 27, 2003; U.S. patent application Ser. No. 10/353,839 entitled “Method and Apparatus for Logical Triggering” filed on Jan. 28, 2003; and U.S. patent application Ser. No. 10/356,666 entitled “Methods For Synthesis of Bio-Active Nanoparticles and Nanocapsules For Use in Optical Bio-Disc Assays and Disc Assembly Including Same” filed Jan. 30, 2003. All of these applications are herein incorporated by reference in their entireties. They thus provide background and related disclosure as support hereof as if fully repeated herein.  
         [heading-0085]     Concluding Summary  
         [0086]     All patents, provisional applications, patent applications, technical specifications, and other publications mentioned in this specification are incorporated herein in their entireties by reference.  
         [0087]     While this invention has been described in detail with reference to certain preferred embodiments, it should be appreciated that the present invention is not limited to those precise embodiments. Rather, in view of the present optical bio-system disclosure that describes the current best mode for practicing the invention, many modifications and variations would present themselves to those of skill in the art without departing from the scope and spirit of this invention. The scope of the invention is, therefore, indicated by the following claims rather than by the foregoing description. All changes, modifications, and variations coming within the meaning and range of equivalency of the claims are to be considered within their scope.  
         [0088]     Furthermore, those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.