Patent Publication Number: US-2003232384-A1

Title: Microarray system utilizing microtiter plates

Description:
FIELD OF THE INVENTION  
       [0001] This invention relates in general to a biological analysis system and more particularly to a biological analysis system which integrates microarray receivers with microtiter plates.  
       BACKGROUND OF THE INVENTION  
       [0002] In recent years, high density arrays formed by spatially addressable bioactive probes on a receiver has greatly enhanced and simplified the process of biological research and development. More specifically, the molecular biology community has developed considerable infrastructure based on microtiter plate technology. Microtiter plates are devices that contain wells that enable molecular biological experiments by combining several chemistries confined in wells. An example of microtiter plates can be found with many manufacturers, including Whatman Philtronics as well as Corning, Inc., to name a few. Microtiter plates are available in many configurations including 96 wells, 192 wells, 384 wells, etc. Plates are available with round wells, square wells, and with filters in the bottom, for example. Since microtiter plate technology has been so widely adopted by the molecular biology community as well as the drug discovery and the drug development communities, a significant amount of robotics technology has been developed to automate the processes that use microtiter plates. In general, robotics are used to move plates from location to location as well as dispense liquids into the wells. Automation provides speed as well as consistency. Array-based ELISAs for High-Throughput Analysis of Human Cytokines, BioTechniques, Vol. 31, No. 1 (2201), 186-194, (Pierce Endogen) describes a technique where antibody solutions at 20 nL/spot were spotted in a 3×3 pattern in well plates. High-Throughput micorarray-based enzyme-Linked Immunosorbent Assay (ELISA), BioTechniques, Vol. 27, No. 4, (1999), 778-788, (Genometrix) demonstrates the potential to conduct multi-analyte assays using 96 well microarray-based ELISA format.  
       [0003] The Corning/Life Sciences web site has extensive information on products used in microtiter plate assay systems. Problems with this technology are the expense and complexity of equipment for forming the biosite arrays in the wells of the microtiter plates.  
       [0004] The following patents disclose alternative microarray techniques that do not adequately solve these problems. U.S. Pat. No. 6,083,763, issued Jul. 4, 2000, inventor Balch, discloses in FIG. 7, a thin film substrate having conductive patterns and biosites deposited thereon which are bonded to a reaction vessel having a matrix of open reaction chambers that are closed off by the thin film substrate. The biosites are disclosed as being deposited by ink-jet, capillary, or photolithiographic techniques. It is also disclosed to bond thin films of plastic or glass to conventional bottomless microtiter plates. Biosites are deposited either before or after the bottoms are bonded to the plate.  
       [0005] U.S. Pat. No. 6,232,066, issued May 15, 2001, inventors Felder et al., discloses a multi-well assemblage including a well separator, a subdivider, and a base which are joined together. Biosite arrays are attached to the base.  
       [0006] U.S. Pat. No. 6,309,828, issued Oct. 30, 2001, inventors Schleifer et al., discloses a technique for applying an array of nucleic acid molecules on a substrate by a device including a synthesis unit in communication with a purification unit in communication with a printing unit. The synthesis, purification, and printing units, are aligned, bottomless microtiter plates.  
       SUMMARY OF THE INVENTION  
       [0007] According to the present invention, there is provided a solution to the problems discussed above.  
       [0008] According to a feature of the present invention, there is provided a biological analysis system comprising: a microarray receiver including a substrate coated with a composition including a population of biological probe modified micro-spheres immobilized in a coating containing a gelling agent or precursor to a gelling agent, at least a sub-population of said population of micro-spheres containing an optical bar code generated from a least one colorant associated with the micro-spheres and including a biological probe; and a microtiter plate having a plurality of wells open at first and second ends in liquid sealing contact with said microarray receiver at said first open ends, each of said wells adapted to receive a fluorescently/chemiluminescently labeled biological target sample which interacts fluorescently/chemiluminescently with said biological probe.  
       ADVANTAGEOUS EFFECT OF THE INVENTION  
       [0009] The invention has the following advantages.  
       [0010] 1. A system is provided incorporating microtiter plates and microarray receivers which is simple and cost efficient.  
       [0011] 2. The microarray receivers can be either patterned or random arrays of biological probes.  
       [0012] 3. The receiver can be opaque or transparent to facilitate detection by reflection emission or transmission detection devices. 
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0013]FIG. 1 is a partial elevational diagrammatic view illustrating an embodiment of the invention.  
     [0014]FIG. 2 is a perspective diagrammatic view illustrating the embodiment of FIG. 1.  
     [0015]FIGS. 3A and 3B are diagrammatic views of microarray receivers showing respectively patterned and randomly distributed biologically active sites.  
     [0016] FIGS.  4 A- 4 C are respective diagrammatic views of optical readout systems for use in conjunction with the present invention.  
     [0017]FIG. 5 is a diagrammatic view of a microarray receiver which can be used in the present invention.  
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
     [0018] In general, according to the present invention there is provided a biological analysis system including the combination of a microtiter plate having an array of open-ended wells and a microarray receiver having random or predetermined array of biologically functional sites which form a repetitive pattern on the receiver and which are in contact with the open ended wells. The microarray receiver is described in U.S. patent application Ser. No. 09/942,241, the contents of which are hereby incorporated by reference. A general description of the microarray receiver will now be given but reference is made to the latter patent application for a more complete description.  
     [0019] The microarray receiver according to the invention, includes a substrate coated with a composition comprising micro-spheres (beads) dispersed in a fluid containing a gelling agent or a precursor to a gelling agent, wherein the micro-spheres are immobilized in a random or ordered position on the substrate. The substrate is free of receptors designed to physically or chemically interact with the micro-spheres. One or more sub-populations of the population of microspheres contain a unique optical bar code generated from at least one colorant associated with the micro-spheres and including a unique biological functionality or probe which react with analytes with which they come in contact.  
     [0020] The distribution or pattern of micro-spheres on the substrate may be entirely random (a spatial distribution showing no reference or bias) or be attracted or held to sites that are pre-marked or predetermined on the substrate. Each micro-sphere in the array has a distinct signature based on color which may be derived from mixing three dyes representing the primary colors Red (R), Green (G), and Blue (B) to create thousands of distinguishable micro-spheres with distinct color addresses (unique RGB values, e.g., R=0, G=204, B=153). The micro-spheres are made with active sites on their surface to which are attached a specific bioactive probe. Therefore, each color address can correspond to a specific bioactive probe.  
     [0021] A microarray or population of micro-spheres can include a few or hundreds or more of sub-populations of micro-spheres, where each sub-population comprises the same color code and the same bio-active probe. Each microarray of micro-spheres occupies a sub-area of the substrate and is repeated in a pattern over the area of the substrate. The dimensional area of the microarray sub-area may be comparable to the dimensional area of a microtiter well or multiple wells may overlay a microarray sub-area.  
     [0022] The micro-spheres are preferably coated onto the substrate as disclosed in U.S. patent application Ser. No. 09/942,241.  
     [0023] In order to use a microarray having bioactive probes to analyze an unknown biological target sample, the sample to be analyzed has to be nonselectively labeled by using fluorescent dyes or chemiluminescent active molecules.  
     [0024] A biological target sample placed into a microtiter plate well comes into contact with the microarray bioactive probes. The fluorescently/chemiluminescently signals which result from the hybridization of the unknown biological target sample with bioactive probes on the surface of the coated micro-spheres are detected and analyzed by an electronic camera/image processor system.  
     [0025] Microtiter plates are available in many configurations including 96 wells, 192 wells, 384 wells, etc. Plates are available with round and square wells and with filters in the bottom.  
     [0026] Referring now to FIG. 1, there is shown an embodiment of the present invention. As shown, biological analysis system  10  includes microtiter plate  12  with microarray receiver  14  integrated to the bottom of plate  12 . Plate  12  and receiver  14  are shown in partial cross section. Plate  12  includes open-ended wells  16  having openings at first and second ends  18 ,  20 . Microarray receiver  14  with areas of biological functionality  17  is in sealing contact with first ends  18  of wells  16 . Preferably microarray receiver  14  has had its biological function established either through ordered deposition or random coating techniques before being combined with plate  12 . In an alternative embodiment, the receiver is sealed to the bottom of plate  12  prior to providing biological functionality which is provided through wells  16  or plate  12  by well known techniques.  
     [0027]FIG. 2 shows the system  10  from the bottom. Microarray receiver  12  has been partially removed to display wells  16  in microtiter plate  14 . Receiver  12  normally extends over the entire bottom of plate  14  covering all wells  16 . In use, the wells  16  are filled with a sample biological target analyte.  
     [0028]FIG. 3A shows a region  18  of a microarray receiver showing sites  20  with biological functionality. The sites  20  are arranged in a predetermined patterned array  22 . Side wall  24  of a well is shown bounding site array  22 .  
     [0029]FIG. 3B shows a region  18  of a microarray receiver showing sites  26  with biological functionality. The sites  26  are arranged in a random array  28 . Side wall  24  of a well is shown bounding site array  28 .  
     [0030] Referring now to FIGS. 4A, 4B,  4 C, there are shown optical readout systems for luminescence, reflection fluorescence and transmission fluorescence, respectively. As shown in FIG. 4A, the hybridization of a target analyte  34  in a well  16  of plate  14  with a biological probe  17  on microarray receiver  12  produces luminescence which is detected by CCD camera  30  with lens  32 .  
     [0031] As shown in FIG. 4B, a source of illumination  40  illuminates the backside of microarray receiver  12 . The hybridization of target analyte  34  with a biological probe  17  fluoresces and the emissions are reflected from reflector  42  to camera  30  with lens  32 . Filter  44  filters out the illuminant light while passing the fluorescent light.  
     [0032] As shown in FIG. 4C, a source of illumination  50  illuminates analytes  34  from the front side of plate  14  and receiver  12 . Since receiver  12  in this case is light transmissive, the fluorescent light emissions from the hybridization of analyte  34  with a biological probe  17  pass through receiver  12  to CCD camera  30  with lens  32  and filter  44 , which filters out the illuminant light but passes the fluorescent light.  
     [0033] It will be understood that the components used in the optical readout systems of FIGS.  4 A- 4 C can be other than those shown. For example, electronic or digital cameras with sensors other than CCD can be used. In addition, the positioning of the camera can result in more complex optical systems than those shown.  
     [0034]FIG. 5 illustrates a microarray receiver that can be used in the present invention. As shown, microarray receiver  12  includes a pattern of 24 regions 60 in a matrix of 4 rows and 6 columns. Each region includes an identical microarray of randomly distributed biological probe sites, a portion of which are shown in the exploded view. In this view, 16 different biological probes attached to micro-spheres are randomly distributed throughout the portion  62  of region  60 . According to the invention, each probe is attached to a micro-sphere of a color unique to that probe so that micro-spheres of 16 different colors are present in portion  16 . If, for example, an analyte containing each of the 16 complimentary targets to the 16 probes is brought into contact with portion  62 , the hybridization between the 16 targets with the 16 probes would produce luminescence or fluorescence of 16 different colors which are detected by an appropriate optical system (FIGS.  4 A- 4 C).  
     [0035] The invention has been described in detail with particular reference to certain preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.  
     PARTS LIST  
     [0036] 10  biological analysis system  
     [0037] 12  microtiter plate  
     [0038] 14  microarray receiver  
     [0039] 16  open-ended wells  
     [0040] 17  biological probe  
     [0041] 18 , 20  first and second ends  
     [0042] 22  patterned array  
     [0043] 24  side wall  
     [0044] 28  site array  
     [0045] 30  CCD camera  
     [0046] 32  lens  
     [0047] 34  target analyte  
     [0048] 40  illumination  
     [0049] 42  reflector  
     [0050] 44  filter  
     [0051] 50  source of illumination  
     [0052] 60  regions  
     [0053] 62  portion