Very large scale immobilized polymer synthesis

Abstract

A synthetic strategy for the creation of large scale chemical diversity. Solid-phase chemistry, photolabile protecting groups, and photolithography are used to achieve light-directed spatially-addressable parallel chemical synthesis. Binary masking techniques are utilized in one embodiment. A reactor system, photoremovable protecting groups, and improved data collection and handling techniques are also disclosed. A technique for screening linker molecules is also provided.

What is claimed is:  
     
         1 . A method of detecting hybridization between biological polymers, comprising the acts of: 
 providing a substrate having a surface including at least one biological polymer and at least one fluorescent label associated with the biological polymer;    generating an excitation laser beam;    scanning said laser beam relative to said surface; collecting fluorescent radiation responsive to said laser beam using optics;    detecting said collected fluorescent radiation; and    autofocusing to bring into focus with respect to said optics at least a portion of said surface including the biological polymer.    
     
     
         2 . The method of  claim 1  wherein said collecting fluorescent radiation includes using an objective lens.  
     
     
         3 . The method of  claim 2  wherein said objective lens has a high numerical aperture.  
     
     
         4 . The method of  claim 1  wherein said scanning includes translating said substrate located on a translation table with respect to said excitation beam.  
     
     
         5 . The method of  claim 1  wherein said autofocusing includes employing a piezoelectric focusing system.  
     
     
         6 . The method of  claim 1  wherein said autofocusing includes employing real-time autofocusing.  
     
     
         7 . The method of  claim 1  wherein said detecting includes confocal detection.  
     
     
         8 . The method of  claim 1  wherein said detecting includes detecting said fluorescent radiation as a function of position of said fluorescent label on said surface.  
     
     
         9 . The method of  claim 1  wherein said detecting includes using a photomultiplier.  
     
     
         10 . The method of  claim 1  wherein said detecting includes using a two-dimensional detector.  
     
     
         11 . The method of  claim 1  wherein said detecting includes detecting said fluorescent radiation about every 1 μm to 100 μm with respect to said surface.  
     
     
         12 . The method of  claim 1  wherein said detecting includes collecting fluorescent radiation from a data collection diameter of about 0.8 μm to 10 μm.  
     
     
         13 . The method of  claim 1  wherein said surface comprises a plurality of different nucleic acids covalently bound thereto, and said fluorescent label is located on nucleic acids capable of hybridizing with one or more of said plurality of different nucleic acids.  
     
     
         14 . The method of  claim 1  including receiving data from said detector and creating an image of said fluorescent radiation emitted from said surface at a plurality of locations where different biological polymers are hybridized.  
     
     
         15 . The method of  claim 14  including detecting presence of a specific biopolymer based on said image.  
     
     
         16 . The method of  claim 13  wherein each of said different nucleic acids are located within an area of about 1 μm 2  to about 1000 μm 2 .  
     
     
         17 . The method of  claim 13  wherein said nucleic acids are present on said surface at a density exceeding 100 different nucleic acids per cm 2 .  
     
     
         18 . The method of  claim 13  wherein said nucleic acids are present on said surface at a density exceeding 400 different nucleic acids per cm 2 .  
     
     
         19 . The method of  claim 13  wherein said nucleic acids are present on said surface at a density exceeding 1000 different nucleic acids per cm 2 .  
     
     
         20 . The system of  claim 13  wherein said nucleic acids are present on said surface at a density exceeding 10,000 different nucleic acids per cm 2 .  
     
     
         21 . A system for detecting hybridization between biological polymers, comprising: 
 a laser source capable of emitting an excitation laser beam;    a translation stage constructed to support a substrate having a surface including at least one biological polymer and at least one fluorescent label bound to the biological polymer;    optics co-operatively arranged with said laser source and a detector;    an autofocus system constructed to bring into focus said surface including the biological polymer with respect to said optics; and    a detector, including a filter, constructed to detect fluorescent radiation emitted by the fluorescent label over an area scanned with respect to said laser beam.    
     
     
         22 . The system of  claim 21  wherein said translation stage comprises an XY translation stage including a stage controller constructed for said scanning.  
     
     
         23 . The system of  claim 22  wherein said stage controller is constructed to receive, as user input, scan dimensions and number of pixels in a region to be scanned.  
     
     
         24 . The system of  claim 23  wherein said stage controller is arranged with said optics to provide said fluorescent radiation to said detector over said pixels being substantially smaller than a synthesis area of the biological polymer.  
     
     
         25 . The system of  claim 21  including a data analysis system.  
     
     
         26 . The system of  claim 25  wherein said data analysis system is arranged to provide array of data indicative of fluorescent intensity.  
     
     
         27 . The system of  claim 21  wherein said autofocus system is constructed for real-time autofocusing.  
     
     
         28 . The system of  claim 21  wherein said optics includes an objective lens having a high numerical aperture and being arranged to collect said fluorescent radiation from said surface.  
     
     
         29 . The system of  claim 21  including a pinhole aperture associated with said detector.  
     
     
         30 . The system of  claim 21  wherein said detector is a photomultiplier.  
     
     
         31 . The system of  claim 21  wherein said detector is a two-dimensional detector.  
     
     
         32 . The system of  claim 21  wherein said detector detects said fluorescent radiation as a function of position of said fluorescent label.  
     
     
         33 . The system of  claim 29  including a data analysis system arranged to generate an image based on said fluorescent radiation detected as a function of position.  
     
     
         34 . The system of  claim 29  wherein said detector acquires said fluorescent radiation about every 1 μm to 100 μm.  
     
     
         35 . The system of  claim 33  wherein said detector and said optics has a data collection diameter of about 0.8 μm to 10 μm.  
     
     
         36 . The system of  claim 21  wherein said surface includes a probe array comprising different immobilized biological polymers.  
     
     
         37 . The system of  claim 35  wherein said biological polymers include nucleic acids.  
     
     
         38 . The system of  claim 36  wherein said nucleic acids are attached to said surface through a linker group.  
     
     
         39 . The system of  claim 37  wherein said nucleic acids are from 2 to 20 nucleotides in length.  
     
     
         40 . The system of  claim 21  wherein each of said polymers is separately located within an area of about 1 μm 2  to about 1000 μm 2 .  
     
     
         41 . The system of  claim 38  wherein said nucleic acids have a density exceeding 100 different nucleic acids per cm 2 .  
     
     
         42 . The system of  claim 38  wherein said nucleic acids have a density exceeding 400 different nucleic acids per cm 2 .  
     
     
         43 . The system of  claim 38 , wherein said nucleic acids have a density exceeding 1000 different nucleic acids per cm 2 .  
     
     
         44 . The system of  claim 35 , wherein said biological polymers include oligonucleotides.  
     
     
         45 . The system of  claim 35 , wherein said biological polymers include proteins or polypeptides.  
     
     
         46 . The system of  claim 35 , wherein said biological polymers include one of the following: agonists, antagonists for cell membrane receptor, toxins, venoms, viral epitopes, hormones, hormone receptors, enzymes, enzyme substrates, cofactors, drugs, lectins, sugars, oligosaccharides, and monoclonal antibodies.  
     
     
         47 . A system for detecting hybridization between nucleic acids, comprising: 
 a laser;    a stage constructed to support a substrate having a surface including a biological polymer and at least one fluorescent label associated with the biological polymer;    an autofocus system;    a detector capable of detecting light emitted from the fluorescent label; and    a data storage system for storing fluoresced light intensity as a function of location on said surface, said data storage system coupled to said stage and said detector.    
     
     
         48 . The system of  claim 46  including a data analysis system.  
     
     
         49 . The system of  claim 46  wherein said data analysis system is arranged to provide array of data indicative of fluorescent intensity over said surface.  
     
     
         50 . The system of  claim 46  wherein said autofocus system is constructed for real-time autofocusing.  
     
     
         51 . The system of  claim 50  including an objective lens having a high numerical aperture and being arranged to collect said fluorescent radiation from said surface.  
     
     
         52 . The system of  claim 46  wherein said autofocus system is constructed to bring into focus said surface including the biological polymer with respect to said objective lens.  
     
     
         53 . The system of  claim 46  wherein said stage comprises an XY translation stage including a stage controller constructed for scanning.  
     
     
         54 . A system for detecting hybridization between nucleic acids, comprising: 
 an excitation radiation source;    a stage constructed to support a substrate having a surface including a biological polymer hybridized to a target and at least one fluorescent label associated with the biological polymer;    an autofocus system;    a confocal detector capable of detecting light emitted from the fluorescent label;    a data storage system for storing fluoresced light intensity as a function of location on said surface, and    a data analysis system provide array of data indicative of binding of a specific target to a specific polymer.    
     
     
         55 . The system of  claim 53  wherein said autofocus system is constructed for real-time autofocusing.  
     
     
         56 . The system of  claim 53  including an objective lens having a high numerical aperture and being arranged to collect said fluorescent radiation from said surface.  
     
     
         57 . The system of  claim 55  wherein said autofocus system is constructed to bring into focus said surface including the biological polymer with respect to said objective lens.  
     
     
         58 . A method of detecting hybridization of between biological polymers, comprising the acts of: 
 providing a substrate having a surface including at least one biological polymer and at least one fluorescent label bound to associated with the biological polymer;    generating an excitation radiation;    directing the excitation radiation to said substrate surface;    detecting said emitted fluorescent radiation; and    autofocusing said irradiated surface including the biological polymer.    
     
     
         59 . The method of  claim 57  wherein said detecting fluorescent radiation includes using an objective lens and a confocal detector.  
     
     
         60 . The method of  claim 58  wherein said objective lens has a high numerical aperture.  
     
     
         61 . The method of  claim 57  wherein said autofocusing includes employing a piezoelectric focusing system for displacing said irradiated surface in real time.  
     
     
         62 . A system for detecting hybridization between nucleic acids, comprising: 
 an excitation radiation source;    a stage constructed to support a substrate having a surface including a biological polymer and at least one fluorescent label bound to the biological polymer;    an autofocus system;    a detector capable of detecting light emitted from the fluorescent label; and    a data storage system for storing fluoresced light intensity as a function of location on said surface, said data storage system coupled to said translation stage and said detector.