Terminal labeled probe array and method of making it

Abstract

A probe array comprises a plurality of probes immobilized at a plurality of matrix sites on a substrate for capturing a target substance, wherein the probes are sequentially synthesized at the matrix sites on the substrate until a desired length, the probes are different from each other, and a labeling compound is coupled to each terminus of the probes in a final step of the synthesis. The probe array of the invention allows sensitive and reliable detection of the target substance. A method of evaluating the amount of the fully synthesized probes at respective matrix sites is also provided.

What is claimed is:  
     
         1 . A method of making a probe array for capturing a target substance comprising the steps of: 
 synthesizing a plurality of probes immobilized at a plurality of matrix sites on a substrate by sequential synthesis where constitutional units are added one by one to a plurality of basal parts immobilized to the substrate until a desired length is obtained; and    coupling a labeling compound to a terminus of the probe of the desired length.    
     
     
         2 . The method according to  claim 1 , wherein the probe is a nucleic acid.  
     
     
         3 . The method according to  claim 2 , wherein the nucleic acid is a DNA, an oligonucleotide, or a peptide nucleic acid.  
     
     
         4 . The method according to  claim 3 , wherein the nucleic acid is a DNA and the sequential synthesis is a phosphoramidite method comprising the steps of: 
 (1) deprotecting hydroxyl groups bonded to the surface of a solid substrate via a linker;    (2) coupling the deprotected hydroxyl group to the 3′phosphorous group of an amidite monomer having a desired base;    (3) capping the hydroxyl groups not reacted in step(2);    (4) oxidizing the coupled amidite in step (3) from phosphate to phosphate;    (5) deprotecting a 5′ hydroxyl moiety of the coupled amidite in step (2);    (6) repeating steps (2) to (5) to obtain an oligonucleotide of a desired length and base sequence in a direction from 3′ to 5′; and    (7) deprotecting the bases.    
     
     
         5 . The method according to  claim 1 , wherein the probe is a protein.  
     
     
         6 . The method according to  claim 1 , wherein the probe is an oligopeptide.  
     
     
         7 . The method according to  claim 1 , wherein the labeled substance coupled to the terminus of the probe is a fluorescent substance.  
     
     
         8 . The method according to  claim 7 , wherein the labeled substance is a fluorescent dye.  
     
     
         9 . The method according to  claim 1 , wherein the labeled substance coupled to the terminus of the probe is different from a labeled substance coupled to the target substance.  
     
     
         10 . A probe array comprising a plurality of probes immobilized at a plurality of matrix sites on a substrate for capturing a target substance, wherein the probes are sequentially synthesized at the matrix sites on the substrate until a desired length, the probes are different from each other, and a labeling compound is coupled to each terminus of the probes in a final step of the synthesis.  
     
     
         11 . A method of measuring an amount of a probe in a probe array wherein the probe array comprises a plurality of probes immobilized at a plurality of matrix sites on a substrate for capturing a target substance, the probes are sequentially synthesized at the matrix sites on the substrate until a desired length, the probes are different from each other, and a labeling compound is coupled to each terminus of the probes in a final step of the synthesis, comprising the step of measuring an amount of the labeling compound at each matrix site.  
     
     
         12 . A method for evaluating an amount of a target substance comprising the steps of: 
 reacting a probe array and a target substance wherein the probe array comprises a plurality of probes immobilized at a plurality of matrix sites on a substrate for capturing a target substance, the probes are sequentially synthesized at the matrix sites on the substrate until a desired length, the probes are different from each other, and a labeling compound is coupled to each terminus of the probes in a final step of the synthesis;    measuring an amount of the labeling compound at each matrix site to determine an amount of the probe at the matrix site;    measuring an amount of a labeled target substance captured by the probe at the matrix site; and    comparing the amount of the probe with the amount of the labeled target substance.    
     
     
         13 . The method according to  claim 12 , wherein the amount of the labeling compound coupled to the probe is compared with an amount of the labeling compound directly bonded to the substrate at a predetermined matrix site on the surface of the substrate during a first step of the sequential synthesis without elongation reaction.  
     
     
         14 . A method of evaluating an amount of a target substance comprising the steps of: 
 reacting a probe array and a target substance, wherein the probe array comprises a plurality of probes immobilized at a plurality of matrix sites on a substrate for capturing a target substance, the probes are sequentially synthesized at the matrix sites on the substrate until a desired length, the probes are different from each other, and a labeling compound is coupled to each terminus of the probes in a final step of the synthesis;    measuring an amount of the labeling compound at each matrix site to determine an amount of the probe at the matrix site;    measuring an amount of a labeled target substance captured by the probe at the matrix site;    measuring an amount of the labeling compound directly bonded to the substrate at a predetermined matrix site on the surface of the substrate during a first step of the sequential synthesis without elongation reaction; and    comparing the amount of the probe, the amount of the labeled target substance, and the amount of the directly bonded labeling compound.