Methods for Detecting Target Analytes

Abstract

This present disclosure provides methods and systems for measuring the concentration of multiple nucleic acid sequences in a sample. The nucleic acid sequences in the sample are simultaneously amplified, for example, using polymerase chain reaction (PCR) in the presence of an array of nucleic acid probes. The amount of amplicon corresponding to the multiple nucleic acid sequences can be measured in real-time during or after each cycle using a real-time microarray. The measured amount of amplicon produced can be used to determine the original amount of the nucleic acid sequences in the sample. Also provided herein are biosensor arrays, systems and methods for affinity based assays that are able to simultaneously obtain high quality measurements of the binding characteristics of multiple analytes, and that are able to determine the amounts of those analytes in solution. The present disclosure also provides a fully integrated bioarray for detecting real-time characteristics of affinity based assays.

What is claimed is: 
     
         1 . A method comprising:
 (a) providing a biosensor comprising:
 (i) a solid substrate comprising an array of semiconductor-based optical sensors; 
 (ii) a molecular recognition layer adjacent to said solid substrate and comprising a plurality of probes corresponding to a plurality of target analytes, wherein said plurality of probes comprises different probes corresponding to different target analytes from said plurality of target analytes, wherein said plurality of probes comprise fluorophores but do not include quenchers; 
   (b) bringing a fluid containing or suspected of containing said plurality of target analytes or derivatives thereof in contact with said molecular recognition layer of said biosensor;   (c) directing excitation light from a location above said molecular recognition layer to said molecular recognition layer, and using said array of semiconductor-based optical sensors to detect signals from said molecular recognition layer by measuring said signals while said fluid is in contact with said solid substrate; and   (d) using said signals to determine a presence or relative amount of said plurality of target analytes.   
     
     
         2 . The method of  claim 1 , wherein (c) comprises measuring fluorescence signals at multiple time points while said fluid is in contact with said solid substrate. 
     
     
         3 . The method of  claim 2 , wherein said fluorescence signals measured at multiple time points are upon binding of said target analytes with said plurality of probes with time, and
 wherein (a)-(d) are performed without washing said fluid when in contact with said solid substrate.   
     
     
         4 . The method of  claim 3 , wherein a given probe of said plurality of probes is coupled to a fluorophore and a given target analyte of said plurality of target analytes is coupled to a quencher. 
     
     
         5 . The method of  claim 1 , wherein said signals are detected upon a non-competitive interaction between a given target analyte of said plurality of target analytes and a given probe of said plurality of probes. 
     
     
         6 . The method of  claim 1 , wherein a given probe of said plurality of probes is adapted to specifically bind to a given target analyte of said plurality of target analytes. 
     
     
         7 . The method of  claim 1 , further comprising:
 measuring said signals at multiple time points to determine a concentration of a given target analyte of said plurality of target analytes.   
     
     
         8 . The method of  claim 1 , further comprising:
 measuring a change in said signals with time to determine a change in an amount of at least a subset of said plurality of target analytes bound to at least a subset of said plurality of probes with time.   
     
     
         9 . The method of  claim 1 , further comprising, prior to (b):
 performing nucleic acid amplification reactions on a plurality of template nucleic acid molecules to yield said plurality of target analytes as amplification products of said plurality of template nucleic acid molecules; and   using said signals to detect hybridization of at least a subset of said plurality of target analytes to at least a subset of said plurality of probes while said fluid is in contact with said molecular recognition layer of said biosensor, to obtain a hybridization measurement.   
     
     
         10 . The method of  claim 9 , further comprising:
 using said hybridization measurement to determine a concentration of each of said subset of said plurality of target analytes.   
     
     
         11 . The method of  claim 9 , further comprising:
 using said hybridization measurement to determine an original amount of each of at least a subset of said plurality of template nucleic acid molecules.   
     
     
         12 . The method of  claim 1 , wherein said biosensor further comprises:
 an optical coupling layer adjacent to said solid substrate, which optical coupling layer is disposed between said molecular recognition layer and said array of semiconductor-based optical sensors.   
     
     
         13 . The method of  claim 12 , wherein said optical coupling layer further comprises a fiber-optic faceplate comprising packed optical fibers. 
     
     
         14 . The method of  claim 1 , wherein said molecular recognition layer comprises a control region that does not contain probes, and wherein (d) further comprises correcting for non-specific binding using signals from said control region. 
     
     
         15 . The method of  claim 1 , wherein a given probe of said plurality of probes is immobilized to said molecular recognition layer at a discrete and independently addressable location of said molecular recognition layer. 
     
     
         16 . The method of  claim 1 , wherein said molecular recognition layer comprises a plurality of discrete and independently addressable locations, wherein a given discrete and independently addressable location of said plurality of discrete and independently addressable locations comprises a given probe of said plurality of probes, wherein said given discrete and independently addressable location receives an excitation photon flux from a source in optical communication with said molecular recognition layer. 
     
     
         17 . The method of  claim 1 , wherein said plurality of target analytes and said plurality of probes are nucleic acid molecules. 
     
     
         18 . The method of  claim 17 , wherein said plurality of probes are nucleic acid molecules comprising different nucleic acid sequences. 
     
     
         19 . The method of  claim 1 , wherein a given probe of said plurality of probes comprises an energy donor. 
     
     
         20 . The method of  claim 1 , wherein a given target analyte of said plurality of target analytes comprises a quencher.