Signal encoding and decoding in multiplexed biochemical assays

Abstract

This disclosure provides methods, systems, compositions, and kits for the multiplexed detection of a plurality of analytes in a sample. In some examples, this disclosure provides methods, systems, compositions, and kits wherein multiple analytes may be detected in a single sample volume by acquiring a cumulative measurement or measurements of at least one quantifiable component of a signal. In some cases, additional components of a signal, or additional signals (or components thereof) are also quantified. Each signal or component of a signal may be used to construct a coding scheme which can then be used to determine the presence or absence of any analyte.

What is claimed is: 
     
         1 . A method of detecting a unique combination of presence or absence of five analytes in a sample without immobilizing the five analytes or using mass spectrometry, the method comprising:
 (a) forming a mixture of the sample and five hybridization probes, wherein each of the five hybridization probes comprises at least one fluorophore and at most four fluorophores;   (b) partitioning the mixture into a plurality of partitions;   (c) exciting each fluorophore to generate a signal if a corresponding analyte is present in any, some, or all of the plurality of partitions;   (d) measuring the signal to generate a cumulative intensity measurement, wherein the cumulative intensity measurement corresponds to the presence of a unique combination of presence or absence of the five analytes in the sample; and   (e) determining whether each of the five analytes is present, in any unique combination of presence or absence, based on the cumulative intensity measurement.   
     
     
         2 . The method of  claim 1 , wherein each analyte is a polynucleotide analyte, a protein, a small molecule, a lipid, a carbohydrate, or mixtures thereof. 
     
     
         3 . The method of  claim 1 , wherein one of the hybridization probes comprises two, three, or four fluorophores. 
     
     
         4 . The method of  claim 1 , wherein, in (b), each partition of the plurality of partitions contains a single hybridization probe. 
     
     
         5 . The method of  claim 1 , wherein, in (b), a partition of the plurality of partitions contains multiple hybridization probes. 
     
     
         6 . The method of  claim 1 , wherein, in (b), a partition of plurality of partitions contains multiple analytes. 
     
     
         7 . The method of  claim 1 , wherein, in (b), a partition of the plurality of partitions contains at most a single analyte. 
     
     
         8 . The method of  claim 1 , wherein the signal is generated during a polymerase chain reaction. 
     
     
         9 . The method of  claim 1 , wherein each of the five analytes comprise cell-free nucleic acid, tumor nucleic acid, fetal nucleic acid, viral nucleic acid, bacterial nucleic acid, or a combination thereof. 
     
     
         10 . The method of  claim 1 , wherein the sample is a biological sample. 
     
     
         11 . The method of  claim 1 , wherein the sample is derived from saliva, plasma, blood, or urine. 
     
     
         12 . The method of  claim 1 , wherein the detecting the unique combination of presence or absence of five analytes indicates the presence of cancer, infection, genetic abnormality, autoimmune disease, cardiorespiratory disease, liver disease, digestive disease, or a combination thereof. 
     
     
         13 . The method of  claim 1 , further comprising transmitting information concerning the presence or absence of the five analytes through a computer network. 
     
     
         14 . The method of  claim 1 , further comprising providing information concerning the presence or absence of the five analytes to a health care professional to enable the health care professional to render a clinical decision based on the information. 
     
     
         15 . The method of  claim 14 , wherein the clinical decision is a treatment decision. 
     
     
         16 . The method of  claim 15 , wherein said treatment decision comprises treatment with an antibiotic, an antiviral, or an anticancer therapeutic. 
     
     
         17 . The method of  claim 1 , wherein at least part of the method is performed using a computer. 
     
     
         18 . The method of  claim 17 , wherein the computer is located on a remote server. 
     
     
         19 . The method of  claim 1 , wherein each hybridization probe is provided at a given concentration such that the cumulative intensity measurement corresponds to detecting the unique combination of presence or absence of five analytes. 
     
     
         20 . The method of  claim 1 , wherein the cumulative intensity measurement is generated without counting a number of partitions that contain two or more of the five analytes.