Patent Publication Number: US-11650202-B2

Title: Methods for single molecule analyte detection

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a Continuation of U.S. patent application Ser. No. 16/727,732, filed on Dec. 26, 2019, which is a Continuation of U.S. patent application Ser. No. 14/443,655, filed on May 18, 2015, which is a U.S. National Stage Entry of International Patent Application No. PCT/US2013/070797, filed on Nov. 19, 2013, which claims the benefit of priority from U.S. Provisional Application No. 61/869,020, filed on Aug. 22, 2013, and from U.S. Provisional Application No. 61/728,067, filed on Nov. 19, 2012, the entire disclosures of which are hereby incorporated by reference in their entirety for all purposes. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 5, 2022 is named 060851-801CO3US_SL_ST25 and is 7.91 KB in size. 
     BACKGROUND 
     Technical Field 
     This disclosure relates to the fields of diagnostics and communications theory, and specifically relates to methods for digital analysis of molecular analytes. 
     Description of the Related Art 
     Multiple molecular and biochemical approaches are available for molecular analyte identification and quantification. Examples include commonly used nucleic acid based assays, such as qPCR (quantitative polymerase chain reaction) and DNA microarray, and protein based approaches, such as immunoassay and mass spectrometry. However, various limitations exist in current analyte analysis technologies. For example, current methods have limitations of sensitivity, especially where analytes are present in biological samples at low copy numbers or in low concentrations. Most of the nucleic acid quantification technologies involve sample amplification for higher sensitivity. However, amplification techniques introduce biases and inaccuracies into the quantification. Moreover, amplification is not possible for protein and peptides. Due to lack of sensitivity, approaches for detection and quantification often require relatively large sample volumes. Current methods are also limited in their capacity for identification and quantification of a large number of analytes. Quantification of all of mRNA and proteins in a sample requires high multiplexity and large dynamic range. In addition, current technologies lack the capability to detect and quantify nucleic acids and proteins simultaneously. 
     Current methods often generate errors during analyte detection and quantification due to conditions such as weak signal detection, false positives, and other mistakes. These errors may result in the misidentification and inaccurate quantification of analytes. 
     Therefore, methods and systems are needed for analyte analysis that allows for high sensitivity with small sample volume, high multiplexity, large dynamic range and the ability to detect protein and nucleic acid molecules in a single assay. More importantly, methods of error correction to correct for analyte detection errors are needed. The present invention addresses these and other limitations of the prior art by introducing sensitive single molecule identification and quantification of biological analytes with a digital readout. 
    
    
     
       BRIEF DESCRIPTIONS OF THE DRAWINGS 
       The disclosed embodiments have other advantages and features which will be more readily apparent from the following detailed description of the invention and the appended claims, when taken in conjunction with the accompanying drawings, in which: 
         FIG.  1    shows a high-level block diagram illustrating an example of a computer, according to one embodiment of the invention. 
         FIG.  2 A  illustrates an example of a probe comprising an antibody and a detectable tag, where the probe binds a target protein, according to one embodiment of the invention. 
         FIG.  2 B  illustrates an example of a probe comprising a primary antibody and a secondary antibody conjugated to detectable tag, according to one embodiment of the invention. 
         FIG.  3    shows a target analyte bound to a probe comprising an aptamer and a tail region, according to one embodiment of the invention. 
         FIG.  4    shows a fluorescent tag attached to a probe comprising an aptamer and a tail region, according to one embodiment of the invention. 
         FIG.  5    shows an example of a probe comprising an antibody linked to a region that can hybridize to a tail region, according to one embodiment of the invention. 
         FIG.  6    illustrates an example of a probe comprising a primary antibody and a secondary antibody conjugated to a tail region, according to one embodiment of the invention. 
         FIG.  7    shows an example of a solid substrate bound with a sample comprising analytes (e.g., proteins, DNA and/or RNA), according to one embodiment of the invention. 
         FIG.  8    shows an example substrate (10×10 array) for binding analytes, according to one embodiment of the invention. 
         FIG.  9    is a top view of a solid substrate with analytes randomly bound to the substrate, according to one embodiment of the invention. 
         FIGS.  10 A- 10 D  illustrate an example of sixteen target proteins arranged on a substrate.  FIGS.  10 B and  10 C  depict examples of images of the substrate after contact with different probe pools, according to one embodiment of the invention. 
         FIG.  11    illustrates an example Reed-Solomon error correction structure, according to one embodiment of the invention. 
         FIG.  12 A  illustrates an example substrate divided into three regions depicting target analyte concentration levels, according to one embodiment of the invention. 
         FIGS.  12 B- 12 C  show graphs of target analyte abundance ranges, according to one embodiment of the invention. 
         FIG.  13    illustrates an example detection assay using a substrate and four analytes using a single color fluorescent tag, single pass, dark counted, and 1 bit per cycle, according to one embodiment of the invention. 
         FIG.  14    shows an example detection assay using a substrate and four analytes using a single color fluorescent tag, four passes per cycle, dark cycle not counted, and 2 bits per cycle, according to one embodiment of the invention. 
         FIG.  15    shows color sequences and IDs for target analytes, and shows scanning results of the target analytes for the probing, binding, and stripping cycles, according to one embodiment of the invention. 
         FIG.  16 A  shows the numbers of specific target analytes identified on different portions of a substrate, according to one embodiment of the invention. 
         FIG.  16 B  shows color sequences and IDs for target analytes, according to one embodiment of the invention. 
         FIG.  17    is an image of single fluor probes hybridized to target analytes bonded to a substrate, according to one embodiment of the invention. 
         FIG.  18    illustrates examples of identification of various target analytes using single fluor detection, according to one embodiment of the invention. 
         FIG.  19    shows color sequences and IDs for two target analytes, and shows scanning results of the target analytes for the probing, binding, and stripping cycles, according to one embodiment of the invention. 
         FIG.  20    is an image of single molecule peptides bound to a substrate, hybridized with conjugated antibodies, according to one embodiment of the invention. 
         FIG.  21    shows a probability plot of estimated concentrations of proteins from the UniProt database, according to one embodiment of the invention. 
         FIG.  22    shows a list of estimated values for various abundance regions of a substrate, according to one embodiment of the invention. 
         FIG.  23    is a simulated image of protein identification across any abundance range, according to one embodiment of the invention. 
         FIG.  24    illustrates a graph of system error rate vs. raw error rate for identifying target analytes, according to one embodiment of the invention. 
     
    
    
     SUMMARY OF THE INVENTION 
     The invention provides systems and methods for detecting a plurality of analytes, comprising: obtaining a plurality of ordered probe reagent sets, each of the ordered probe reagent sets comprising one or more probes directed to a defined subset of N distinct target analytes, wherein the N distinct target analytes are immobilized on spatially separate regions of a substrate, and each of the probes is detectably labeled. The method also includes steps for performing at least M cycles of probe binding and signal detection, each cycle comprising one or more passes, wherein a pass comprises use of at least one of the ordered probe reagent sets. The method comprises detecting from the at least M cycles a presence or an absence of a plurality of signals from the spatially separate regions of the substrate. 
     The method includes determining from the plurality of signals at least K bits of information per cycle for one or more of the N distinct target analytes, wherein the at least K bits of information are used to determine L total bits of information, wherein K×M=L bits of information and L&gt;log2 (N), and wherein the L bits of information are used to determine a presence or an absence of one or more of the N distinct target analytes. 
     In some embodiments, L&gt;log2 (N), and L comprises bits of information for target identification. In other embodiments, L&gt;log2 (N), and L comprises bits of information that are ordered in a predetermined order. 
     In one embodiment, the predetermined order is a random order. In another embodiment, L&gt;log2 (N), and L comprises bits of information comprising a key for decoding an order of the plurality of ordered probe reagent sets. 
     The method also includes digitizing the plurality of signals to expand a dynamic range of detection of the plurality of signals. In some embodiments, the at least K bits of information comprise information about the number of passes in a cycle. In another embodiment, the at least K bits of information comprise information about the absence of a signal for one of the N distinct target analytes. 
     In one embodiment, the detectable label is a fluorescent label. In another embodiment, the probe comprises an antibody. In one embodiment, the antibody is conjugated directly to a label. The antibody can also be bound to a secondary antibody conjugated to a label. In other embodiments, the probe comprises an aptamer. In one embodiment, the aptamer comprises a homopolymeric base region. In other embodiments, the plurality of analytes comprises a protein, a peptide aptamer, or a nucleic acid molecule. 
     The method can include detecting from the at least M cycles a presence or an absence of a plurality of optical signals. The method can also include detecting from the at least M cycles a presence or an absence of a plurality of electrical signals. 
     In one embodiment, the method is computer implemented. In another embodiment, K is one bit of information per cycle. In other embodiments, K is two bits of information per cycle. K can also be three or more bits of information per cycle. 
     In another embodiment, the method includes determining from the L bits of information an error correction for the plurality of output signals. The error correction method can be a Reed-Solomon code. 
     In one embodiment, the method comprises determining a number of ordered probe reagent sets based on the number of N distinct target analytes. The method can also include determining a type of probe reagent sets based on the type of N distinct target analytes. 
     In an embodiment, the N distinct target analytes are present in a sample, which is divided into a plurality of aliquots diluted to a plurality of distinct final dilutions, and each of the plurality of aliquots is immobilized onto a distinct section of the substrate. In another embodiment, one of the distinct final dilutions is determined based on a probable naturally-occurring concentration of at least one of the N distinct target analytes. In another embodiment, a concentration of one of the N distinct target analytes is determined by counting the occurrences of the target analyte within one of the distinct sections and adjusting the count according to the dilution of the respective aliquot. 
     The invention includes a kit for detecting a plurality of analytes, comprising: a plurality of ordered probe reagent sets, each of the ordered probe reagent sets comprising one or more probes directed to a defined subset of N distinct target analytes, wherein the N distinct target analytes are immobilized on spatially separate regions of a substrate, and each of the probes is detectably labeled. The kit includes instructions for detecting said N distinct analytes based on a plurality of detectable signals. The kit include instructions for performing at least M cycles of probe binding and signal detection, each cycle comprising one or more passes, wherein a pass comprises use of at least one of the ordered probe reagent sets. The kit includes instructions for detecting from the at least M cycles a presence or an absence of a plurality of signals from the spatially separate regions of said substrate. The kit also includes instructions for determining from the plurality of signals at least K bits of information per cycle for one or more of said N distinct target analytes, wherein the at least K bits of information are used to determine L total bits of information, wherein K×M=L bits of information and L&gt;log2 (N), and wherein said L bits of information are used to determine a presence or an absence of one or more of the N distinct target analytes. 
     In some embodiments, the kit includes one or more probes that comprise an antibody. In other embodiments, the label is a fluorescent label. In another embodiment, the probe is an antibody. In one embodiment, the antibody is conjugated directly to a label. In yet another embodiment, the antibody is bound to a secondary antibody conjugated to a label. In other embodiments, the probe comprises an aptamer. The aptamer can comprise a homopolymeric base region. In some embodiments, the plurality of analytes comprises a protein, a peptide aptamer, or a nucleic acid molecule. 
     In other embodiments, L&gt;log2 (N). In another embodiment, M≤N. The kit can also include instructions for determining an identification of each of the N distinct target analytes using the L bits of information, wherein L comprises bits of information for target identification. 
     The kit can include instructions for determining an order of said plurality of ordered probe reagent sets using the L bits of information, wherein L comprises bits of information that are ordered in a predetermined order. The predetermined order can be a random order. The kit can also include instructions for using a key for decoding an order of the plurality of ordered probe reagent sets. 
     DETAILED DESCRIPTION 
     The figures and the following description relate to various embodiments of the invention by way of illustration only. It should be noted that from the following discussion, alternative embodiments of the structures and methods disclosed herein will be readily recognized as viable alternatives that may be employed without departing from the principles of what is claimed. 
     Reference will now be made in detail to several embodiments, examples of which are illustrated in the accompanying figures. It is noted that wherever practicable similar or like reference numbers may be used in the figures and may indicate similar or like functionality. The figures depict embodiments of the disclosed system (or method) for purposes of illustration only. One skilled in the art will readily recognize from the following description that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles described herein. 
     DEFINITIONS 
     A “target analyte” or “analyte” refers to a molecule, compound, substance or component that is to be identified, quantified, and otherwise characterized. A target analyte can comprise by way of example, but not limitation to, an atom, a compound, a molecule (of any molecular size), a polypeptide, a protein (folded or unfolded), an oligonucleotide molecule (RNA, cDNA, or DNA), a fragment thereof, a modified molecule thereof, such as a modified nucleic acid, or a combination thereof. In an embodiment, a target analyte polypeptide or protein is about nine amino acids in length. Generally, a target analyte can be at any of a wide range of concentrations (e.g., from the mg/mL to ag/mL range), in any volume of solution (e.g., as low as the picoliter range). For example, samples of blood, serum, formalin-fixed paraffin embedded (FFPE) tissue, saliva, or urine could contain various target analytes. The target analytes are recognized by probes, which are used to identify and quantify the target analytes using electrical or optical detection methods. 
     Modifications to a target protein, for example, can include post-translational modifications, such as attaching to a protein other biochemical functional groups (such as acetate, phosphate, various lipids and carbohydrates), changing the chemical nature of an amino acid (e.g. citrullination), or making structural changes (e.g. formation of disulfide bridges). Examples of post-translational modifications also include, but are not limited to, addition of hydrophobic groups for membrane localization (e.g., myristoylation, palmitoylation), addition of cofactors for enhanced enzymatic activity (e.g., lipolyation), modifications of translation factors (e.g., diphthamide formation), addition of chemical groups (e.g., acylation, alkylation, amide bond formation, glycosylation, oxidation), sugar modifications (glycation), addition of other proteins or peptides (ubiquination), or changes to the chemical nature of amino acids (e.g., deamidation, carbamylation). 
     In other embodiments, target analytes are oligonucleotides that have been modified. Examples of DNA modifications include DNA methylation and histone modification. 
     A “probe” as used herein refers to a molecule that is capable of binding to other molecules (e.g., oligonucleotides comprising DNA or RNA, polypeptides or full-length proteins, etc.), cellular components or structures (lipids, cell walls, etc.), or cells for detecting or assessing the properties of the molecules, cellular components or structures, or cells. The probe comprises a structure or component that binds to the target analyte. In some embodiments, multiple probes may recognize different parts of the same target analyte. Examples of probes include, but are not limited to, an aptamer, an antibody, a polypeptide, an oligonucleotide (DNA, RNA), or any combination thereof. Antibodies, aptamers, oligonucleotide sequences and combinations thereof as probes are also described in detail below. 
     The probe can comprise a tag that is used to detect the presence of the target analyte. The tag can be directly or indirectly bound to, hybridized to, conjugated to, or covalently linked to the target analyte binding component. In some embodiments, the tag is a detectable label, such as a fluorescent molecule or a chemiluminescent molecule. In other embodiments, the tag comprises an oligonucleotide sequence that has a homopolymeric base region (e.g., a poly-A tail). The probe can be detected electrically, optically, or chemically via the tag. 
     As used herein, the term “tag” refers to a molecule capable of detecting a target analyte). The tag can be an oligonucleotide sequence that has a homopolymeric base region (e.g., a poly-A tail). In other embodiments, the tag is a label, such as a fluorescent label. The tag can comprise, but is not limited to, a fluorescent molecule, chemiluminescent molecule, chromophore, enzyme, enzyme substrate, enzyme cofactor, enzyme inhibitor, dye, metal ion, metal sol, ligand (e.g., biotin, avidin, streptavidin or haptens), radioactive isotope, and the like. The tag can be directly or indirectly bound to, hybridizes to, conjugated to, or covalently linked to the probe. 
     A “protein” or “polypeptide” or “peptide” refers to a molecule of two or more amino acids, amino acid analogs, or other peptidomimetics. The protein can be folded or unfolded (denatured). The polypeptide or peptide can have a secondary structure, such as an α-helix, β sheet, or other conformation. As used herein, the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. A peptide can be two or more amino acids in length. Longer length peptides are often referred to as polypeptides. A protein can refer to full-length proteins, analogs, and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the protein or polypeptide, for example, glycosylation, acetylation, phosphorylation and the like. Furthermore, as ionizable amino and carboxyl groups are present in the molecule, a particular polypeptide may be obtained as an acidic or basic salt, or in neutral form. A protein or polypeptide may be obtained directly from the source organism, or may be recombinantly or synthetically produced. 
     Proteins can be identified and characterized by a peptide sequence, side-chain modifications, and/or tertiary structure. Side-chain modifications include phosphorylation, acetylation, sugars, etc. Phosphorylation of hydroxyl groups from serine, threonine and tyrosine amino acids are particularly important modifications of interest. 
     The term “in vivo” refers to processes that occur in a living organism. 
     The term “mammal” as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines. 
     “Sample” as used herein includes a specimen, culture, or collection from a biological material. Samples may be derived from or taken from a mammal, including, but not limited to, humans, monkey, rat, or mice. Samples may be include materials such as, but not limited to, cultures, blood, tissue, formalin-fixed paraffin embedded (FFPE) tissue, saliva, hair, feces, urine, and the like. These examples are not to be construed as limiting the sample types applicable to the present invention. 
     A “bit” as used herein refers to a basic unit of information in computing and digital communications. A bit can have only one of two values. The most common representations of these values are 0 and 1. The term bit is a contraction of binary digit. In one example, a system that uses 4 bits of information can create 16 different values (as shown in Table 1A). All single digit hexadecimal numbers can be written with 4 bits. Binary-coded decimal is a digital encoding method for numbers using decimal notation, with each decimal digit represented by four bits. In another example, a calculation using 8 bits, there are 2 8  (or 256) possible values. 
     
       
         
           
               
             
               
                 TABLE 1A 
               
             
            
               
                   
               
               
                 Example bit values 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Binary 
                 Octal 
                 Decimal 
                 Hexadecimal 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0000 
                 0 
                 0 
                 0 
               
               
                   
                 0001 
                 1 
                 1 
                 1 
               
               
                   
                 0010 
                 2 
                 2 
                 2 
               
               
                   
                 0011 
                 3 
                 3 
                 3 
               
               
                   
                 0100 
                 4 
                 4 
                 4 
               
               
                   
                 0101 
                 5 
                 5 
                 5 
               
               
                   
                 0110 
                 6 
                 6 
                 6 
               
               
                   
                 0111 
                 7 
                 7 
                 7 
               
               
                   
                 1000 
                 10 
                 8 
                 8 
               
               
                   
                 1001 
                 11 
                 9 
                 9 
               
               
                   
                 1010 
                 12 
                 10 
                 A 
               
               
                   
                 1011 
                 13 
                 11 
                 B 
               
               
                   
                 1100 
                 14 
                 12 
                 C 
               
               
                   
                 1101 
                 15 
                 13 
                 D 
               
               
                   
                 1110 
                 16 
                 14 
                 E 
               
               
                   
                 1111 
                 17 
                 15 
                 F 
               
               
                   
               
            
           
         
       
     
     A “pass” in a detection assay refers to a process where a plurality of probes are introduced to the bound analytes, selective binding occurs between the probes and distinct target analytes, and a plurality of signals are detected from the probes. A pass includes introduction of a set of antibodies that bind specifically to a target analyte. There can be multiple passes of different sets of probes before the substrate is stripped of all probes. 
     A “cycle” is defined by completion of one or more passes and stripping of the probes from the substrate. Subsequent cycles of one or more passes per cycle can be performed. Multiple cycles can be performed on a single substrate or sample. For proteins, multiple cycles will require that the probe removal (stripping) conditions either maintain proteins folded in their proper configuration, or that the probes used are chosen to bind to peptide sequences so that the binding efficiency is independent of the protein fold configuration. 
     It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. 
     Overview 
     Detection techniques for highly multiplexed single molecule identification and quantification of analytes using both optical and electrical systems are disclosed. Analytes can include, but are not limited to, a protein, a peptide, DNA and RNA molecules, with and without modifications. Electrical detection is accomplished using ion sensitive field effect transistors (ISFET) integrated with MEMS (micro-electrical mechanical systems) structures for enhanced sensitivity. Techniques include poly-A tags with and without differential stops, complementary specific and non-specific probes for detailed characterization of analytes, highly multiplexed single molecule identification and quantification using antibody probes. Optical detection is accomplished by detection of fluorescent or luminescent tags. 
     1. Computer System 
       FIG.  1    is a high-level block diagram illustrating an example of a computer  100  for use in analyzing molecular analytes, in accordance with one embodiment. Illustrated are at least one processor  102  coupled to a chipset  104 . The chipset  104  includes a memory controller hub  120  and an input/output (I/O) controller hub  122 . A memory  106  and a graphics adapter  112  are coupled to the memory controller hub  122 , and a display device  118  is coupled to the graphics adapter  112 . A storage device  108 , keyboard  110 , pointing device  114 , and network adapter  116  are coupled to the I/O controller hub  122 . Other embodiments of the computer  100  have different architectures. For example, the memory  106  is directly coupled to the processor  102  in some embodiments. 
     The storage device  108  is a non-transitory computer-readable storage medium such as a hard drive, compact disk read-only memory (CD-ROM), DVD, or a solid-state memory device. The memory  106  holds instructions and data used by the processor  102 . The pointing device  114  is used in combination with the keyboard  110  to input data into the computer system  100 . The graphics adapter  112  displays images and other information on the display device  118 . In some embodiments, the display device  118  includes a touch screen capability for receiving user input and selections. The network adapter  116  couples the computer system  100  to the network. Some embodiments of the computer  100  have different and/or other components than those shown in  FIG.  1   . For example, the server can be formed of multiple blade servers and lack a display device, keyboard, and other components. 
     The computer  100  is adapted to execute computer program modules for providing functionality described herein. As used herein, the term “module” refers to computer program instructions and other logic used to provide the specified functionality. Thus, a module can be implemented in hardware, firmware, and/or software. In one embodiment, program modules formed of executable computer program instructions are stored on the storage device  108 , loaded into the memory  106 , and executed by the processor  102 . 
     2. Compositions 
     Compositions are provided that bind and tag analytes, such as DNA, RNA, protein, and peptides, in a specific manner, such that individual molecules can be detected and counted. 
     Antibodies as Probes 
     In some embodiments, the probe comprises antibodies that can be used as probes to detect target analytes in a sample. As described below, antibodies are immunoglobulins that specifically bind to target proteins or polypeptides. In a preferred embodiment, antibodies used in the invention are monoclonal and can bind specifically to folded or unfolded proteins. 
     “Antibody” refers to an immunoglobulin that specifically binds to, and is thereby defined as complementary with, another molecule. The antibody is a glycoprotein produced by B-cells that is used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, called an antigen. Antibodies are typically made of basic structural units: two large heavy chains and two small light chains. The antibody can be monoclonal or polyclonal, and can be naturally occurring, modified or recombinant. Antibodies can be prepared by techniques that are well known in the art, such as immunization of a host and collection of sera (polyclonal), or by preparing continuous hybrid cell lines and collecting the secreted protein (monoclonal), or by cloning and expressing nucleotide sequences or mutagenized versions thereof coding at least for the amino acid sequences required for specific binding of natural antibodies. Antibodies can include a complete immunoglobulin or fragment thereof, which immunoglobulins include the various classes and isotypes, such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof may include Fab, Fv and F(ab′)2, Fab′, and the like. 
     A “monoclonal antibody” (mAB) is an immunoglobulin produced by a single clone of lymphocytes, i.e. the progeny of a single B cell, which recognizes only a single epitope on an antigen. In addition, aggregates, polymers, and conjugates of immunoglobulins or their fragments can be used where appropriate so long as binding affinity for a particular target is maintained. An antibody (primary antibody) can be covalently linked to a detectable label (e.g., fluorescent label). In other embodiments, a primary antibody binds to a secondary antibody that is covalently linked to a detectable label. In some embodiments, the primary antibody is conjugated to a labeled oligonucleotide molecule, as described in U.S. Pat. No. 7,122,319 to Liu et al. filed on Nov. 5, 2003, which is incorporated by reference in its entirety. 
       FIG.  2 A  illustrates an example of a probe comprising an antibody  132  and a detectable tag  134 , and the probe binds a target analyte  130 . In  FIG.  2 B , an example is shown of a probe comprising a primary antibody  132  and a secondary antibody  210 . The secondary antibody  210  is conjugated to a detectable label  134 . 
     Aptamers 
     An “aptamer” as used herein refers to a nucleic acid molecule or a peptide molecule that binds to a target analyte. An aptamer can be a component of a probe. In some embodiments, nucleic acid aptamers are nucleic acid molecules that have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. See Tuerk C &amp; Gold L (1990). Other methods of aptamer generation include SAAB (selected and amplified binding site) and CASTing (cyclic amplification and selection of targets). Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 249:505-510; M. Svobodová, A. Pinto, P. Nadal and C. K. O&#39;Sullivan. (2012) Comparison of different methods for generation of single-stranded DNA for SELEX processes. “Anal Bioanal Chem” (2012) 404:835-842. Aptamers can bind to a unique n-mer sequence found in a protein (e.g., denatured or folded protein) or polypeptide. In one embodiment, the aptamer binds to a unique 9-mer sequence. In some embodiments, aptamer can bind to a tag, such as an oligonucleotide strand comprising a homopolymeric base region (e.g., a poly-A tail). 
     In some embodiments, the probe comprises an aptamer and a tail region. An aptamer is an oligonucleotide or peptide molecule that binds to a specific target analyte.  FIG.  3    shows a target analyte  130  that is bound to an aptamer  300 . The aptamer  300  includes a probe region  320 , which is configured to specifically bind to the target analyte  130 . The probe region  320  can comprise a protein, peptide, or nucleic acid, and the probe region  320  recognizes and binds to the target analyte. Each probe region  320  can be coupled to a tag. In some embodiments, the tag is a tail region  310 . The tail region  310  is an oligonucleotide molecule of at least 25 nucleotides and serves as a template for polynucleotide synthesis. The tail region  310  is generally a single-stranded DNA molecule, but could also be an RNA molecule. In one embodiment, the tail region  310  is covalently linked to the probe region  330  through a nucleic acid backbone. 
     In another embodiment, a portion of the tail region  310  specifically binds to a linker region  330 . The linker region  330  is covalently linked to the probe region  320  through a nucleic acid backbone. The linker region  330  can be configured to specifically bind to a portion of one tail region  310 , or portions of multiple tail regions  310 . In an embodiment, the linker region  330  comprises at least  10  nucleotides. In another embodiment, the linker region  330  comprises  20 - 25  nucleotides. A probe region  320  can be covalently linked to a single linker region  330 , or can be covalently linked to multiple distinct linker regions  330  that each specifically binds to a portion of a distinct tail region  310 . 
     The tail region  310  provides a template for polynucleotide synthesis. During polynucleotide synthesis, one hydrogen ion is released for each nucleotide incorporated along the tail region template. A plurality of these hydrogen ions can be detected as an electrical output signal by a transistor. A minimum threshold number of hydrogen ions must be released for the transistor to detect an electrical output signal. For example, the minimum threshold number could be 25 depending on details of the detector configuration. In that case, the tail region  310  must be at least 25 nucleotides long. In some embodiments, the tail region  310  is at least 25, 100, 200, 1000, or 10,000 nucleotides in length. The tail region  310  can include one or more homopolymeric base regions. For example, the tail region  310  can be a poly-A, poly-C, poly-G, or a poly-T tail. In another embodiment, the tail region  310  comprises a homopolymeric base region followed by a different homopolymeric base region, for example a poly-A tail followed by a poly-G tail. In one embodiment, the tail region  310  is a DNA-based poly-A tail that is 100 nucleotides in length. Nucleotides (dTTP&#39;s) are added under conditions that promote polynucleotide synthesis, and the nucleotides are incorporated to transcribe the tail region, thereby releasing hydrogen ions. If the minimum threshold number of hydrogen ions for the transistor to detect an electrical output signal is 100 nucleotides or less, a transistor will detect an electrical output signal. This signal is used to identify the target analyte associated with the poly-A tail region and potentially determine the concentration of the target analyte in the solution. 
     In some embodiments, the tail region  310  comprises a homopolymeric base region that includes one or more stop bases.  FIG.  3    illustrates a single stop base  330  that is flanked by two homopolymeric base regions. A stop base  330  is a portion of a tail region  310  comprising at least one nucleotide adjacent to a homopolymeric base region, such that the at least one nucleotide is composed of a base that is distinct from the bases within the homopolymeric base region. In one embodiment, the stop base  330  is one nucleotide. In other embodiments, the stop base  330  comprises a plurality of nucleotides. Generally, the stop base  330  is flanked by two homopolymeric base regions. In an embodiment, the two homopolymeric base regions flanking a stop base  330  are composed of the same base. In another embodiment, the two homopolymeric base regions are composed of two different bases. In another embodiment, the tail region  310  contains more than one stop base  330 . 
     Further details about aptamers and tail regions as probes for differential detection of small molecules is described in U.S. Provisional Application No. 61/868,988. 
     Molecular Tags 
     In some embodiments, the probe comprises a molecular tag for detection of the target analyte. Tags can be attached chemically or covalently to other regions of the probe. In some embodiments, the tags are fluorescent molecules. Fluorescent molecules can be fluorescent proteins or can be a reactive derivative of a fluorescent molecule known as a fluorophore.  FIG.  4    illustrates a fluorescent tag  402  attached to a probe  320 . Fluorophores are fluorescent chemical compounds that emit light upon light excitation. In some embodiments, the fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Examples of fluorescent tags include, but are not limited to, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), fluorescein, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), cyanine (Cy3), phycoerythrin (R-PE) 5,6-carboxymethyl fluorescein, (5-carboxyfluorescein-N-hydroxysuccinimide ester), Texas red, nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride, and rhodamine (5,6-tetramethyl rhodamine). 
     Other exemplary fluorescent tags are listed below in Table 1B. 
     
       
         
           
               
             
               
                 TABLE 1B 
               
               
                   
               
               
                 Fluorescent Tags 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 Hydroxycoumarin 
                 Cy7 
                 mKeima-Red 
                 Katushka 
               
               
                   
                   
                   
                 (TurboFP635) 
               
               
                 Aminocoumarin 
                 DyLight 350 
                 TagCFP 
                 mKate 
               
               
                   
                   
                   
                 (TagFP635) 
               
               
                 Methoxycoumarin 
                 DyLight 405 
                 AmCyan1 
                 TurboFP635 
               
               
                 Cascade Blue 
                 DyLight 488 
                 mTFP1 (Teal) 
                 mPlum 
               
               
                 Pacific Blue 
                 DyLight 549 
                 S65A 
                 mRaspberry 
               
               
                 Pacific Orange 
                 DyLight 594 
                 Midoriishi- 
                 mNeptune 
               
               
                   
                   
                 Cyan 
                   
               
               
                 Lucifer yellow 
                 DyLight 633 
                 Wild Type 
                 E2-Crimson 
               
               
                   
                   
                 GFP 
                   
               
               
                 NBD 
                 DyLight 649 
                 S65C 
                 Mono- 
               
               
                   
                   
                   
                 chlorobimane 
               
               
                 R-Phycoerythrin 
                 DyLight 680 
                 TurboGFP 
                 Calcein 
               
               
                 (PE) 
                   
                   
                   
               
               
                 PE-Cy5 conjugates 
                 DyLight 750 
                 TagGFP 
                 HyPer 
               
               
                 PE-Cy7 conjugates 
                 DyLight 800 
                 TagGFP2 
                   
               
               
                 Red 613 
                 Hoechst 33342 
                 AcGFP1 
                   
               
               
                 PerCP 
                 DAPI 
                 S65L 
                   
               
               
                 TruRed 
                 Hoechst 33258 
                 Emerald 
                   
               
               
                 FluorX 
                 SYTOX Blue 
                 S65T 
                   
               
               
                 Fluorescein 
                 Chromomycin 
                 EGFP 
                   
               
               
                   
                 A3 
                   
                   
               
               
                 BODIPY-FL 
                 Mithramycin 
                 Azami-Green 
                   
               
               
                 TRITC 
                 YOYO-1 
                 ZsGreen1 
                   
               
               
                 X-Rhodamine 
                 Ethidium 
                 Dronpa-Green 
                   
               
               
                   
                 Bromide 
                   
                   
               
               
                 Lissamine 
                 Acridine 
                 TagYFP 
                   
               
               
                 Rhodamine B 
                 Orange 
                   
                   
               
               
                 Texas Red 
                 SYTOX Green 
                 EYFP 
                   
               
               
                 Allophycocyanin 
                 TOTO-1, TO- 
                 Topaz 
                   
               
               
                 (APC) 
                 PRO-1 
                   
                   
               
               
                 APC-Cy7 
                 Thiazole 
                 Venus 
                   
               
               
                 conjugates 
                 Orange 
                   
                   
               
               
                 Alexa Fluor 350 
                 Propidium 
                 mCitrine 
                   
               
               
                   
                 Iodide (PI) 
                   
                   
               
               
                 Alexa Fluor 405 
                 LDS 751 
                 YPet 
                   
               
               
                 Alexa Fluor 430 
                 7-AAD 
                 TurboYFP 
                   
               
               
                 Alexa Fluor 488 
                 SYTOX 
                 PhiYFP 
                   
               
               
                   
                 Orange 
                   
                   
               
               
                 Alexa Fluor 500 
                 TOTO-3, TO- 
                 PhiYFP-m 
                   
               
               
                   
                 PRO-3 
                   
                   
               
               
                 Alexa Fluor 514 
                 DRAQ5 
                 ZsYellow1 
                   
               
               
                 Alexa Fluor 532 
                 Indo-1 
                 mBanana 
                   
               
               
                 Alexa Fluor 546 
                 Fluo-3 
                 Kusabira- 
                   
               
               
                   
                   
                 Orange 
                   
               
               
                 Alexa Fluor 555 
                 DCFH 
                 mOrange 
                   
               
               
                 Alexa Fluor 568 
                 DHR 
                 mOrange2 
                   
               
               
                 Alexa Fluor 594 
                 SNARF 
                 mKO 
                   
               
               
                 Alexa Fluor 610 
                 Y66H 
                 TurboRFP 
                   
               
               
                 Alexa Fluor 633 
                 Y66F 
                 tdTomato 
                   
               
               
                 Alexa Fluor 647 
                 EBFP 
                 DsRed- 
                   
               
               
                   
                   
                 Express2 
                   
               
               
                 Alexa Fluor 660 
                 EBFP2 
                 TagRFP 
                   
               
               
                 Alexa Fluor 680 
                 Azurite 
                 DsRed 
                   
               
               
                   
                   
                 monomer 
                   
               
               
                 Alexa Fluor 700 
                 GFPuv 
                 DsRed2 
                   
               
               
                   
                   
                 (“RFP”) 
                   
               
               
                 Alexa Fluor 750 
                 T-Sapphire 
                 mStrawberry 
                   
               
               
                 Alexa Fluor 790 
                 TagBFP 
                 TurboFP602 
                   
               
               
                 Cy2 
                 Cerulean 
                 AsRed2 
                   
               
               
                 Cy3 
                 mCFP 
                 mRFP1 
               
               
                   
               
            
           
         
       
     
     Probes Including Antibodies and Oligonucleotides 
     As shown in  FIG.  5   , the probe can comprise an antibody  132  linked to a region  410  that can hybridize to an oligonucleotide tail region  310 . The oligonucleotide tail region  310  can be bound to the antibody  132  via a linking region  410 , such as a polyethylene glycol (PEG) chain, ethylene oxide subunits, or other similar chains that can link the antibody  132  to the oligonucleotide tail  310 . In some embodiments, the linking region can include an oligonucleotide that is linked to the antibody peptide using standard chemical methods such as, e.g., NHS ester-maleimide mediated conjugation chemistry where N-terminal Cys incorporated peptide reacts with a maleimide active ted oligo. In other embodiments, linking is accomplished through an internal Cys via oxime formation through a hydroxyl-amine modified peptide reaction with an aldehyde modified oligonucleotide. Such methods are known to the ordinarily skilled artisan. The oligonucleotide sequence in the linking region  410  can hybridize to a portion of the oligonucleotide tail region  310 . The oligonucleotide tail region  310  can comprise an oligonucleotide sequence that is used as a template for polynucleotide synthesis and electrical detection, as described above. U.S. Pat. No. 7,122,319, filed on Nov. 5, 2003 to Liu et al. describes various embodiments for analyte binding agents (e.g., antibodies) linked to oligonucleotide tags, and is incorporated by reference in its entirety. 
     As shown in  FIG.  6   , the probe comprises a primary antibody  132  and a secondary antibody  210 . The primary antibody  132  binds the target analyte  130 , and the secondary antibody  210  binds the primary antibody  132 . The secondary antibody  210  is conjugated to a linker region  410  that hybridizes to an oligonucleotide tail region  310 . The tail region  310  acts as a detectable tag in electrical detection of the target analyte  130 . 
     3. Methods 
     I. Substrate and Sample Preparation 
     The present invention provides methods for identifying and quantifying a wide range of analytes, from a single analyte up to tens of thousands of analytes simultaneously over many orders of magnitude of dynamic range, while accounting for errors in the detection assay. 
     As shown in  FIG.  7   , a sample comprising analytes  610  (e.g., proteins, peptides, DNA and/or RNA) are bound to a solid substrate  600 . The substrate  600  can comprise a glass slide, silicon surface, solid membrane, plate, or the like used as a surface for immobilizing the analytes  610 . In one embodiment, the substrate  600  comprises a coating that binds the analytes  610  to the surface. In another embodiment, the substrate  600  comprises capture antibodies or beads for binding the analytes  610  to the surface. The analytes  610  can be bound randomly to the substrate  600  and can be spatially separated on the substrate  600 . The sample can be in aqueous solution and washed over the substrate, such that the analytes  610  bind to the substrate  600 . In one embodiment, the proteins in the sample are denatured and/or digested using enzymes before binding to the substrate  600 . In some embodiments, the analytes  610  can be covalently attached to the substrate. In another embodiment, selected labeled probes are randomly bound to the solid substrate  600 , and the analytes  610  are washed across the substrate. 
       FIG.  8    shows an example substrate  600  (10×10 array) for binding analytes  610 , where each array insert  700  has 11×11 (110) target arrays. 
       FIG.  9    is a top view of a solid substrate  600  with analytes randomly bound to the substrate  600 . Different analytes are labeled as A, B, C, and D. For optical detection of the analytes, the imaging system requires that the analytes are spatially separated on the solid substrate  600 , so that there is no overlap of fluorescent signals. For a random array, this means that multiple pixels will be needed for each fluorescent spot. 
     The number of pixels can be as few as 1 and as many as hundreds of pixels per spot. It is expected that the optimal amount of pixels per fluorescent spot is between 5 and 20 pixels. In one example, an imaging system has 224 nm pixels. For a system with 10 pixels per fluorescent spot on average, there is a surface density of 2 fluorescent pixels/μm 2 . This does not mean that the protein surface density needs to be this low. If probes are only chosen for low abundance proteins, then the amount of protein on the surface may be much higher. For instance, if there are, on average, 20,000 proteins per μm 2  on the surface, and probes are chosen only for the rarest 0.01% (as an integrated sum) proteins, then the fluorescent protein surface density will be 2 fluorescent pixels/μm 2 . In another embodiment, the imaging system has 163 nm pixels. In another embodiment, the imaging system has 224 nm pixels. In a preferred embodiment, the imaging system has 325 nm pixels. In other embodiments, the imaging system has as large as 500 nm pixels. 
     II. Optical Detection Methods 
     Optical detection methods can be used to quantify and identify a large number of analytes simultaneously in a sample. 
     In one embodiment, optical detection of fluorescently-tagged single molecules can be achieved by frequency-modulated absorption and laser-induced fluorescence. Fluorescence can be more sensitive because it is intrinsically amplified as each fluorophore emits thousands to perhaps a million photons before it is photobleached. Fluorescence emission usually occurs in a four-step cycle: 1) electronic transition from the ground-electronic state to an excited-electronic state, the rate of which is a linear function of excitation power, b) internal relaxation in the excited-electronic state, c) radiative or non-radiative decay from the excited state to the ground state as determined by the excited state lifetime, and d) internal relaxation in the ground state. Single molecule fluorescence measurements are considered digital in nature because the measurement relies on a signal/no signal readout independent of the intensity of the signal. 
     Optical detection requires an optical detection instrument or reader to detect the signal from the labeled probes. U.S. Pat. Nos. 8,428,454 and 8,175,452, which are incorporated by reference in their entireties, describe exemplary imaging systems that can be used and methods to improve the systems to achieve sub-pixel alignment tolerances. In some embodiments, methods of aptamer-based microarray technology can be used. See Optimization of Aptamer Microarray Technology for Multiple Protein Targets,  Analytica Chimica Acta  564 (2006). 
     A. Optical Detection of Multiple Analytes Using Tagged Antibodies 
     The method includes optical detection of analytes using tagged antibodies as probes. For a known target analyte (protein) in the sample, an antibody is selected that specifically binds to the target analyte. Selected antibodies can be those developed for ELISA and comparable systems as single molecule probes. There are hundreds to thousands of existing and qualified primary and secondary antibodies that are readily available. In some embodiments, primary antibodies are selected that are conjugated to a tag, such as a fluorophore. In other embodiments, primary antibodies are selected that bind to secondary antibodies, and the secondary antibodies are conjugated to a fluorescent molecule. 
     In one embodiment, the method includes selecting a primary antibody that has a known, specific target protein in the sample. The primary antibody is tagged with a detectable tag, such as a fluorescent molecule. The selected primary antibodies are introduced and washed across the substrate. The primary antibodies bind to their target analytes, and signals from the tags are detected. 
     In another embodiment, a primary antibody and a secondary antibody conjugated to a detectable tag are selected. The selected primary antibodies are introduced and washed across the substrate. The primary antibodies bind to their target analytes. Next, secondary antibodies are washed across the substrate and bind to the primary antibodies. The tags produce a detectable signal, and the signals are detected and analyzed, preferably using a computer, to determine whether a signal is detected at a defined location, and in some embodiments additional information about the nature of the signal (e.g., the color of the label). 
     A pass comprises a binding step and a signal detection step. There can be a number of passes per cycle, where each pass includes binding of a set of tagged antibodies to a different target protein and detection and analysis of signals from the tagged antibodies. There can be multiple passes of different tagged antibodies before the substrate is stripped of all tagged antibodies. A cycle concludes when one or more passes are completed, and the tagged antibodies are stripped from the substrate. Subsequent cycles of one or more passes per cycle can be performed with the same substrate and sample of bound analytes. 
     An optical detection instrument or reader is used to optically detect each of the signals from the labeled antibodies. The number of signals, location of the signal, and presence or absence of the signal can be recorded and stored. Details about the quantification and identification of the analytes based on the detected optical signals are described below. 
     1. Multiple Tags for Multiple Analytes 
     In one embodiment, a plurality of antibodies conjugated to fluorescent tags is used to detect individual proteins bound to a substrate. Each distinct type of protein is tagged with a limited number of fluorescent tags. For example, in a single pass, antibodies are introduced that are tagged with a red fluorescent tag and selectively bind to protein A. The number of red fluors on the substrate is counted after binding. The number of tags counted is proportional to the concentration of protein A. 
     Each subsequent pass introduces a different fluorescent tag (different color) for detecting a different protein (e.g., blue fluorescent tag for protein B, yellow fluorescent tag for protein C, etc.). The presence of each fluorescent tag is counted at each pass and recorded.  FIG.  9    illustrates a solid substrate comprising analytes A, B, C, and D. At each pass, a different analyte can be detected with a different fluorescent tag and counted accordingly. 
     In some embodiments, a “dark level” is used in the detection and analysis of the analyte. A dark level exists where there is no tag present in the pass and no positive signal is counted, which is referred to as a “dark pass.” The absence of any signal is considered to be a level (i.e., dark cycle counted). Incorporating a dark level allows the number of probes per cycle to be reduced by one. In some embodiments, it is preferred to have a positive signal and not use a dark level because the use of dark levels can be more susceptible to errors. One example embodiment is shown in  FIG.  13   . In other embodiments, where the raw system error rate is low and the number of probes per cycle is low, the use of a dark level can significantly increase the amount of information transferred per cycle. 
     A specific case in which the use of a dark level is helpful is where a primary antibody probe is hybridized to an analyte bound to a substrate, and in which a fluorescently or electrically tagged secondary antibody is bound to the first antibody. The secondary antibody can bind non-specifically to all antibodies so that only one level of information is possible per cycle for a single pass system. In this case, the use of a dark level (i.e., not including a primary antibody in the cycle) is required to achieve 1 bit of information per cycle. 
     To eliminate the use of a dark level when using secondary antibodies, either the use of two or more types of secondary antibodies which have high affinities to a predetermined set of probes of primary antibodies and have low affinities to other predetermined sets of probes of primary antibodies or at least two passes per cycle are required. 
     2. Single Tag for Multiple Analytes 
     In another embodiment, a plurality of antibodies conjugated to fluorescent tags is used to detect individual proteins bound to a substrate. Each type of protein can be tagged with the same fluorescent tag (same color). For example, in one pass, antibody probes tagged with a red fluorescent tag selectively bind to protein A, and the number of red fluors on the substrate is counted. On a second pass, antibody probes tagged with a red fluorescent molecule that specifically bind to protein B are introduced, and the presence of the additional red fluorescent tags at additional locations on the substrate is counted and recorded. Multiple passes can be performed using antibodies labeled with the same fluorescent label that specifically bind different target proteins. The presence of additional red fluorescent tags detected on the substrate at each pass are counted and recorded. One example embodiment is shown in  FIG.  14   . 
     B. Methods for Optical Detection of Analytes 
     The high dynamic-range analyte quantification methods of the invention allow the measurement of over 10,000 analytes from a biological sample. The method can quantify analytes with concentrations from about 1 ag/mL to about 50 mg/mL and produce a dynamic range of more than 10 10 . The optical signals are digitized, and analytes are identified based on a code (ID code) of digital signals for each analyte. 
     As described above, analytes are bound to a solid substrate, and probes are bound to the analytes. Each of the probes comprises tags and specifically binds to a target analyte. In some embodiments, the tags are fluorescent molecules that emit the same fluorescent color, and the signals for additional fluors are detected at each subsequent pass. During a pass, a set of probes comprising tags are contacted with the substrate allowing them to bind to their targets. An image of the substrate is captured, and the detectable signals are analyzed from the image obtained after each pass. The information about the presence and/or absence of detectable signals is recorded for each detected position (e.g., target analyte) on the substrate. 
     In some embodiments, the invention comprises methods that include steps for detecting optical signals emitted from the probes comprising tags, counting the signals emitted during multiple passes and/or multiple cycles at various positions on the substrate, and analyzing the signals as digital information using a K-bit based calculation to identify each target analyte on the substrate. Error correction can be used to account for errors in the optically-detected signals, as described below. 
     In some embodiments, a substrate is bound with analytes comprising N target analytes. To detect N target analytes, M cycles of probe binding and signal detection are chosen. Each of the M cycles includes 1 or more passes, and each pass includes N sets of probes, such that each set of probes specifically binds to one of the N target analytes. In certain embodiments, there are N sets of probes for the N target analytes. 
     In each cycle, there is a predetermined order for introducing the sets of probes for each pass. In some embodiments, the predetermined order for the sets of probes is a randomized order. In other embodiments, the predetermined order for the sets of probes is a non-randomized order. In one embodiment, the non-random order can be chosen by a computer processor. The predetermined order is represented in a key for each target analyte. A key is generated that includes the order of the sets of probes, and the order of the probes is digitized in a code to identify each of the target analytes. 
     In some embodiments, each set of ordered probes is associated with a distinct tag for detecting the target analyte, and the number of distinct tags is less than the number of N target analytes. In that case, each N target analyte is matched with a sequence of M tags for the M cycles. The ordered sequence of tags is associated with the target analyte as an identifying code. 
     In one example, there are 16 target proteins and 16 distinct probes for each of the target proteins, but only four fluorescent tags (red, blue, green, and yellow).  FIG.  10 A  illustrates an example of the 16 target proteins (labeled P1, P2, P3, etc.) arranged on a substrate. The assay can be set up with two cycles and one pass per cycle. Accordingly, two ordered sets of pools are created (one ordered set per cycle). Each probe pool uses the four tags to label the 16 target proteins in a unique 2-color sequence. 
     Table 2 below shows the 16 target analytes and corresponding probe numbers. Table 3 shows the four fluorescent tags (labeled 0 through 3). Tables 4 and 5 show two probe pools where each of the 16 target analytes are labeled with a first fluorescent tag in probe pool 1 and a second fluorescent tag in probe pool 2.  FIG.  10 B  illustrates the substrate of  FIG.  10 A  that has been contacted with probe pool 1.  FIG.  10 C  illustrates the substrate of  FIG.  10 A  that has been contacted with probe pool 2. For example, Probe A2 is tagged with a blue fluorescent tag in probe pool 1, and a red fluorescent tag in probe pool 2. Accordingly, in cycle 1, probe A2 (bound to analyte P2) will emit a blue color, and in cycle 2, probe A2 will emit a red color. In another example, illustrated by  FIG.  10 D , probe A7 has a green (GRN) tag in probe pool 1 (or cycle number 1). In probe pool 2, probe A7 has a blue (BLU) tag. In each probe pool, several probes share the same tag color, but the sequence of colors across the two pools is unique for each analyte. In probe pool #1, for example, probes A4 and A8 are both tagged yellow. Only probe A9, however, is tagged red in probe pool #1 and green in probe pool #2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Analyte and Probe 
               
            
           
           
               
               
               
            
               
                   
                 Analyte Number 
                 Probe Number 
               
               
                   
               
               
                   
                 P1 
                 A1 
               
               
                   
                 P2 
                 A2 
               
               
                   
                 P3 
                 A3 
               
               
                   
                 P4 
                 A4 
               
               
                   
                 P5 
                 A5 
               
               
                   
                 P6 
                 A6 
               
               
                   
                 P7 
                 A7 
               
               
                   
                 P8 
                 A8 
               
               
                   
                 P9 
                 A9 
               
               
                   
                 P10 
                 A10 
               
               
                   
                 P11 
                 A11 
               
               
                   
                 P12 
                 A12 
               
               
                   
                 P13 
                 A13 
               
               
                   
                 P14 
                 A14 
               
               
                   
                 P15 
                 A15 
               
               
                   
                 P16 
                 A16 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Tag Number and Color 
               
            
           
           
               
               
            
               
                 Tag Number 
                 Tag Color 
               
               
                   
               
            
           
           
               
               
               
            
               
                 0 
                 Red 
                 RED 
               
               
                 1 
                 Blue 
                 BLU 
               
               
                 2 
                 Green 
                 GRN 
               
               
                 3 
                 Yellow 
                 YLW 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Probe Pool 1 
               
               
                 Probe Pool #1 
               
            
           
           
               
               
               
            
               
                   
                 Probe Number 
                 Tag Color 
               
               
                   
               
               
                   
                 A1 
                 RED 
               
               
                   
                 A2 
                 BLU 
               
               
                   
                 A3 
                 GRN 
               
               
                   
                 A4 
                 YLW 
               
               
                   
                 A5 
                 RED 
               
               
                   
                 A6 
                 BLU 
               
               
                   
                 A7 
                 GRN 
               
               
                   
                 A8 
                 YLW 
               
               
                   
                 A9 
                 RED 
               
               
                   
                 A10 
                 BLU 
               
               
                   
                 A11 
                 GRN 
               
               
                   
                 A12 
                 YLW 
               
               
                   
                 A13 
                 RED 
               
               
                   
                 A14 
                 BLU 
               
               
                   
                 A15 
                 GRN 
               
               
                   
                 A16 
                 YLW 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Probe Pool 2 
               
               
                 Probe Pool #2 
               
            
           
           
               
               
               
            
               
                   
                 Probe Number 
                 Tag Color 
               
               
                   
               
               
                   
                 A1 
                 RED 
               
               
                   
                 A2 
                 RED 
               
               
                   
                 A3 
                 RED 
               
               
                   
                 A4 
                 RED 
               
               
                   
                 A5 
                 BLU 
               
               
                   
                 A6 
                 BLU 
               
               
                   
                 A7 
                 BLU 
               
               
                   
                 A8 
                 BLU 
               
               
                   
                 A9 
                 GRN 
               
               
                   
                 A10 
                 GRN 
               
               
                   
                 A11 
                 GRN 
               
               
                   
                 A12 
                 GRN 
               
               
                   
                 A13 
                 YLW 
               
               
                   
                 A14 
                 YLW 
               
               
                   
                 A15 
                 YLW 
               
               
                   
                 A16 
                 YLW 
               
               
                   
               
            
           
         
       
     
     Table 6 shows an example of a key comprising an ID (identification) code for each target analyte based on color sequence. The table shows N protein targets by name, a corresponding base-10 number (1 to 10,000), a base-M number (e.g., base 4 with 7 digits shown here), and a color sequence. The color sequence is the order and type of detected signal (red, blue, green, yellow) that was emitted for a particular analyte. The key provides a corresponding base-M number (e.g., base 4, 7 digits) and the identity of the target analyte that corresponds with each color sequence. Accordingly, the base-4 calculation allows for an ordered color sequence of 7 signals, and identification of over 10,000 different target analytes, each having its own identifying color sequence. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Key of protein targets by name, base-10 number, 
               
               
                 base-M number and color sequence 
               
            
           
           
               
               
               
               
            
               
                   
                 Target list 
                 Target list 
                 Target list 
               
               
                 Target list 
                 by base-10 
                 by base-M 
                 by color 
               
               
                 by name 
                 number 
                 number 
                 sequence 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Alpha-1-acid 
                 1 
                 0000001 
                 RRRRRRB 
               
               
                 Glycoprotein 
                   
                   
                   
               
               
                   
               
               
                 Apolipo- 
                 2 
                 0000002 
                 RRRRRRG 
               
               
                 protein B 
                   
                   
                   
               
               
                   
               
               
                 Myoglobin 
                 3 
                 0000003 
                 RRRRRRY 
               
               
                   
               
               
                 . . . 
                   
                   
                   
               
               
                   
               
               
                 L-Selectin 
                 6,751 
                 1221133 
                 BGGBBYY 
               
               
                   
               
               
                 . . . 
                   
                   
                   
               
               
                   
               
               
                 MMP9 
                 9,999 
                 2130033 
                 GBYRRYY 
               
               
                   
               
               
                 Troponin T 
                 10,000 
                 2130100 
                 GBYRBRR 
               
               
                   
               
            
           
         
       
     
     In one embodiment, the method includes the following steps for labeling probe pools to count N different kinds of target analytes on a substrate using fluorescently tagged probes of X different colors: 
     1. Number a list of the N targets (or their probes) using base-X numbers. 
     2. Associate fluorescent tags with base-X digits from 0 to X−1. (For example, 0, 1, 2, 3 correspond to red, blue, green, yellow.) 
     3. Find C such that X C&gt;N.    
     4. At least C probe pools are needed to identify the N targets. Label the C probe pools by an index k=1 to C. 
     5. In the k th  probe pool, label each probe with a fluorescent tag of the color that corresponds to the k th  base-X digit of the base-X number that identifies the probe&#39;s target in the list created in Step 1. 
     For example, if one has N=10,000 target analytes and four fluorescent tags, a base 4 can be chosen. The 4 fluorescent tag colors designated with the numbers 0, 1, 2, and 3, respectively. For example, numbers 0, 1, 2, 3 correspond to red, blue, green, and yellow. 
     When base 4 is chosen, each fluorescent color is represented by 2 bits (0 and 1, where 0=no signal and 1=signal present), and there are 7 colors that are used as a code to identify a target analyte. For example, protein A may be identified with the code of “1221133” that represents the color combination and order of “blue, green, green, blue, blue, yellow, yellow.” For the 7 possible colors, there are a total of 14 bits of information for the target analyte (7×2=14 bits). 
     Next, C is chosen such that 4 C &gt;10,000. In this case, C can be 7 such that there are 7 probe pools to identify 10,000 targets (4 7 =16,384, which is greater than 10,000). A color sequence of length C means that C different probe pools must be constructed. The 7 probe pools are labeled from k=1 to 7. Then each probe is labeled with a fluorescent tag that corresponds to the kth base and X-digit. For example, the third probe in the code “1221133” will be the 3 rd  base-4 th  digit and corresponds to green. 
     C. Quantification of Optically-Detected Probes 
     After the detection process, the signals from each probe pool are counted, and the presence or absence of a signal and the color of the signal can be recorded for each position on the substrate. 
     From the detectable signals, K bits of information are obtained in each of M cycles for the N distinct target analytes. The K bits of information are used to determine L total bits of information, such that K×M=L bits of information and L≥log 2  (N). The L bits of information are used to determine the identity (and presence) of N distinct target analytes. If only one cycle (M=1) is performed, then K×1=L. However, multiple cycles (M&gt;1) can be performed to generate more total bits of information L per analyte. Each subsequent cycle provides additional optical signal information that is used to identify the target analyte. 
     In practice, errors in the signals occur, and this confounds the accuracy of the identification of target analytes. For instance, probes may bind the wrong targets (e.g., false positives) or fail to bind the correct targets (e.g., false negatives). Methods are provided, as described below, to account for errors in optical and electrical signal detection. 
     III. Electrical Detection Methods 
     In other embodiments, electrical detection methods are used to detect the presence of target analytes on a substrate. Target analytes are tagged with oligonucleotide tail regions and the oligonucleotide tags are detected using ion-sensitive field-effect transistors (ISFET, or a pH sensor), which measures hydrogen ion concentrations in solution. ISFETs are described in further detail in U.S. Pat. No. 7,948,015, filed on Dec. 14, 2007, to Rothberg et al., and U.S. Publication No. 2010/0301398, filed on May 29, 2009, to Rothberg et al., which are both incorporated by reference in their entireties. 
     ISFETs present a sensitive and specific electrical detection system for the identification and characterization of analytes. In one embodiment, the electrical detection methods disclosed herein are carried out by a computer (e.g., a processor). The ionic concentration of a solution can be converted to a logarithmic electrical potential by an electrode of an ISFET, and the electrical output signal can be detected and measured. 
     ISFETs have previously been used to facilitate DNA sequencing. During the enzymatic conversion of single-stranded DNA into double-stranded DNA, hydrogen ions are released as each nucleotide is added to the DNA molecule. An ISFET detects these released hydrogen ions and can determine when a nucleotide has been added to the DNA molecule. By synchronizing the incorporation of the nucleoside triphosphates (dATP, dCTP, dGTP, and dTTP), the DNA sequence may also be determined. For example, if no electrical output signal is detected when the single-stranded DNA template is exposed to dATP&#39;s, but an electrical output signal is detected in the presence of dGTP&#39;s, the DNA sequence is composed of a complementary cytosine base at the position in question. 
     In one embodiment, an ISFET is used to detect a tail region of a probe and then identify corresponding target analyte. For example, a target analyte can be immobilized on a substrate, such as an integrated-circuit chip that contains one or more ISFETs. When the corresponding probe (e.g., aptamer and tail region) is added and specifically binds to the target analyte, nucleotides and enzymes (polymerase) are added for transcription of the tail region. The ISFET detects the release hydrogen ions as electrical output signals and measures the change in ion concentration when the dNTP&#39;s are incorporated into the tail region. The amount of hydrogen ions released corresponds to the lengths and stops of the tail region, and this information about the tail regions can be used to differentiate among various tags. 
     The simplest type of tail region is one composed entirely of one homopolymeric base region. In this case, there are four possible tail regions: a poly-A tail, a poly-C tail, a poly-G tail, and a poly-T tail. However, it is often desirable to have a great diversity in tail regions. 
     One method of generating diversity in tail regions is by providing stop bases within a homopolymeric base region of a tail region. A stop base is a portion of a tail region comprising at least one nucleotide adjacent to a homopolymeric base region, such that the at least one nucleotide is composed of a base that is distinct from the bases within the homopolymeric base region. In one embodiment, the stop base is one nucleotide. In other embodiments, the stop base comprises a plurality of nucleotides. Generally, the stop base is flanked by two homopolymeric base regions. In an embodiment, the two homopolymeric base regions flanking a stop base are composed of the same base. In another embodiment, the two homopolymeric base regions are composed of two different bases. In another embodiment, the tail region contains more than one stop base. 
     In one example, an ISFET can detect a minimum threshold number of 100 hydrogen ions. Target Analyte 1 is bound to a composition with a tail region composed of a 100-nucleotide poly-A tail, followed by one cytosine base, followed by another 100-nucleotide poly-A tail, for a tail region length total of 201 nucleotides. Target Analyte 2 is bound to a composition with a tail region composed of a 200-nucleotide poly-A tail. Upon the addition of dTTP&#39;s and under conditions conducive to polynucleotide synthesis, synthesis on the tail region associated with Target Analyte 1 will release 100 hydrogen ions, which can be distinguished from polynucleotide synthesis on the tail region associated with Target Analyte 2, which will release 200 hydrogen ions. The ISFET will detect a different electrical output signal for each tail region. Furthermore, if dGTP&#39;s are added, followed by more dTTP&#39;s, the tail region associated with Target Analyte 1 will then release one, then 100 more hydrogen ions due to further polynucleotide synthesis. The distinct electrical output signals generated from the addition of specific nucleoside triphosphates based on tail region compositions allow the ISFET to detect hydrogen ions from each of the tail regions, and that information can be used to identify the tail regions and their corresponding target analytes. 
     Various lengths of the homopolymeric base regions, stop bases, and combinations thereof can be used to uniquely tag each analyte in a sample. Additional description about electrical detection of aptamers and tail regions to identify target analytes in a substrate are described in U.S. Provisional Application No. 61/868,988, which is incorporated by reference in its entirety. 
     In other embodiments, antibodies are used as probes in the electrical detection method described above. The antibodies may be primary or secondary antibodies that bind via a linker region to an oligonucleotide tail region that acts as tag. Examples of such probes are shown in  FIGS.  2 ,  5  and  6   . 
     These electrical detection methods can be used for the simultaneous detection of hundreds (or even thousands) of distinct target analytes. Each target analyte can be associated with a digital identifier, such that the number of distinct digital identifiers is proportional to the number of distinct target analytes in a sample. The identifier may be represented by a number of bits of digital information and is encoded within an ordered tail region set. Each tail region in an ordered tail region set is sequentially made to specifically bind a linker region of a probe region that is specifically bound to the target analyte. Alternatively, if the tail regions are covalently bonded to their corresponding probe regions, each tail region in an ordered tail region set is sequentially made to specifically bind a target analyte. 
     In one embodiment, one cycle is represented by a binding and stripping of a tail region to a linker region, such that polynucleotide synthesis occurs and releases hydrogen ions, which are detected as an electrical output signal. Thus, number of cycles for the identification of a target analyte is equal to the number of tail regions in an ordered tail region set. The number of tail regions in an ordered tail region set is dependent on the number of target analytes to be identified, as well as the total number of bits of information to be generated. In another embodiment, one cycle is represented by a tail region covalently bonded to a probe region specifically binding and being stripped from the target analyte. 
     The electrical output signal detected from each cycle is digitized into bits of information, so that after all cycles have been performed to bind each tail region to its corresponding linker region, the total bits of obtained digital information can be used to identify and characterize the target analyte in question. The total number of bits is dependent on a number of identification bits for identification of the target analyte, plus a number of bits for error correction. The number of bits for error correction is selected based on the desired robustness and accuracy of the electrical output signal. Generally, the number of error correction bits will be 2 or 3 times the number of identification bits. 
     IV. Decoding the Order and Identity of Detected Analytes 
     The probes used to detect the analytes are introduced to the substrate in an ordered manner in each cycle. A key is generated that encodes information about the order of the probes for each target analyte. The signals detected for each analyte can be digitized into bits of information. The order of the signals provides a code for identifying each analyte, which can be encoded in bits of information. 
     In one example for optical detection of analytes, using 1-bit of information, each analyte is associated with an ordered set of probes. Table 7 below illustrates that each target analyte is associated with a predetermined order of a set of probes introduced over 7 cycles, and the order of the signals emitted from the ordered set of probes is used as a code for identifying the target analyte. For example, for alpha-1-acid glycoprotein, the identifying code is an ordered set of probes of six red (R) signals followed by a final blue (B) signal. When a set of signals is received for a target analyte that reads “RRRRRRB,” the key is used to find a match between the identifying code of an order for the probes and the obtained signals from the analyte. Accordingly, the code is used to determine that the target analyte is alpha-1-acid glycoprotein. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Key For Target Analytes Based On An Ordered Set Of  
               
               
                 Probes Over 7 Cycles and Corresponding Identifying Code 
               
            
           
           
               
               
               
               
            
               
                   
                 Cycles 
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Target Analyte 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 Signal 
                 Code 
               
               
                   
               
               
                 Alpha-1-acid 
                 R 
                 R 
                 R 
                 R 
                 R 
                 R 
                 B 
                 RRRRRRB 
                 0000001 
               
               
                 Glycoprotein 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Apolipoprotein B 
                 R 
                 R 
                 R 
                 R 
                 R 
                 R 
                 G 
                 RRRRRRG 
                 0000002 
               
               
                 Myoglobin 
                 R 
                 R 
                 R 
                 R 
                 R 
                 R 
                 Y 
                 RRRRRRY 
                 0000003 
               
               
                 L-Selectin 
                 B 
                 G 
                 G 
                 B 
                 B 
                 Y 
                 Y 
                 BGGBBYY 
                 1221133 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the user of a kit comprising the ordered set of probes and instructions for using the probe does not have access to the code, such that he or she cannot match the ordered set of signals to the corresponding target analyte. In one embodiment, the kit does not include the key for decoding the results, and the user sends the data to a third party for processing of the data using the code. In another embodiment, the key with ID codes is provided to a user of the kit, and the user can decipher the ordered set of signals to the target analyte. 
     In a second example, each color (fluorescent signal) can be represented by a 2-bit sequence, and a 2-color sequence can be represented by a 4-bit data symbol. Table 8 provides an example of four colors (red, blue, green and yellow) and their corresponding bit values. For example, a color sequence “BGGBBYY” for a particular analyte may be encoded in 14 bits as 01101001011111 according to the bit scheme shown in Table 8. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 2-Bit Assignments for Fluorescent Labels 
               
            
           
           
               
               
               
            
               
                   
                 Color 
                 Bits 
               
               
                   
               
               
                   
                 R 
                 00 
               
               
                   
                 B 
                 01 
               
               
                   
                 G 
                 10 
               
               
                   
                 Y 
                 11 
               
               
                   
               
            
           
         
       
     
     The order of the probes can be different for each analyte for each new cycle (when a cycle includes multiple passes) or for each set of cycles. The key used to identify an analyte in one set of cycles does not have to be used again in a second assay. The codes for the target analytes can be altered for each assay. 
     In some embodiments, the predetermined order of the ordered set of probes is chosen randomly. In other embodiments, the predetermined order is not random. In one embodiment, the computer software is used to specify the order. 
     V. Error-Correction Methods 
     In optical and electrical detection methods described above, errors can occur in binding and/or detection of signals. In some cases, the error rate can be as high as one in five (e.g., one out of five fluorescent signals is incorrect). This equates to one error in every five-cycle sequence. Actual error rates may not be as high as 20%, but error rates of a few percent are possible. In general, the error rate depends on many factors including the type of analytes in the sample and the type of probes used. In an electrical detection method, for example, a tail region may not properly bind to the corresponding probe region on an aptamer during a cycle. In an optical detection method, an antibody probe may not bind to its target or bind to the wrong target. 
     Additional cycles are generated to account for errors in the detected signals and to obtain additional bits of information, such as parity bits. The additional bits of information are used to correct errors using an error correcting code. In one embodiment, the error correcting code is a Reed-Solomon code, which is a non-binary cyclic code used to detect and correct errors in a system. In other embodiments, various other error correcting codes can be used. Other error correcting codes include, for example, block codes, convolution codes, Golay codes, Hamming codes, BCH codes, AN codes, Reed-Muller codes, Goppa codes, Hadamard codes, Walsh codes, Hagelbarger codes, polar codes, repetition codes, repeat-accumulate codes, erasure codes, online codes, group codes, expander codes, constant-weight codes, tornado codes, low-density parity check codes, maximum distance codes, burst error codes, luby transform codes, fountain codes, and raptor codes. See Error Control Coding, 2 nd  Ed., S. Lin and D J Costello, Prentice Hall, New York, 2004. Examples are also provided below that demonstrate the method for error-correction by adding cycles and obtaining additional bits of information. 
     One example of a Reed-Solomon code includes a RS(15,9) code with 4-bit symbols, where n=15, k=9, s=4, and t=3, and n=2 s −1 and k=n−2t, “n” being the number of symbols, “k” being the number of data symbols, “s” being the size of each symbol in bits, and “t” being the number of errors that can be corrected, and “2t” being the number of parity symbols. There are nine data symbols (k=9) and six parity symbols (2t=6). If base-X numbers are used, and X=4, then each fluorescent color is represented by two bits (0 and 1). A pair of colors may be represented by a four-bit symbol that includes two high bits and two low bits. 
       FIG.  11    illustrates the RS(15,9) example structure. Since base-4 was chosen, seven probe pools, or a sequence of seven colors, are used to identify each target analyte. This sequence is represented by 3½, 4-bit symbols. The remaining 5½ data symbols are set to zero. A Reed-Solomon RS(15,9) encoder then generates the six parity symbols, represented by 12 additional probe pools. Thus, a total of 19 probe pools (7+12) are required to obtain error correction for t=3 symbols. 
     Monte Carlo simulations of error-correcting code performance have been performed assuming seven probe pools, to identify up to 16,384 distinct targets. Using these simulations, the maximum permissible raw error rate (associated with identifying a fluorescent label) to achieve a corrected error rate of 10 −5  was determined for different numbers of parity bits. Table 10A below illustrates these findings. 
     
       
         
           
               
             
               
                 TABLE 10A 
               
             
            
               
                   
               
               
                 Error rates 
               
            
           
           
               
               
               
            
               
                   
                 Reed-Solomon parity 
                 Maximum permissible raw error rate to 
               
               
                   
                 symbol number 
                 ensure a correct error rate &lt;10 −5   
               
               
                   
               
            
           
           
               
               
               
            
               
                   
                 6 
                 2% 
               
               
                   
                 8 
                 5% 
               
               
                   
                 10 
                 10%  
               
               
                   
               
            
           
         
       
     
     In some embodiments, a key is generated that includes the expected bits of information associated with an analyte (e.g., the expected order of probes and types of signals for the analyte). These expected bits of information for a particular analyte are compared with the actual L bits of information that are obtained from the target analyte. Using the Reed-Solomon approach, an allowance of up to t errors in the signals can be tolerated in the comparison of the expected bits of information and the actual L bits of information. 
     In some embodiments, a Reed-Solomon decoder is used to compare the expected signal sequence with an observed signal sequence from a particular probe. For example, seven probe pools may be used to identify a target analyte, the expected color sequence being BGGBBYY, represented by 14 bits. Additional parity pools may then be used for error correction. For example, six 4-bit parity symbols may be used. Then, as shown below in Table 10B, the expected signal sequence is compared against the observed signal sequence, and a decoded signal sequence is generated from the comparison. 
     
       
         
           
               
               
             
               
                 TABLE 10B 
               
               
                   
               
             
            
               
                 EXPECTED SIGNAL 
                 BGGBBYYYYRYBBYGBYGR 
               
               
                 SEQUENCE 
                   
               
               
                   
               
               
                 OBSERVED SIGNAL 
                 BGG  BYYYY  YBBYGBYGR 
               
               
                 SEQUENCE 
                   
               
               
                   
               
               
                 DECODED SIGNAL 
                 BGGBBYYYYRYBBYGBYGR 
               
               
                 SEQUENCE 
               
               
                   
               
            
           
         
       
     
     The observed signal sequence has 2 errors in an ordered sequence of 19 signals. When the received probe sequence is decoded by a Reed-Solomon decoder, the original, transmitted probe sequence is recovered. The expected signal sequence is the sequence that is designed to identify one type of analyte. The observed signal sequence is the sequence of fluorescent signals received at a particular location on a solid substrate. The decoded sequence is the recovered sequence after decoding by an error correcting code decoder. 
     In another embodiment, using electrical detection of analytes, the probes and selected bits of information used in the electrical detection method follow error correction calculations as shown in Table 11 below. In Example 1, 3 bits of ID are chosen, which corresponds to a total of 8 target analytes and 8 ID numbers (2 3  =8). In addition, the error factor is calculated to be the number of bits of error divided by the number of bits of ID. Here, the number of bits used for error correction in this example is 9 (3×3=9), and the error factor would be 3 (9/3=3). The total of bits per run is 12 (sum of 3 bits of ID and 9 bits of error correction). The number of bits per cycle can be chosen as 3 and the number of probes per cycle is determined to be 8 (2 3  =8). Next, the number of cycles is calculated to be 4 based on the number of bits, error factor, and bits per cycle. The equation used is ((bits×(1+error factor)/bits per cycle). Here, the calculation is (3×(1+3))/3)=4 cycles. In this example, one stop is used per electrical tag. The number of detectable probes can be increased based on selection of higher bits, as shown in examples 2-5 in Table 11. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Summary of Example Assays Using Various Number of Bits, Number of 
               
               
                 Targets, Number of Probes, Cycles and Stop Types 
               
            
           
           
               
               
               
            
               
                   
                 Example #  
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 1 
                 2 
                 3 
                 4 
                 5 
                 Equation 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 # Bits ID 
                 3 
                 4 
                 8 
                 12 
                 16 
                 bits 
               
               
                 # ID&#39;s (# of Simultaneous Targets) 
                 8 
                 16 
                 256 
                 4,096 
                 65,536 
                 2 bits   
               
               
                 Error Factor (# Bits Error/# Bits ID) 
                 3 
                 3 
                 3 
                 3 
                 3 
                 err 
               
               
                 # Bits Error Correction 
                 9 
                 12 
                 24 
                 36 
                 48 
                 err * bits 
               
               
                 Total # of Bits per Run 
                 12 
                 16 
                 32 
                 48 
                 64 
                 bits * (1 + err) 
               
               
                 # Bits per Cycle 
                 3 
                 4 
                 4 
                 6 
                 8 
                 bpc 
               
               
                 # Probes per Cycle 
                 8 
                 16 
                 16 
                 64 
                 256 
                 2 bpc   
               
               
                 # Cycles 
                 4 
                 4 
                 8 
                 8 
                 8 
                 (bits * (1 + err))/bpc 
               
               
                 # Stops 
                 1 
                 1 
                 2 
                 2 
                 2 
                 stp 
               
               
                 # Stop Types 
                 1 
                 3 
                 3 
                 2 
                 3 
                 typ 
               
               
                 # Flow Sequences per Cycle 
                 3 
                 7 
                 13 
                 9 
                 13 
                 1 + 2 * stp * typ 
               
               
                 # Levels 
                 9 
                 7 
                 4 
                 8 
                 10 
                   
               
               
                 Max # of Probes 
                 8 
                 18 
                 27 
                 84 
                 324 
               
               
                   
               
            
           
         
       
     
     Additional description about electrical detection methods are found in U.S. Provisional Application No. 61/868,988, which is incorporated by reference in its entirety. 
     VI. Dynamic Range 
     The concentrations of analytes such as proteins in samples such as human serum can vary by factors of greater than 10 10 . The dynamic ranges of likely concentrations for particular proteins are generally smaller. For example, Ferritin is normally found between 10 4 -10 5  pg/mL in human serum. Most protein concentrations do not vary by more than a factor of 10 3  from one human serum sample to another. 
     Because it is difficult to detect fluorescent labels corresponding to target analytes at a large dynamic range of concentrations, a substrate containing target analytes can be divided into concentration regions. For example,  FIG.  12 A  shows an example substrate that has been divided into regions “HIGH,” “MED,” and “LOW.” The same target analytes may be distributed throughout each of the three regions. However, the target analytes are diluted to different concentration samples before distribution: one sample each of high concentration (HIGH), medium concentration (MED), and low concentration (LOW). In one embodiment, the target analytes in the “HIGH” region of the substrate are distributed at a concentration of around 1 protein per square micron, the target analytes in the “MED” region are distributed at around 10 2  proteins per square micron, and the target analytes in the “LOW” region are distributed at around 10 4  proteins per square micron. In a further embodiment, the dilutions are adjusted such that the density of fluorescent labels in each concentration region is around one tag per 10-25 pixels in an image of the substrate. 
       FIG.  12 B  is a graph showing an example of abundance ranges of target analytes from a sample, located in different concentration regions (Low, Med, and High) of a substrate.  FIG.  12 C  is also a graph showing abundance ranges of target analytes, with a fourth concentration region: “Rare.” Overlapping abundance ranges demonstrate that certain target analytes may be detected in more than one concentration region. These graphs ( FIGS.  12 B and  12 C ) were generated from simulations performed in Example 5 (below). 
     In one embodiment, particular target analytes within a sample may be separated from the sample to increase the dynamic range even further. For example, in a sample of human serum, it may be desirable to remove albumin, a highly abundant protein. Any separation technique may be used, including high-performance liquid chromatography. 
     Once the different dilutions of target analyte samples have been attached to the substrate, probes may be applied to selectively bind to the target analytes. In an embodiment, the probes may be prepared at varying concentrations so that they selectively bind to the target analytes of medium abundance in the “MED” region of the substrate. 
     EXAMPLES 
     Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for. 
     The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton,  Proteins: Structures and Molecular Properties  (W.H. Freeman and Company, 1993); A. L. Lehninger,  Biochemistry  (Worth Publishers, Inc., current addition); Sambrook, et al.,  Molecular Cloning: A Laboratory Manual  (2nd Edition, 1989);  Methods In Enzymology  (S. Colowick and N. Kaplan eds., Academic Press, Inc.);  Remington&#39;s Pharmaceutical Sciences,  18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey and Sundberg  Advanced Organic Chemistry  3 rd    Ed . (Plenum Press) Vols A and B(1992). 
     Example 1: Optical Detection Assay for Multiple Analytes Using a Single Fluorescent Tag, Single Pass, Dark Counted, and 1 Bit per Cycle 
     In one example, the method is performed using the following parameters: Single Fluorescent Tag (Single Color), Single Pass, Dark Counted, and 1 Bit per Cycle.  FIG.  9    illustrates this example where the fluorescent surface density is lower than the deposited protein surface density. The Figure shows four different types of target proteins mixed with other non-target proteins shown randomly deposited on the surface. 
     Table 12 below shows how a total of four bits of information can be obtained using four cycles of hybridization and stripping, such that there is one pass per cycle. The signals obtained from the four cycles are digitized into bits of information. 
     As illustrated in  FIG.  13   , probes are introduced for Analyte A in Cycle 1, and the presence of the analyte is indicated by a “1.” In Cycle 2, the probes for Analyte A are not added, and the analyte is not detected, indicated by a “0.” After four cycles, Analyte A can be associated with a binary code of “1010.” In a base-2 system, the code is represented as “1010”. In a base-10 system, the same code is represented as “10” (e.g., (2 3 ×1)+(2 2× 0)+(2 1 ×1)+(2 0 ×0)=10). 
     In the first cycle, only antibody probes for targets A and B are included in the probe pool. The imaging system measure a single color image for the first cycle, where A and B molecules fluoresce, but C and D are dark (no probes and no signal). The probes for targets A and B are stripped. For the second cycle, antibody probes for targets C and D are introduced and are imaged and then the antibody probes for C and D are stripped. For the third cycle, antibody probes targets A and C are introduced and imaged. The antibody probes for targets A and C are then stripped. For the fourth cycle, antibody probes for targets B and D are introduced, and the fluorescent molecules are imaged. After imaging multiple cycles, the ID (code of fluorescent signals) for the target molecule at each position is determined. Only 2 cycles are necessary for identification of 4 molecules. In some embodiments, additional cycles can be used for error correction information, which is described below, or to identify more than 4 molecules. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Single Fluorescent Tag, Single Pass,  
               
               
                 Dark Counted, and 1 Bit per Cycle 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 ID- 
                 ID- 
               
               
                   
                 Cycle 1 
                 Cycle 2 
                 Cycle 3 
                 Cycle 4 
                 Binary 
                 Decimal 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Analyte A 
                 1 
                 0 
                 1 
                 0 
                 1010 
                 10 
               
               
                 Analyte B 
                 1 
                 0 
                 0 
                 1 
                 1001 
                 9 
               
               
                 Analyte C 
                 0 
                 1 
                 1 
                 0 
                 0110 
                 6 
               
               
                 Analyte D 
                 0 
                 1 
                 0 
                 1 
                 0101 
                 5 
               
               
                   
               
            
           
         
       
     
     Example 2: Optical Detection Assay for Multiple Analytes Using Single Color, Four Passes Per Cycle, Dark Pass Not Counted, and 2 Bits Per Cycle 
     In another example, the following parameters are used: Single Color, Four Passes per Cycle, Dark Pass Not Counted, and 2 bits per Cycle. 
     In  FIG.  14   , four passes are shown for a single cycle. The first pass includes probes for target analyte A. The probes for target A hybridize, and the detectable signals are imaged. For example, the probes comprise a green fluorescent molecule and emit a green color. The probes for target A are not stripped from the substrate in this example. In pass 2, the probes for target B are hybridized, and the probes for target B have the same fluorescent color as the probes for target A. The additional signals for target B (green fluors) are detected, and both of the signals for targets A and B are imaged. The probes for A and B are not stripped from the substrate. 
     In pass 3, probes for target C are introduced and hybridize to target C. Probes for target C emit the same fluorescent color as targets A and B. The signals emitted from the probes for targets A, B and C are imaged. In pass 4, probes for target D are hybridized, and the signals emitted from targets A, B, C and D are imaged. Finally, all probes are stripped, and the first cycle is completed. 
     Multiple cycles can be performed to increase the number of targets to be quantified. It is not necessary to have probes for every target in every pass, and there may be many more than four molecules observed. 
     Table 13 below shows how signals obtained from one cycle with four passes are digitized and represented as two bits of information per cycle. Over the period of four cycles, a total of 8 bits of information per analyte can be obtained. Table 14 provides the key for the digital output for four passes in a cycle. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Single Color, Four Pass, Dark Not Counted, 2 Bits per Cycle 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Cycle 1 
                 Cycle 2 
                 Cycle 3 
                 Cycle 4 
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Pass 
                 ID- 
                 ID- 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 1 
                 2 
                 3 
                 4 
                 1 
                 2 
                 3 
                 4 
                 1 
                 2 
                 3 
                 4 
                 1 
                 2 
                 3 
                 4 
                 Hex 
                 Decimal 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Analyte A 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0210 
                 36 
               
               
                 Analyte B 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1011 
                 69 
               
               
                 Analyte C 
                 0 
                 0 
                 1 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 2320 
                 184 
               
               
                 Analyte D 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 3132 
                 222 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Analyte A 
                 0 
                 2 
                 1 
                 0 
                 0210 
                 36 
               
               
                 Analyte B 
                 1 
                 0 
                 1 
                 1 
                 1011 
                 69 
               
               
                 Analyte C 
                 2 
                 3 
                 2 
                 0 
                 2320 
                 184 
               
               
                 Analyte D 
                 3 
                 1 
                 3 
                 2 
                 3132 
                 222 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Key for Example 2 Assay 
               
            
           
           
               
               
               
            
               
                   
                 Pass 
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 1 
                 2 
                 3 
                 4 
                 ID - Hex 
                 ID - Decimal 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
               
               
                   
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
               
               
                   
                 0 
                 0 
                 1 
                 1 
                 2 
                 2 
               
               
                   
                 0 
                 0 
                 0 
                 1 
                 3 
                 3 
               
               
                   
                   
               
            
           
         
       
     
     It is possible to use multiple fluors on the secondary analytes instead of performing successive hybridizations to achieve more bits of information per cycle. For instance, four colors of fluorescent tags on secondary antibodies would allow for one hybridization step and one stripping step per cycle to achieve two bits of information per cycle. 
     A combination of using multiple colors and also performing multiple hybridization steps per cycle could increase the number of bits measureable per cycle. For instance, using a four-color imaging system and performing 4 steps of hybridization per cycle would allow up to four bits of information per cycle to be achieved. 
     Example 3: Optical Detection Assay for Multiple Analytes Using Five Colors, Three Passes Per Cycle, Dark Pass Counted, Four Bits Per Cycle 
     In another example, the following parameters are used: Five Colors, Three Passes per Cycle, Dark Pass Counted, Four bits per Cycle. 
     The following tables illustrate an assay with a five color system with 3 passes of hybridization per cycle. A total of 16 levels or equivalently four bits of information is possible per cycle if the absence of any signal is considered to be a level (i.e., dark cycle counted). Table 16 provides a key for the ID code for each analyte. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                   
                 Cycle 1 
                 Cycle 2 
                 Cycle 3 
                 Cycle 4 
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Pass 
                 ID- 
                 ID- 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 1 
                 2 
                 3 
                 1 
                 2 
                 3 
                 1 
                 2 
                 3 
                 1 
                 2 
                 3 
                 Hex 
                 Decimal 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Analyte A 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 5 
                 5 
                 5 
                 2 
                 2 
                 2 
                 13D4 
                 5,076 
               
               
                 Analyte B 
                 0 
                 2 
                 2 
                 0 
                 4 
                 4 
                 0 
                 5 
                 5 
                 0 
                 0 
                 3 
                 5BE9 
                 23,529 
               
               
                 Analyte C 
                 0 
                 0 
                 3 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                 4 
                 4 
                 4 
                 906A 
                 36,970 
               
               
                 Analyte D 
                 0 
                 0 
                 0 
                 0 
                 5 
                 5 
                 0 
                 3 
                 3 
                 0 
                 0 
                 4 
                 0E8C 
                 3,724 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Analyte A 
                 1 
                 3 
                 D 
                 4 
                 13D4 
                 5,076 
               
               
                 Analyte B 
                 5 
                 B 
                 E 
                 9 
                 5BE9 
                 23,529 
               
               
                 Analyte C 
                 9 
                 0 
                 6 
                 A 
                 906A 
                 36,970 
               
               
                 Analyte D 
                 0 
                 E 
                 8 
                 C 
                 0E8C 
                 3,724 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Pass 
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 2 
                 3 
                 ID - Hex 
                 ID - Decimal 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 1 
                 1 
                 1 
                 1 
               
               
                 0 
                 1 
                 1 
                 2 
                 2 
               
               
                 0 
                 0 
                 1 
                 3 
                 3 
               
               
                 2 
                 2 
                 2 
                 4 
                 4 
               
               
                 0 
                 2 
                 2 
                 5 
                 5 
               
               
                 0 
                 0 
                 2 
                 6 
                 6 
               
               
                 3 
                 3 
                 3 
                 7 
                 7 
               
               
                 0 
                 3 
                 3 
                 8 
                 8 
               
               
                 0 
                 0 
                 3 
                 9 
                 9 
               
               
                 4 
                 4 
                 4 
                 A 
                 10 
               
               
                 0 
                 4 
                 4 
                 B 
                 11 
               
               
                 0 
                 0 
                 4 
                 C 
                 12 
               
               
                 5 
                 5 
                 5 
                 D 
                 13 
               
               
                 0 
                 5 
                 5 
                 E 
                 14 
               
               
                 0 
                 0 
                 5 
                 F 
                 15 
               
               
                   
               
            
           
         
       
     
     Table 17 below shows the number of bits per cycle for a multi-color, multi-pass hybridization for optical detection, with and without the absence of signal considered to be a level (dark cycle counted/dark cycle not counted). 
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Number of Bits per Cycle for Multi-Color, Multi- 
               
               
                 Pass Hybridization for Optical Detection 
               
            
           
           
               
               
               
               
               
               
            
               
                 # Colors 
                 # Passes 
                 # of Levels 
                 # Bits  
                 # of Levels 
                 # Bits  
               
               
                 Per 
                 (Hybridizations) 
                 Per Cycle 
                 Per Cycle 
                 Per Cycle 
                 Per Cycle 
               
            
           
           
               
               
               
               
            
               
                 Cycle 
                 Per Cycle 
                 Dark Not Counted 
                 Dark Counted 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 1 
                 1 
                 1 
                 0.00 
                 2 
                 1.00 
               
               
                 1 
                 2 
                 2 
                 1.00 
                 3 
                 1.58 
               
               
                 1 
                 3 
                 3 
                 1.58 
                 4 
                 2.00 
               
               
                 1 
                 4 
                 4 
                 2.00 
                 5 
                 2.32 
               
               
                 1 
                 5 
                 5 
                 2.32 
                 6 
                 2.58 
               
               
                 2 
                 1 
                 2 
                 1.00 
                 3 
                 1.58 
               
               
                 2 
                 2 
                 4 
                 2.00 
                 5 
                 2.32 
               
               
                 2 
                 3 
                 6 
                 2.58 
                 7 
                 2.81 
               
               
                 2 
                 4 
                 8 
                 3.00 
                 9 
                 3.17 
               
               
                 2 
                 5 
                 10 
                 3.32 
                 11 
                 3.46 
               
               
                 3 
                 1 
                 3 
                 1.58 
                 4 
                 2.00 
               
               
                 3 
                 2 
                 6 
                 2.58 
                 7 
                 2.81 
               
               
                 3 
                 3 
                 9 
                 3.17 
                 10 
                 3.32 
               
               
                 3 
                 4 
                 12 
                 3.58 
                 13 
                 3.70 
               
               
                 3 
                 5 
                 15 
                 3.91 
                 16 
                 4.00 
               
               
                 4 
                 1 
                 4 
                 2.00 
                 5 
                 2.32 
               
               
                 4 
                 2 
                 8 
                 3.00 
                 9 
                 3.17 
               
               
                 4 
                 3 
                 12 
                 3.58 
                 13 
                 3.70 
               
               
                 4 
                 4 
                 16 
                 4.00 
                 17 
                 4.09 
               
               
                 4 
                 5 
                 20 
                 4.32 
                 21 
                 4.39 
               
               
                 5 
                 1 
                 5 
                 2.32 
                 6 
                 2.58 
               
               
                 5 
                 2 
                 10 
                 3.32 
                 11 
                 3.46 
               
               
                 5 
                 3 
                 15 
                 3.91 
                 16 
                 4.00 
               
               
                 5 
                 4 
                 20 
                 4.32 
                 21 
                 4.39 
               
               
                 5 
                 5 
                 25 
                 4.64 
                 26 
                 4.70 
               
               
                   
               
            
           
         
       
     
     Example 4: Demonstrating DNA Probe and Target Hybridization and Stripping at a Bulk Level 
     Nucleic acids were used to demonstrate APTIQ probe/target hybridization and stripping cycles at a bulk level. Oligonucleotides (Table 18) were purchased from IDT Integrated DNA Technologies (Coralville, Iowa). Oligos were dissolved in molecular grade water at a final concentration of 100 μM and were stored at −20° C. 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Oligo and Probe Sequences 
               
            
           
           
               
               
            
               
                   
                 Sequence 
               
               
                   
               
               
                 Sequence Name 
                   
               
               
                 Oligo B1 
                 /5AmMC6/d(A) 40  GCA CCC TTG GTC TCC TCC A 
               
               
                 Oligo B2 
                 /5AmMC6/ d(A) 40 CT CAG CAG CAT CTC AGG GCC A 
               
               
                 Oligo B3 
                 /5AmMC6/ d(A) 40 GCT GCA TGC ACG CAC ACA CA 
               
               
                   
               
               
                 Probe Name 
                   
               
               
                 Cy3-anti-B1 
                 /5Cy3/TGG AGG AGA CCA AGG GTG CAG T 
               
               
                 Cy3-anti-B2 
                 /5Cy3/TGG CCC TGA GAT GCT GCT GAG T 
               
               
                 Cy3-anti-B3 
                 /5Cy3/TGT GTG TGC GTG CAT GCA GC 
               
               
                 Cy5-anti-B1 
                 /5Cy5/TGG AGG AGA CCA AGG GTG CAG T 
               
               
                 Cy5-anti-B2 
                 /5Cy5/TGG CCC TGA GAT GCT GCT GAG T 
               
               
                 Cy5-anti-B3 
                 /5Cy5/TGT GTG TGC GTG CAT GCA GC 
               
               
                   
               
            
           
         
       
     
     Oligos with C6-amino linkers were printed on microarrays at ArrayIt (Sunnyvale, Calif.). Unless otherwise specified, all reagents and equipment used in these Examples were purchased from ArrayIt. Oligos were printed at 50 μM final concentration in 1× MSP buffer (Cat ID: MSP) on SuperEpoxy 2 Microarray Substrates (Cat ID: SME2), on a NanoPrint Microarrayer using SMP3 Microarray Printing Pin. Printed microarrays were dried overnight. 
     Prior to use, a substrate slide was blocked for 1.5 hours in Blockit Blocking Buffer (Cat ID: BKT) at room temperature with gentle agitation at 350 rpm, followed by washing 3 times, 1 minute at a time, with Wash Buffers 1, 2, 3 at 1× (Cat ID: WB1, WB2, WB3) in a square petri dish, 30 ml volume at 350 RMP 2 mm orbit. The slide was then spin dried for 10 seconds with a Microarray Centrifuge (Cat ID: MHC110). 
     A Gasket (Cat ID: GAHC4x24) was blocked in Blockit blocking buffer for at least 1 hour, rinsed using distilled water, dried using Microarray Cleanroom Wipe (Cat ID: MCW), and loaded into the lid of the cassette (Cat ID: AHC4x24). Hybit hybridization buffer (Cat ID: HHS2) was used for hybridization at 1×. Cy3 or Cy5 labeled probes (Table 18) (corresponding to color R—red, or G—green, respectively) were mixed in the probe pools in 1× hybridization buffer. 75 μl of hybridization probe pools were loaded on the microarray and incubated for 15 minutes at 37 C°, RMP 350 on Arrayit Array Plate Hyb Station (Cat ID: MMHS110V). 
     100 ul of wash buffer (at 37° C.) 1 was added to each well and then incubated for 1 minute at RMP 350. The wash buffer was then taken out by expelling the wash buffer into the waste. Wash buffer 1 was used two more times, wash buffer 2 was used three times and then wash buffer 3 was used three times. 
     The slide was removed from the gasket submerged in wash buffer 3 in a container, and spin dried in the Microarray Centrifuge. The slide was scanned in an Axon scanner 4200A with setting of PMT250 for both 532 and 635 Lasers. The slide was incubated with spots side up in a square petri dish containing 30 ml of Stripping Buffer A at 350 RMP for 10 minutes. Stripping Buffer A was removed and immediately followed by addition of 30 ml of Stripping Buffer B. The procedure was repeated with Stripping Buffer B and Stripping Buffer C. The slide was dried in the microarray centrifuge and prepared for the next cycle of hybridization. The slide was scanned after stripping to confirm the efficiency of the stripping. 
       FIG.  15    illustrates the scanning results: three oligo targets, (B1, B2, and B3) were identified by probing binding and stripping for 6 cycles. The color sequence of each oligo target was correctly identified. 
     Example 5: Using DNA to Demonstrate Single Molecule Counting 
     We describe a method for the identification and quantification of single molecules. Oligonucleotides (Table 19) were purchased from IDT Integrated DNA Technologies. Oligos were dissolved in molecular grade water at final concentration 100 uM and were stored at −20° C. 
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 Oligo and Probe Sequences 
               
            
           
           
               
               
               
            
               
                   
                 Sequence 
                 SEQ ID NO 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Sequence Name 
                   
                   
               
               
                 C6-P53 
                 /5AmMC6/AAA AAA ACT GCA CCC TTG GTC TCC TCC A 
                 13 
               
               
                 C6-BRAF 
                 /5AmMC6/AAA AAA ACT CAG CAG CAT CTC AGG GCC A 
                 14 
               
               
                 C6-EGFR 
                 /5AmMC6/AAA AAA GCT GCA TGC ACG CAC ACA CA 
                 15 
               
               
                 C6-KRAS 
                 /5AmMC6/AAA AAA ATC CCA GCA CCA CCA CTA CCG A 
                 16 
               
               
                   
               
               
                 Probe Name 
                   
                   
               
               
                 Cy3-anti-P53 
                 /5Cy3/TGG AGG AGA CCA AGG GTG CAG T 
                 7 
               
               
                 Cy3-anti-BRAF 
                 /5Cy3/TGG CCC TGA GAT GCT GCT GAG T 
                 8 
               
               
                 Cy3-anti-EGFR 
                 /5Cy3/TGT GTG TGC GTG CAT GCA GC 
                 9 
               
               
                 Cy3-anti-KRAS 
                 /5Cy3/TCG GTA GTG GTG GTG CTG GGA T 
                 17 
               
               
                 Cy5-anti-P53 
                 /5Cy5/TGG AGG AGA CCA AGG GTG CAG T 
                 10 
               
               
                 Cy5-anti-BRAF 
                 /5Cy5/TGG CCC TGA GAT GCT GCT GAG T 
                 11 
               
               
                 Cy5-anti-EGFR 
                 /5Cy5/TGT GTG TGC GTG CAT GCA GC 
                 12 
               
               
                 Cy5-anti-KRAS 
                 /5Cy5/TCG GTA GTG GTG GTG CTG GGA T 
                 18 
               
               
                   
               
            
           
         
       
     
     Silicon slides were purchased from University Wafer (Boston, Mass.), diced (American Precision Dicing Inc., San Jose, Calif.), and coated with SuperEpoxy substrate (ArrayIt). The single crystal silicon chips were prepared as 25 mm×75 mm substrate slides. The thickness of the silicon chips used were 500 μm, 675 μm, and 1000 μm. A thermal oxide was grown on the silicon chips of 100 nm and then diced into slides. 
     A slide was incubated in a solution of 4 DNA oligos (Table 19), each oligo ending in a C6 molecule. The sequences of the 4 oligos were A, B, C &amp; D corresponding to the genes encoding for KRAS, EGFR, BRAF and P53. The 4 oligos with C6-amino linker were mixed at 100 nM per oligo in 1× micro spotting solution (Cat ID: MSS, ArrayIt) and then incubated on the epoxy coated silicon slide in a container at room temperature overnight. During incubation, a reaction between the epoxy coating and the C6 oligos covalently bonded the single stranded DNA to the surface. The slide was then washed with molecule grade water for 5 minutes, 3 times, followed by incubation in ArrayIt BlockIt blocking solution for 1 hour at room temperature with gentle agitation at 350 rpm, followed by washing 3 times for 1 minute each time with Wash Buffers 1, 2, 3 at 1× in a square petri dish, 30 ml volume at 350 RMP 2 mm orbit. The slide was spin dried for 10 seconds with the Microarray Centrifuge. 
     The chip was fabricated with glue into a biochip consisting of 3 parts, silicon chip, peek frame, and a 170 μm-thick coverslip glass. The coverslip (Nexterion, Tempe, Ariz.) was glued on the silicon slide with Bostik glue mixed with 50 uM beads (Gelest, Morrisville, Pa.) on an in-house developed device. The glue and beads was packed in 3 cc syringe (Hamilton Company, Reno, Nev.) and centrifuged in EFD ProcessMate centrifuge (Nordson, Westlake, Ohio) and then delivered by Nordson EFD Ultimus I glue dispenser. 
     Cy3 or Cy5 labeled probes (Table 19) were mixed in the probe pools in 1×HybIt hybridization buffer. Hybridization solution from pool #1 was delivered in the biochip and incubated for 15 minutes at room temperature. The chip was then washed with washing buffer 1, 2 and 3 (ArrayIt), 8 times with each buffer. 15% glycerol in 1×SSPE (150 mM NaCl, 10 mMNaH 2 PO4, 1 mMEDTA) was added to the chip before imaging. Successive probe pools of probes 1, 2, 3, 4 were hybridized and stripped. After each hybridization step, an imaging system imaged 12 regions of the slide, each region being 100 μm×100 μm. The camera used was a Hamamatsu Orca 4.0 with a 40× magnification system using Olympus part # UAPON40XW. 
     After imaging, the chip was rinsed with molecular grade water and then stripped in the stripping buffer A, B, C (ArrayIt), 8 times each buffer. 15% glycerol in 1×SSPE was added to the chip before imaging. After cycle 1 which includes hybridization probe pool #1, imaging, stripping, imaging, the cycle 2 starts with hybridization with probe pool #2. 
     Data was taken on two slides (slides #177 and #179,  FIG.  16 A ), each slide containing twelve fields. Each field was  100  μm× 100  μm. A two-color imaging system was used with CY3 and CY5 filters. For each slide, at least 12-14 cycles of data were collected, with the analysis using 9 to 10 cycles of data. The mapping of target identification ID to color sequence is illustrated in Table 19. In  FIG.  16 A , each color maps to a sequence such that a probe is labeled with CY5 or CY3, corresponding respectively to color R (red) or G (green), which maps to 1 or 0 with 1 bit of information being acquired per cycle ( FIG.  16 B ). For this case, the error correction scheme was conservative and required zero errors per target, an error being defined as a positive identification in a sequence where it was not expected. Up to five missing sequences were allowed per molecule. Missing sequences are cases where a molecule is not identified in a cycle. These are not classified as errors. 
     Slides #177 &amp; #179 were measured under similar conditions. A small portion of each slide was measured (measuring the entire slide is an implementation of scale and automation).  FIG.  16 A  shows the number of molecules in each field with the number of each gene that was identified. The percentages of identified molecules were 12%-13%. 
       FIG.  17    illustrates an image taken with the prototype imager of single fluor DNA probes that have been hybridized to the DNA target oligos covalently bonded to the surface. Between 10% -15% of the identified molecules had multiple fluors per spot due to aggregation that occurred during sample attachment. 
       FIG.  18    illustrates representative examples of identification of each of the four targets from slide #177 ( FIG.  16 A ). Each spot in the circle is aligned to center on the target. The targets are identified with single fluor detection. Approximately 10% -15% of the targets on the image were clustered molecular species where more than a single fluor was bound. Inspecting the data shows that for both experiments, greater levels of P53 and KRAS were observed than BRAF and EGFR. The total number of molecules identified was under 2000 in both cases, demonstrating the potential high sensitivity of the method in detecting and identifying low numbers of molecules. 
     Example 6: Demonstrating Peptide Probe and Target Hybridization and Stripping at a Bulk Level 
     Peptides were used to demonstrate APTIQ probe/target binding and stripping cycles at bulk level. Peptide MUC1 (Sequence: APDTRPAPG) was purchased from American Peptide (Sunnyvale, Calif.). MUC1 was dissolved at lmg/ml in 1× peptide printing buffer 1 (Cat ID: PEP, ArrayIt). Peptide MUC16 at 0.2 mg/ml, monoclonal antibodies against mouse anti-MUC1 C595 [Cat ID: NCRC48], and rabbit anti-MUC16 [Cat ID: EPSISR23] were purchased from Abcam (Cambridge, Mass.). The following secondary antibodies were also purchased from Abcam: goat anti-mouse IgG Cy3 (Cat ID: ab97035), goat anti-rabbit IgG Cy3 (Cat ID: ab6939), goat anti-mouse IgG Cy5 (Cat ID: ab97037), goat anti-rabbit IgG Cy5 (Cat ID: ab6564). 
     Peptides were printed on microarrays at ArrayIt (Sunnyvale, Calif.). MUC1 peptide was printed at 0.5 mg/ml final concentration and MUC16 at 0.1 mg/ml in 1× peptide printing buffer 2 (Cat ID: PEP, ArrayIt) on SuperEpoxy 2 Microarray Substrates on a NanoPrint Microarrayer using SMP3 Microarray Printing Pin. Printed microarrays were dried overnight. 
     Prior to use, the slide was blocked for 1.5 hours in Blockit Plus Blocking Buffer (Cat ID: BKTP, ArrayIt) at room temperature with gentle agitation at 350 rpm, followed by washing 3 times 1 minute each with 1× PBS in a square petri dish, 30 ml volume at 350 RMP 2 mm orbit. The slide was spin dried for 10 seconds with the ArrayIt Microarray Centrifuge. 
     Anti-MUC1 and anti-MUC 16 primary antibodies were diluted 250 fold in 1×PBS buffer (137 mM NaCl; 2.7 mM KCl; 10 mM Na 2 HPO4; 2 mM KH 2 PO4, pH 7.4). Secondary antibodies were diluted 10000 fold in 1×PBS. Cy3 or Cy5 labeled antibodies were mixed in the 2 pools in 1×PBS: Pool #1: anti-mouse Cy3 and anti-rabbit Cy5; Pool #2: anti-rabbit Cy5 and anti-rabbit Cy3. 
     5 ml of the mixture of primary probe pools were added to the slide and incubated for 1 hour at room temperature in a container. The slide was then washed with 1×PBS, 3 times, 5 minutes each time with gentle shaking at 450 rpm. 
     Secondary antibody pool #1 was added to the slide and incubated for 1 hour at room temperature. The slide was then washed with 1×PBS, 3 times, 5 minutes each time with gentle shaking at 450 rpm. The slide was removed from the container and dried in the Microarray Centrifuge. The slide was scanned in Axon 4200A with settings at PMT250 for both 532 and 635 Lasers. 
     The slide was incubated with spots side up in a square petri dish containing 5ml of Stripping Buffer (Cat ID: 21028, Fisher Scientific, Rockford, Ill.) at 300 RMP for 1 hour. The slide was then washed with distilled water 3 times, for 5 minutes each time. The slide was dried in the microarray centrifuge and then was prepared for the next cycle of antibody binding and stripping. The slide was scanned after stripping to make sure the stripping was efficient. 
       FIG.  19    illustrates the scanning results: two oligo targets, (MUC1 and MUC16) were identified by probing binding and stripping for 4 cycles. The color sequence of each oligo target was correctly identified. 
     Example 7: Using Peptides to Demonstrate Single Molecule Counting 
     Preparation of peptides was performed using the same technique as in Example 4. Peptide MUC1 (20 ng/ml) and MUC 16 (4 ng/ml) were diluted in ArrayIt 1× peptide printing buffer 2 (ArrayIt, Sunnyvale, Calif.) and then incubated on a silicon slide in a container at room temperature overnight. The slide was then washed with molecule grade water for 5 minutes, 3 times, followed by incubation in ArrayIt BlockIt plus blocking solution for 1 hour at room temperature with gentle agitation at 300 rpm. The chip was subsequently washed with molecular grade water for 5 minutes, 3 times. The slide was spin dried in microarray high-speed centrifuge. The slide was then built in biochip following the same procedures as above. 
     Primary antibodies are diluted 250 fold in 1xPBS. Secondary antibodies are diluted 10,000 fold in 1×PBS. A mixture of primary antibodies against MUC1 and MUC16 was delivered in the biochip and incubated for 60 minutes at room temperature. The chip was then washed with 8× with 1×PBS. A mixture of secondary antibodies (either pool #1 containing anti-mouse Cy3 and anti-rabbit Cy5 or pool #2 containing anti-rabbit Cy5 and anti-rabbit Cy3) was delivered in the biochip and incubated for 60 minutes at room temperature. The chip was then washed with washing 8× with 1×PBS. 15% glycerol in 1×SSPE was added to the chip before imaging. 
       FIG.  20    shows an image of single molecule peptides, such that conjugated antibodies (CY5 and CY3) were bound to isolated peptides which in turn were covalently bonded to the chip surface. Multiple fluors may be bound to a given antibody which creates a spread in the intensity of each observed molecule. These molecules were measured to be somewhat brighter than the DNA single molecule measurements where a single fluor was conjugated to each DNA probe. A total of six cycles were run of two proteins in single molecule mode. The molecules were bound and removed with high yield. Similar techniques can be used for scaling the system up to many more proteins and DNA/RNA targets. 
     After imaging, the biochip was rinsed with molecular grade water and then stripped in the stripping buffer (Cat ID: 21028, Fisher Scientific, Rockford, Ill.) for 1 hour followed by washing 8× with 1×PBS. 15% glycerol in 1×SSPE was added to the biochip before imaging. After cycle 1 which includes hybridization probe pool #1, imaging, stripping, imaging, the cycle 2 started with hybridization with probe pool #2. 
     Example 8: Quantifying the Human Plasma Proteome 
     A system model was created to demonstrate the feasibility of measuring the concentration of the ˜10,000 proteins in the human plasma proteome across 12 logs of dynamic range using single-molecule identification with Reed-Solomon error correction encoding. For this model, the proteins in the plasma proteome were divided into three concentration regions as shown in  FIG.  12 B , referred to as low, medium and high-concentration regions. Theoretically, the total range of protein concentrations in the human plasma proteome is over 10 logs of dynamic range, but the concentration of each protein does not vary by more than a few logs.  FIG.  12 B  depicts overlapping concentration regions. For each of the regions, probes are selected for proteins that are expected to fall within that particular region but not exceed the maximum concentration allowable for that region. Greater overlap can be achieved by adding another concentration range, as shown in  FIG.  12 C , with the trade-off being that a greater area of the substrate chip is used. 
     The data used in the model came from the UniProt database (uniprot.org, FASTA file for organism 9606), “Toward a Human Blood Serum Proteome,” Joshua Adkins et al., “The Human Plasma Proteome,” N. Leigh Anderson et al. and “A High-Confidence Human Plasma Proteome Reference Set with Estimated Concentrations in PeptideAtlas,” T. Farrah et al. Because not all proteins in the UniProt database are associated with a published concentration, random concentrations were assigned without changing the well-known highly abundant protein concentrations or the overall concentration.  FIG.  21    shows a probability plot of the estimated concentrations. Concentrations between 10 5  and 10 11  pg/mL use published values. All published values found in the lower range were used. The remaining protein concentrations were estimated using a log normal approximation, with an estimated Gaussian distribution over a log normal space of 6 orders of magnitude from 10 −1  pg/mL to 10 4  pg/mL. 
       FIG.  22    lists specific estimated values for each of the abundance regions used. For each abundance region, the same target protein density (dTP) was assumed of 1.5 target proteins per μm 2 . However, in the low abundance region, the number of high abundance region proteins per number of target proteins (rHT) was 25,000 to 1, and 250 to 1 in the medium abundance region. The number of high abundance proteins per pixel (rHP) ranged from 1,000 to 0.04. The number of pixels per target protein was constant for each of the three regions. 
     For the model, a four color imaging system is assumed giving 2 bits of information per cycle.  FIG.  23    shows what a simulated image would be expected to look like for any of the colors across any of the abundance ranges, except for the case of albumin in the high abundance range when at any given time half of the target molecules are expected to be the same color. A field is the imaging region of the camera for a given exposure, in this case a 2,000 by 2,000 pixel camera is assumed with 40× magnification giving 163 nm pixels. A total of 2,500 fields (nF) are required for the low abundance region, but only 250 fields each are required for the high and medium abundance regions. 
     With the system model optimized, it was determined that the lowest abundance region would interrogate 9,575 proteins out of 9,719 or 98.5% of the proteome. At the other extreme, the high abundance region interrogates only the top 2.9% of the proteome. This is because there is only a small percentage of the proteins in the plasma proteome that make up the high abundance region. The measurable concentration ranges vary depending on the concentration region. The low abundance concentration region measures concentrations between 30 fg/mL and 300 ng/mL. The medium abundance concentration region measures concentrations between 82 pg/mL and 85 ug/mL. The high abundance concentration region measures concentrations between 20 ng/mL and 100 mg/mL. The total chip area required for this measurement is 320 mm 2 , or a chip with dimensions of 18 mm×18 mm. 
     An analysis was conducted to determine the efficacy of Reed Solomon error correction in the plasma proteome measurement across 12 logs of dynamic range. There is an intrinsic error rate for each measurement cycle for each counted molecule. Since each molecule is spread out over a slide (particularly significant for the low abundance molecules), there will be other molecules nearby that should not cross-react with probes but still do. A robust system will allow for this to occur and will be able to correct these errors and give the correct identification of a molecule. The rate at which incorrect binding occurs per molecule per cycle is the raw error rate. The system error rate is the per molecule identification error rate after correction has been performed. 
       FIG.  24    illustrates a chart of the expected system error rate vs. the raw error rate for cases varying raw error rates and varying numbers of imaging cycles. The data for the chart was generated using a Monte Carlo simulation in which a large number of system configurations was simulated. To identify 16,384 proteins, a total of 7 data cycles are required for a four color system (4 7  =16,384). The maximum allowable error rate is calculated by dividing the allowable number of system errors by the number of molecules identified. In this case, for an average of one error per protein the allowable number of errors is 16,384. The number of molecules identified is 4.0×10 8  (2,500 fields×1.6×10 5  molecules per field) with an allowable error rate calculate to be 4.1×10 −5 . 
     Reed Solomon encoding requires parity cycles to improve the raw error rate. Assuming a Reed Solomon system over a Galois Field of 4 (mm=4), each symbol (or word) is a 4-bit symbol that can be represented by two 2-bit symbols (i.e. 2 cycles of a 4 color system that obtains 2 bits per cycle). For a Reed Solomon system, the length of the symbol (or code word) is nn=2 mm−1 , or 15 4-bit symbols or equivalently 30 2-bit symbols. This means that up to 30 cycles may be processed by a four-color fluidics/imaging system. The number of errors that can be corrected is 3, 4 or 5 per target which corresponds to tt={3, 4, or 5} parity symbols. Four imaging cycles are required per parity cycle. This gives a total of 7 data cycles for the ID and 12, 16, or 20 imaging cycles for the error correction. This means that the total number of cycles required to identify 16,384 simultaneous proteins is 19, 23 and 27 cycles for 3, 4 and 5 allowable errors per molecule. As previously calculated, the maximum system error rate of 4.1×10 −5  allows one error per protein. If more errors per protein are allowed, then the maximum system error rate can drop proportionately. 
       FIG.  24    shows the contours of raw error rate vs. system error rate. For 19 imaging cycles a maximum raw error rate allowable is 3%, for 23 cycles the maximum raw error rate allowable is 6%, and for 27 cycles the maximum raw error rate allowable is 13%. It is expected that the raw error rate will be less than 5%, although for all but the rarest proteins raw error rates of up to 20% appear to be well within the acceptable range of this technology. 
     Since the maximum number of cycles allowable is 30 cycles, more data cycles could be included. In particular, if three more data cycles were included, the number of identifiable targets would increase by 4{circumflex over ( )}3, or 64× resulting in a maximum possible identifiable targets of 1,048,576, a number higher than realistic probe concentrations will allow. However, this illustrates that the technique is scalable to an arbitrarily large number of molecules limited only by biology. 
     SUMMARY 
     The foregoing description of the embodiments of the invention has been presented for the purpose of illustration; it is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Persons skilled in the relevant art can appreciate that many modifications and variations are possible in light of the above disclosure. 
     Some portions of this description describe the embodiments of the invention in terms of algorithms and symbolic representations of operations on information. These algorithmic descriptions and representations are commonly used by those skilled in the data processing arts to convey the substance of their work effectively to others skilled in the art. These operations, while described functionally, computationally, or logically, are understood to be implemented by computer programs or equivalent electrical circuits, microcode, or the like. Furthermore, it has also proven convenient at times, to refer to these arrangements of operations as modules, without loss of generality. The described operations and their associated modules may be embodied in software, firmware, hardware, or any combinations thereof. 
     Any of the steps, operations, or processes described herein may be performed or implemented with one or more hardware or software modules, alone or in combination with other devices. In one embodiment, a software module is implemented with a computer program product comprising a computer-readable medium containing computer program code, which can be executed by a computer processor for performing any or all of the steps, operations, or processes described. 
     Embodiments of the invention may also relate to an apparatus for performing the operations herein. This apparatus may be specially constructed for the required purposes, and/or it may comprise a general-purpose computing device selectively activated or reconfigured by a computer program stored in the computer. Such a computer program may be stored in a non-transitory, tangible computer readable storage medium, or any type of media suitable for storing electronic instructions, which may be coupled to a computer system bus. Furthermore, any computing systems referred to in the specification may include a single processor or may be architectures employing multiple processor designs for increased computing capability. 
     Embodiments of the invention may also relate to a product that is produced by a computing process described herein. Such a product may comprise information resulting from a computing process, where the information is stored on a non-transitory, tangible computer readable storage medium and may include any embodiment of a computer program product or other data combination described herein. 
     Finally, the language used in the specification has been principally selected for readability and instructional purposes, and it may not have been selected to delineate or circumscribe the inventive subject matter. It is therefore intended that the scope of the invention be limited not by this detailed description, but rather by any claims that issue on an application based hereon. Accordingly, the disclosure of the embodiments of the invention is intended to be illustrative, but not limiting, of the scope of the invention, which is set forth in the following claims. 
     All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.