Fluorogenic substrates and methods of use

Abstract

The present disclosure relate to methods and fluorogenic substrates for analysis of analyte(s) in a sample. The methods include detecting presence of an analyte of interest on capture particles by contacting the capture particles present in nano-chambers with a fluorogenic substrate. The fluorogenic substrate may be of formula (I): (I) wherein: X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation; Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate.

Description

FLUOROGENIC SUBSTRATES AND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATIONSPursuant to 35 U.S.C. § 119(e), this application claims priority to the filing date of the United States Provisional Patent Application Serial No. 63 / 601,591, filed November 21, 2023, the disclosure of which application is herein incorporated by reference.INTRODUCTIONMethods for accurately analyzing analyte(s) of interest in a sample are essential for diagnostics, prognostics, environmental assessment, food safety, detection of chemical or biological warfare agents, and the like. Such methods not only need to be precise and sensitive but are also advantageous when a sample is to be analyzed quickly and with minimal instrumentation. As such, there is an interest in methods with improved sample analysis capabilities.SUMMARYEmbodiments of the present disclosure relate to methods and fluorogenic substrates for analysis of analyte(s) of interest in a sample. In certain embodiments, the sample may be a biological sample, an environmental sample, a food sample, water sample, etc.In certain embodiments, the analyte of interest, if present in a sample, is immobilized on capture particles and the capture particles are assessed for presence or absence of the analyte of interest.The fluorogenic substrate may be of formula (I):wherein: X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation; Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate.The method of detecting presence of an analyte of interest on capture particles comprises contacting the capture particles present in micro-chambers with a fluorogenic substrate of formula (I). The micro-chambers are sized to contain only one capture particle per micro-chamber; the analyte of interest is associated with a phosphatase enzyme; the phosphatase enzyme converts the fluorogenic substrate into a fluorogenic enzymatic product; the fluorogenic substrate emits a first fluorescent signal and the fluorogenic enzymatic product emits a second fluorescent signal distinct from the first fluorescent signal; detecting presence or absence of the second fluorescent signal in the micro-chambers; wherein presence of the second fluorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers. The micro-chambers may be micro wells or micro-droplets.BRIEF DESCRIPTION OF THE FIGURESFIG. 1 shows comparison of pyranine-O-phosphate (also referred to as PyPhos) and fluorescein diphosphate (FDP) using digital detection in the single enzyme molecule detection.FIG. 2 shows comparison of pyranine-O-phosphate and fluorescein diphosphate (FDP) in the single enzyme molecule detection.FIG. 3 shows comparison of pyranine-O-phosphate and FDP (fluorescein diphosphate) in Digital ELISA for HBsAg.FIG. 4 shows dual-color imaging for pyranine-O-phosphate system.FIG. 5 shows dynamic range expansion using the dual-emission properties of pyranine- O-phosphate system.FIG. 6 shows monitoring of thermal stability of pyranine-O-phosphate and FDP.FIG. 7 shows half-life of the substrate in the storage solution at room temperature.FIG. 8 shows rapid detection test using pyranine-O-phosphate. (A-C) Fluorescent images of the nano-chamber array containing single enzyme molecules of alkaline-phosphatase (ALP).FIG. 9 shows output signal for the demonstration using an actual Digital ELISA assay.FIG. 10 shows theoretical curve for the result of HBsAg assay.FIG. 11 shows residual pyranine present in pyranine phosphate salts with either Tris or TEA counterion was measured by fluorescence spectroscopy.FIG. 12 shows a solution of pyranine phosphate was converted to pyranine using excess alkaline phosphatase.FIG. 13 shows enzymatic reaction rate of pyranine phosphate with calf intestinal alkaline phosphatase (BBI ALP21G).FIG. 14 shows signal from dipyranine phosphate incubated with calf intestinal alkaline phosphatase (BBI ALP21G) for 18 hours.DETAILED DESCRIPTIONMethods and substrates for detecting presence of analyte(s) in a sample are provided. In certain embodiments, the analyte of interest, if present in a sample, is immobilized on capture particles and the capture particles are assessed for presence or absence of the analyte of interest. The fluorogenic substrate may be of formula (I):wherein: X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation; Y is selected from a group consisting of diphosphate, triphosphate, oxy methyl phosphate, or phosphate. The method of detecting presence of an analyte of interest on capture particles comprises contacting the capture particles present in micro-chambers with a fluorogenic substrate of formula (I). The micro-chambers are sized to contain only one capture particle per micro-chamber; the analyte of interest is associated with a phosphatase enzyme; the phosphatase enzyme converts the fluorogenic substrate into a fluorogenic enzymatic product; the fluorogenic substrate emits a first fluorescent signal and the fluorogenic enzymatic product emits a second fluorescent signal distinct from the first fluorescent signal; detecting presence or absence of the second fluorescent signal in the micro-chambers; wherein presence of the second fluorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers.Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, representative illustrative methods and materials are now described.All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unlessexpressly formulated under 35 U.S.C. §112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.DEFINITIONSBefore the embodiments of the present disclosure are described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.“Comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context. When used in the context of a range, the modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the range of from about “2 to about 10” also discloses the range “from 2 to 10.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9- 1.1.“Affinity” and “binding affinity” as used interchangeably herein refer to the tendency or strength of binding of the binding member to the analyte. For example, the binding affinity may be represented by the equilibrium dissociation constant (KD), the dissociation rate (kd), or the association rate (ka).As used herein, “analyte”, “target analyte”, “analyte of interest” are used interchangeably and refer to the analyte being measured in the methods and devices disclosed herein. In someaspects, the analyte is a biomolecule. Non-limiting examples of biomolecules include macromolecules such as, proteins, lipids, and carbohydrates. Analytes of interest are further described below. Analytes of interest are further described below.An “amount” as used herein refers to a quantity specified (e.g., high or low) or a number e.g., where the number is a level, such as a position on a real or imaginary scale of amount or quantity, or a concentration, such as, for example, a relative amount of a given substance contained within a solution or in a particular volume of space, e.g., the amount of solute per unit volume of solution.“Antibody” and “antibodies” as used herein refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”) antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual-variable domain immunoglobulins and methods for making them are described in Wu, C., el al., Nature Biotechnology, 25(11): 1290-1297 (2007) and PCT International Application WO 2001 / 058956, the contents of each of which are herein incorporated by reference), and functionally active epitope-binding fragments of any of the above. Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. For the sake of simplicity, an antibody against an analyte is frequently referred to herein as being either an “anti-analyte antibody” or merely an “analyte antibody”.“Antibody fragment” as used herein refers to a portion of an intact antibody comprising the antigen-binding site or variable region. The portion does not include the constant heavy chain domains (i.e., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chainvariable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and singlechain polypeptides containing the three CDRs of the heavy chain variable region.“Analog” as used herein refers to a molecule that has a similar structure to a molecule of interest (e.g., nucleoside analog, nucleotide analog, sugar phosphate analog, analyte analog, etc.). An analyte analog is a molecule that is structurally similar to an analyte but for which the binding member has a different affinity.“Aptamer” as used herein refers to an oligonucleotide or peptide molecule that can bind to pre-selected targets including small molecules, proteins, and peptides among others with high affinity and specificity. Aptamers may assume a variety of shapes due to their propensity to form helices and single- stranded loops. An oligonucleotide or nucleic acid aptamer can be a singlestranded DNA or RNA (ssDNA or ssRNA) molecule. A peptide aptamer can include a short variable peptide domain, attached at both ends to a protein scaffold.“Bead” and “particle” are used herein interchangeably and refer to a substantially spherical solid support.“Cation” refers to a positively charged ion with fewer electrons than protons.“Component,” “components,” or “at least one component,” refer generally to a capture antibody, a detection reagent or conjugate, a calibrator, a control, a sensitivity panel, a container, a buffer, a diluent, a salt, an enzyme, a co-factor for an enzyme, a detection reagent, a pretreatment reagent / solution, a substrate (e.g., as a solution), a stop solution, and the like that can be included in a kit for assay of a test sample, such as a patient urine, serum, whole blood, tissue aspirate, or plasma sample, in accordance with the methods described herein and other methods known in the art. Some components can be in solution or lyophilized for reconstitution for use in an assay.“Control” as used herein refers to a reference standard for an analyte such as is known or accepted in the art, or determined empirically using acceptable means such as are commonly employed. A “reference standard” is a standardized substance which is used as a measurement base for a similar substance. For example, there are documented reference standards published in the U.S. Pharmacopeial Convention (USP-NF), Food Chemicals Codex, and Dietary Supplements Compendium (all of which are available at http: / / www.usp.org), and other well-known sources. Methods for standardizing references are described in the literature. Also well-known are means for quantifying the amounts of analyte present by use of a calibration curve for analyte or by comparison to an alternate reference standard. A standard curve can be generated using serial dilutions or solutions of known concentrations of analyte, by mass spectroscopy, gravimetric methods, and by other techniques known in the art. Alternate reference standards that have beendescribed in the literature include standard addition (also known as the method of standard addition), or digital polymerase chain reaction.“Contacting” and grammatical equivalents thereof as used herein refer to any type of combining action which brings a binding member into sufficiently close proximity with the analyte of interest in the sample such that a binding interaction will occur if the analyte of interest specific for the binding member is present in the sample. Contacting may be achieved in a variety of different ways, including combining the sample with a binding member, exposing a target analyte to a binding member by introducing the binding member in close proximity to the analyte, and the like.“Digital microfluidics (DMF),” “digital microfluidic module (DMF module),” or “digital microfluidic device (DMF device)” as used interchangeably herein refer to a module or device that utilizes digital or droplet-based microfluidic techniques to provide for manipulation of discrete and small volumes of liquids in the form of droplets. Digital microfluidics uses the principles of emulsion science to create fluid-fluid dispersion into channels (principally water-in- oil emulsion). It allows the production of monodisperse drops / bubbles or with a very low polydispersity. Digital microfluidics is based upon the micromanipulation of discontinuous fluid droplets within a reconfigurable network. Complex instructions can be programmed by combining the basic operations of droplet formation, translocation, splitting, and merging.Digital microfluidics operates on discrete volumes of fluids that can be manipulated by binary electrical signals. By using discrete unit-volume droplets, a microfluidic operation may be defined as a set of repeated basic operations, i.e., moving one unit of fluid over one unit of distance. Droplets may be formed using surface tension properties of the liquid. Actuation of a droplet is based on the presence of electrostatic forces generated by electrodes placed beneath the bottom surface on which the droplet is located. Different types of electrostatic forces can be used to control the shape and motion of the droplets. One technique that can be used to create the foregoing electrostatic forces is based on dielectrophoresis which relies on the difference of electrical permittivity between the droplet and surrounding medium and may utilize high- frequency AC electric fields. Another technique that can be used to create the foregoing electrostatic forces is based on electrowetting, which relies on the dependence of surface tension between a liquid droplet present on a surface and the surface on the electric field applied to the surface.As used herein, “immobilized” refers to a stable association of the analyte with a surface of a capture particle. By “stable association” is meant a physical association between two entitiesin which the mean half-life of association is at least 10 minutes, at least 30 minutes, at least 1 hour, at least 3 hours, at least 10 hours, at least 18 hours, at least one day or more, e.g., under physiological conditions. According to certain embodiments, the stable association arises from a covalent bond between the two entities, a non-covalent bond between the two entities (e.g., an ionic or metallic bond), or other forms of chemical attraction, such as hydrogen bonding, Van der Waals forces, and the like, or a combination thereof.“Label” or “detectable label” as used interchangeably herein refers to a tag attached to a specific binding member or analyte by a cleavable or non-cleavable linker.“Polynucleotides” or “oligonucleotides” refer to nucleobase polymers or oligomers in which the nucleobases are connected by sugar phosphate linkages (sugar- phosphate backbone). Exemplary poly- and oligonucleotides include polymers of 2'-deoxyribonucleotides (DNA) and polymers of ribonucleotides (RNA). A polynucleotide may be composed entirely of ribonucleotides, entirely of 2'-deoxyribonucleotides or combinations thereof. “Nucleic acid” encompasses “polynucleotide” and “oligonucleotides” and includes single stranded and double stranded polymers of nucleotide monomers.“Polynucleotide analog” or “oligonucleotide analog” refers to nucleobase polymers or oligomers in which the nucleobases are connected by a sugar phosphate backbone comprising one or more sugar phosphate analogs. Typical sugar phosphate analogs include, but are not limited to, sugar alkylphosphonates, sugar phosphoramidites, sugar alkyl- or substituted alkylphosphotriesters, sugar phosphorothioates, sugar phosphorodithioates, sugar phosphates and sugar phosphate analogs in which the sugar is other than 2'-deoxyribose or ribose, nucleobase polymers having positively charged sugar-guanidyl interlinkages such as those described in U.S. Patent No. 6,013,785 and U.S. Patent No. 5,696,253.“Receptor” as used herein refers to a protein-molecule that recognizes and responds to endogenous-chemical signals. When such endogenous-chemical signals bind to a receptor, they cause some form of cellular / tissue-response. Examples of receptors include, but are not limited to, neural receptors, hormonal receptors, nutrient receptors, and cell surface receptors.As used herein, “spacer” refers to a chemical moiety that provides linkage between the binding member and the support. In some embodiments, one or more spacers may be included at the N-terminus or C-terminus of a polypeptide or nucleotide-based tag or label in order to distance optimally the sequences from the specific binding member. Spacers may include but are not limited to 6-aminocaproic acid, 6-aminohexanoic acid; 1,3-diamino propane; 1,3-diamino ethane; polyethylene glycol (PEG) polymer groups, short amino acid sequences, and such as polyglycine sequences, of 1 to 5 amino acids. In some embodiments, the spacer is a nitrobenzyl group,dithioethylamino, 6 carbon spacer, 12 carbon spacer, or 3-(9-((3- carboxypropyl)(tosyl)carbamoyl)acridin-10-ium-10-yl)propane-l-sulfonate.“Specific binding partner” or “specific binding member” as used interchangeably herein refers to one of two or more different molecules that specifically recognize the other molecule compared to substantially less recognition of other molecules. The one of two different molecules has an area on the surface or in a cavity, which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule. The molecules may be members of a specific binding pair. For example, a specific binding member may include, but is not limited to, a protein, such as a receptor, an enzyme, and an antibody, or a ligand.“Threshold” as used herein refers to an empirically determined and subjective cutoff level above which acquired data is considered “signal”, and below which acquired data is considered “noise”. A computer program based on CUSUM (Cumulative Sums Algorithm) is employed to process acquired data and detect events based on threshold input from the user. Variation between users is avoided by detection of as many events as possible followed by filtering the data afterwards for specific purposes. With a “loose” threshold a lesser number of events will be counted as signal. With a “tight” threshold a greater number of events will be counted as signal. Setting the threshold as loose or tight is a subjective choice based on the desired sensitivity or specificity for an assay, and whether in a given assessment false positives or false negatives would be preferred.An “analog assay” as used herein refers to an assay in which the presence of and / or amount of an analyte in a test sample is determined by measuring the total signal produced (e.g., fluorescence, color, etc.) by the analyte in an entire reaction mixture (e.g., a single reaction vessel). In an analog assay, the noise is indistinguishable from the signal. An example of an analog assay is an assay in which the presence of and / or amount of an analyte is determined by measuring the total signal produced from a plurality of beads or microparticles contained in a single reaction vessel.“Digital assay” as used herein refers to an assay in which an analyte is captured and a molecule of the analyte segregated and interrogated (e.g., to detect the presence and / or amount of the analyte in a sample). In a digital assay, noise is separated from signal. In a digital assay, the results are assigned a value of 1 or 0. Examples of digital assays include one or more of the following (which may overlap but are not mutually exclusive): single molecule detection assay, a nanowell assay, a single molecule enzyme linked immunosorbent assay, a direct capture counting assay, etc.“Dynamic range” as used herein refers to range over which an assay readout is proportional to the amount of target molecule or analyte in the sample being analyzed.“Microparticle(s)(s)” and “microbead(s)” are used interchangeably herein and refer to a microbead or microparticle that is allowed to occupy or settle in an array of wells, such as, for example, in an array of wells. The microparticle may include at least one specific binding member that binds to an analyte of interest.As used herein, a “positive well” refers to a well that has a signal related to presence of a bead / particle / analyte molecule, which signal is above a threshold value. As used herein, a “negative well” refers to a well that may not have a signal related to presence of a bead / particle / analyte molecule. In certain embodiments, the signal from a negative well may be at a background level, i.e., below a threshold value.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety to disclose and describe the methods and / or materials in connection with which the publications are cited. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.SUBSTRATESProvided herein are Anorogenic substrates. In certain embodiments, the Anorogenic substrate is a compound of formula (I) as below:wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, phosphate.In certain embodiments of formula (I), X is H+. In certain embodiments of formula (I), X is K+. In certain embodiments of formula (I), X is Ca2+. In certain embodiments of formula (I), X is tris or tetra tris. In certain embodiments of formula (I), X is triethylammonium.In certain embodiments of formula (I), Y is phosphate. In certain embodiments of formula (I), Y is diphosphate. In certain embodiments of formula (I), Y is triphosphate. In certain embodiments of formula (I), Y is oxymethyl phosphate.Provided herein is fluorogenic substrates of formula (la) as below:wherein:X is selected from a group consisting of H+, K+, Ca2+, tetra tris, triethylammonium, or any other cation.In certain embodiments of formula (la), X is H+. In certain embodiments of formula (I), X is K+. In certain embodiments of formula (I), X is Ca2+. In certain embodiments of formula (I), X is tris or tetra tris. In certain embodiments of formula (I), X is triethylammonium.Provided herein is fluorogenic substrates of formula (lb) as below:wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is selected from a group consisting of diphosphate, triphosphate, or phosphate.In certain embodiments of formula (lb), X is H+. In certain embodiments of formula (I), X is K+. In certain embodiments of formula (I), X is Ca2+. In certain embodiments of formula (I), X is tris or tetra tris. In certain embodiments of formula (I), X is triethylammonium. In certain embodiments of formula (lb), Y is phosphate. In certain embodiments of formula(I), Y is diphosphate. In certain embodiments of formula (1), Y is triphosphate.In certain embodiments, compound of formula (I), formula (la) or formula (lb) are selected from:1-7METHODSProvided herein are methods for sample analysis using a phosphatase enzyme and a single type of Anorogenic substrate or a mixture of two types of Auorogenic substrates. A single type of Auorogenic substrate, such as, pyranine-O-phosphate (also referred to as PyPhos) may be used for detection of an analyte present at low concentrations in a sample in a digital detection method. A mixture of two types of Auorogenic substrates, may be used for detection of an analyte present at medium to high concentrations in a sample in an analog detection method or a combined digital and analog detection method.In certain embodiments, the method may involve single molecule counting using a digital detection method. In certain embodiments, a method for analyte analysis may involve assessing an analyte present in a sample. In certain embodiments, the assessing may be used for determining presence of and / or concentration of an analyte in a sample. In certain embodiments, the method may also be used for determining presence of and / or concentration of a plurality of different analytes present in a sample.Provided herein are methods for measuring or detecting an analyte present in a biological sample. The method of detecting presence of an analyte of interest on capture particles comprises contacting the capture particles present in micro-chambers with a Anorogenic substrate. The Auorogenic substrate may be of formula (I):wherein: X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation; Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate. The micro-chambers are sized to contain only one capture particle per micro-chamber; the analyte of interest is associated with a phosphatase enzyme; the phosphatase enzyme converts the Auorogenic substrate into a Auorogenic enzymatic product; the Auorogenic substrate emits a first Auorescent signal and the Auorogenic enzymatic product emits a second fluorescent signal distinct from the first Auorescent signal; detecting presence or absence of the second Auorescent signal in the micro-chambers; wherein presence of the second Auorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers.In some embodiments, the Auorogenic substrate molecule is pyranine-O-phosphate and the Auorogenic enzymatic product is pyranine. In some embodiments, the Auorogenic substrate is present a buffer, e.g., an alkaline buffer. For example, the method comprises contacting the capture particles with an alkaline buffer comprising the Auorogenic substrate. The concentration of the Auorogenic substrate may be selected based on the level of phosphatase enzyme immobilized on the capture particles. The concentration of the Auorogenic substrate may be about 1 pM to 5 mM,e.g., 1 |1M-1O |1M, 10 I1M-100 |1M, 100 |lM-500 flM, 500 |lM-lmM, or 1-3 mM. The pH of the alkaline buffer may be 8-12, e.g., 8.5-11, 9-10.5, or 9-10.In some embodiments, the phosphatase enzyme is an alkaline phosphatase enzyme. In some embodiments, the phosphatase enzyme is an acidic phosphatase enzyme. In some embodiments, the phosphatase enzyme is a commercially available phosphatase enzyme. In some embodiments, the phosphatase enzyme is conjugated to a binding member, where the binding member is a secondary antibody that binds to a primary antibody bound to the analyte captured on the capture particles. For example, the primary antibody may be a mouse or a goat antibody and the second antibody may be an anti-mouse or an anti-goat antibody.In some embodiments, each capture particle has up to 10 molecules of the analyte immobilized thereon and wherein one molecule of the phosphatase enzyme is associated with one analyte molecule.The phosphatase enzyme may be associated to an analyte molecule by any suitable means. For example, the phosphatase enzyme may be conjugated to a binding member that binds to the analyte captured on the capture particles. In some embodiments, the binding member may bind to the analyte captured on the capture particles indirectly via another binding member. In one example, the analyte may be a protein or peptide, and the phosphatase enzyme may be conjugated to another protein or peptide that binds to the analyte.In some embodiments, each capture particle has 1 or 0 molecules of the analyte immobilized on the capture particle. The capture particle having a surface on which the analyte is immobilized may be any convenient surface, such an exterior surface of a bead or an interior and exterior surface of a porous bead. For example, the analyte may be attached covalently or non- covalently to a bead, e.g., latex, agarose, sepharose, streptavidin, tosyl-activated, epoxy, polystyrene, amino bead, amine bead, carboxyl bead, silica bead, or the like.In certain embodiments, the capture particle may be a microparticle, also referred to as microbead. In some embodiments, the microparticle may be between about 0.1 nm and about 10 microns, between about 50 nm and about 5 microns, between about 100 nm and about 1 micron, between about 0.1 nm and about 700 nm, between about 500 nm and about 10 microns, between about 500 nm and about 5 microns, between about 500 nm and about 3 microns, between about 100 nm and 700 nm, or between about 500 nm and 700 nm in diameter. For example, the microparticle may be about 4-6 microns, about 2-3 microns, or about 0.5-1.5 microns in diameter. Particles less than about 500 nm diameter are sometimes considered nanoparticles. Thus, the microparticle optionally may be a nanoparticle between about 0.1 nm and about 500 nm, betweenabout 10 nm and about 500 nm, between about 50 nm and about 500 nm, between about 100 nm and about 500 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, or about 500 nm in diameter. The particle may be roughly spherical in shape and the diameter may be the diameter across the largest dimension of the particle. While the size of the capture particles are described in terms of diameter, the particles may not necessarily be spherical in size and can be amorphous in shape. For amorphous particles, the diameter is the largest distance from one side to the diametrically opposite side. In certain embodiments, the capture particles may be substantially spherical.In certain embodiments, the size of the capture particle is chosen based on size of the microchambers. For example, one capture particle per microchamber.In certain embodiments, the bead may be a magnetic bead, also referred to as a magnetic particle. Magnetic beads / particles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic or ferrofluidic. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrCh, MnAs, MnBi, EuO, NiO / Fe. Examples of ferrimagnetic materials include NiFe2O4, CoFe2O4, FesOa (or FeO.Fe2O3). Beads can have a solid core portion that is magnetic and is surrounded by one or more non-magnetic layers. Alternately, the magnetic portion can be a layer around a non-magnetic core. Exemplary magnetic particles include those available from commercial sources, such as, Dynabeads® Magnetic beads provided by Invitrogen, Estapor® SuperParamagnetic Microspheres and PureProteome™ Magnetic Beads by Merck Millipore, BcMag™ by Bioclone Inc., ProMag™ and BioMag® from Bangslabs, SupraMag™ by Polymicrospheres Inc., TurboBeads® by Turbobeads Lie., and SPHERO™ Polystyrene Magnetic Particles by Spherotech and the like. Superparamagnetic beads are available from Sigma- Aldrich and Thermo Scientific. The capture particle on which the analyte is immobilized may be stored in dry form or in a liquid. The magnetic beads may be subjected to a magnetic field prior to or after contacting with the sample with a magnetic bead on which the first binding member is immobilized.In certain embodiments, the capture particles are coated with one or more molecules of a binding member that specifically binds to the analyte of interest. In a multiplex assay, a population of first capture particles coated with one or more molecules of a first binding member that specifically binds to a first analyte of interest and a population of second capture particles coated with one or more molecules of a second binding member that specifically binds to a second analyte of interest may be used. The first capture particles and / or second capture particles may be barcoded to aid in identification of first and / or second capture particles. If an analyte is present in the sample, the capture particles bind to the analyte via the binding member. An optional washstep may be performed to dislodge non-specifically bound analytes. The capture particles may then be contacted with a second binding member that binds to the analyte bound to the capture particles. The second binding member may be conjugated to a phosphatase enzyme. The contacting steps may be in a binding buffer that facilitates the specific binding interaction. The capture particles may then be contacted with the Anorogenic substrate.The binding buffer may include molecules standard for antigen-antibody binding buffers such as, albumin (e.g., BSA), non-ionic detergents (Tween-20, Triton X-100), and / or protease inhibitors (e.g., PMSF). In certain cases, capture particles with the first binding member immobilized thereon may be present in a binding buffer prior to contacting with a sample comprising or suspected of comprising an analyte. The length of time for binding interaction between the binding member and analyte to occur may be determined empirically and may depend on the binding affinity and binding avidity between the binding member and the analyte. In certain embodiments, the contacting or incubating may be for a period of about 5 sec to 1 hour, such as, about 10 sec-30 minutes, about 30 sec -1 minute, about 1 minute-15 minutes, about 5 minutes-10 minutes, e.g., about 10 sec, about 15 sec, about 30 sec, about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour or about 2 hours. Other conditions for the binding interaction, such as, temperature, salt concentration, may also be determined empirically or may be based on manufacturer’s instructions. For example, the contacting may be carried out at room temperature (about 21°C-28°C, e.g., about 23°C - 25°C), about 37 °C, or about 4°C. In certain embodiments, an optional mixing of the sample with the capture particles may be carried out during the contacting step.In some embodiments, the detecting step is performed within 5 minutes after the contacting step with the Anorogenic substrate. In some embodiments, the detecting step is performed within 10 seconds to 2 minutes after the contacting step with the Auorogenic substrate.In some embodiments, the analyte of interest is a peptide or protein, and the capture particles comprise a first binding member that specifically binds to the peptide or protein and wherein the peptide or protein is associated with the phosphatase enzyme via a second binding member that specifically binds to the peptide or protein and wherein the second binding member is conjugated to the phosphatase enzyme.In some embodiments, the analyte of interest is a nucleic acid, and the capture particles comprise a first binding member that specifically binds to the nucleic acid and wherein the nucleic acid is associated with the phosphatase enzyme via a second binding member that specifically binds to the nucleic acid and wherein the second binding member is conjugated to the phosphatase enzyme.In some embodiments, the micro-chambers are micro-wells. In some embodiments, the micro-wells may be present as an array of micro-wells. The size and shape of the micro-wells is compatible with the capture particles. For example, the micro-wells may have a substantially circular opening having a diameter larger than the diameter of the capture particles. The depth of the micro-wells may be more than the diameter of the capture particles. The number of microwells in an array may be selected based on the number of capture particles being analyzed. The array of wells may include a plurality of wells that may range from 109to 10 in number per 1mm2. In certain cases, an array of about 100,000 to 500,000 wells (e.g., femtoliter wells) covering an area approximately 12 mm2may be fabricated. Each well may measure about 4.2 pm wide X 3.2 pm deep (volume approximately 50 femtoliters), and may be capable of holding a single bead / particle (about 3 pm diameter). At this density, the femtoliter wells are spaced at a distance of approx. 7.4 pm from each other. In some examples, the microwell array may be fabricated to have individual wells with a diameter of 10 pm to 10,000 pm. For example, the array of microwells may be array of femtoliter wells, array of nanoliter wells, or array of microliter wells. In certain embodiments, the micro-wells in an array may all have substantially the same volume. The array of micro-wells may have a volume up to 100 pl, e.g., about 0.1 femtoliter, 1 femtoliter, 10 femtoliter, 25 femtoliter, 50 femtoliter, 100 femtoliter, 0.1 pL, 1 pL, 10 pL, 25 pL, 50 pL, 100 pL, 0.1 nL, 1 nL, 10 nL, 25 nL, 50 nL, 100 nL, 0.1 microliter, 1 microliter, 10 microliter, 25 microliter, 50 microliter, or 100 microliter.The placement of single capture particle per well allows for either a digital readout or analog readout. For example, for a low number of positive wells (<~70% positive) Poisson statistics can be used to quantitate the analyte concentration in a digital format; for high numbers of positive wells (>~70%) the relative intensities of signal-bearing wells are compared to the signal intensity generated from a single capture particle, respectively, and used to generate an analog signal. A digital signal may be used for lower analyte concentrations, whereas an analog signal may be used for higher analyte concentrations. A combination of digital and analog quantitation may be used, which may expand the linear dynamic range.The wells may be any of a variety of shapes, such as, cylindrical with a flat bottom surface, cylindrical with a rounded bottom surface, cubical, cuboidal, frustoconical, inverted frustoconical, or conical. In certain cases, the wells may include a sidewall that may be oriented to facilitate the receiving and retaining of capture particles that have been moved over the micro-well array.In some embodiments, the micro-chambers are micro-droplets. Digital microfluidics comprising an array of electrodes to manipulate droplets in a microfluidics device, e.g., movedroplets, split droplets, merge droplets, etc. in a small space may be used for analysis of the droplets. The terms droplet(s) and fluidic droplet(s) are used interchangeably to refer to a discreet volume of liquid that is roughly spherical in shape and is bounded on at least one side by a wall or substrate of a microfluidics device. Roughly spherical in the context of the droplet refers to shapes such as spherical, partially flattened sphere, e.g., disc shaped, slug shaped, truncated sphere, ellipsoid, hemispherical, or ovoid. The volume of the micro-droplets may range from aboutlO pl to about 5 pL, such as, 10 pl - 1 pL, 7.5 pl -10 pL, 5 pl -1 nL, 2.5 pl - 10 nL, or Ipl - 100 nL, e.g., 10 pl, 5 pl, 1 pl, 800 nL, 500 nL, or lesser.Micro-chambers, such as, micro- wells and micro-droplets may have a diameter between about 0.1 nm and about 10 microns, between about 50 nm and about 5 microns, between about 100 nm and about 1 micron, between about 0.1 nm and about 700 nm, between about 500 nm and about 10 microns, between about 500 nm and about 5 microns, between about 500 nm and about 3 microns, between about 100 nm and 700 nm, or between about 500 nm and 700 nm.Also provided herein is a method of detecting presence of an analyte of interest on capture particles, the method comprising contacting the capture particles present in nano-chambers with a fluorogenic substrate of formula (I):wherein: X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation; Y is selected from a group consisting of diphosphate, triphosphate, oxy methyl phosphate, or phosphate. The micro-chambers are sized to contain only one capture particle per nano-chamber, the analyte of interest is associated with a phosphatase enzyme, the phosphatase enzyme converts the fluorogenic substrate into a fluorogenic enzymatic product, the fluorogenic substrate emits a first fluorescent signal and the fluorogenic enzymatic product emits a second fluorescent signal distinct from the first fluorescent signal; detecting presence or absence of the second fluorescent signal in the micro-chambers, wherein presence of the second fluorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers.Also provided herein is a method of detecting presence of an analyte of interest on capture particles, the method comprising contacting the capture particles deposited into micro-chambers with a first fluorogenic substrate and a second fluorogenic substrate, wherein the first fluorogenic substrate has the structure of formula (la):and the second fluorogenic substrate has the structure of formula (lb):wherein: X is selected from a group consisting of H+, K+, Ca2+, tris, tetra tris, triethylammonium, or any other cation; Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate, the micro-chambers are sized to contain only one capture particle per micro-chamber, the analyte of interest is associated with a phosphatase enzyme, the phosphatase enzyme converts the first and second fluorogenic substrates into a fluorogenic enzymatic product, the first and second fluorogenic substrate emit a first fluorescent signal and the fluorogenic enzymatic product emits a second fluorescent signal distinct from the first fluorescent signal; detecting presence or absence of the second fluorescent signal in the micro-chambers, wherein presence of the second fluorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers.Also provided herein is a method of detecting presence of an analyte of interest on capture particles, the method comprising contacting the capture particles deposited into nano-chambers with a first fluorogenic substrate and a second fluorogenic substrate, wherein the first fluorogenic substrate has the structure of formula (la):and the second fluorogenic substrate has the structure of formula (lb):wherein: X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation; Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate, the micro-chambers are sized to contain only one capture particle per nano-chamber, the analyte of interest is associated with a phosphatase enzyme, the phosphatase enzyme converts the first and second fluorogenic substrates into a fluorogenic enzymatic product, the first and second fluorogenic substrate emit a first fluorescent signal and the fluorogenic enzymatic product emits a second fluorescent signal distinct from the first fluorescent signal; detecting presence or absence of the second fluorescent signal in the micro-chambers, wherein presence of the second fluorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers.In some embodiments, the micro-chambers are micro-wells. In some embodiments, the micro- wells may be present as an array of micro-wells. In some embodiments, the micro-chambers are nano-wells. In some embodiments, the nano-wells may be present as an array of nano-wells. The size and shape of the nano-wells is compatible with the capture particles. For example, the nano-wells may have a substantially circular opening having a diameter larger than the diameter of the capture particles. The depth of the nano-wells may be more than the diameter of the capture particles. The number of nano-wells in an array may be selected based on the number of capture particles being analyzed. The array of wells may include a plurality of wells that may range from 109to 10 in number per 1mm2. In certain cases, an array of about 100,000 to 500,000 wells (e.g.,femtoliter wells) covering an area approximately 12 mm2may be fabricated. Each well may measure about 4.2 pm wide X 3.2 pm deep (volume approximately 50 femtoliters), and may be capable of holding a single bead / particle (about 3 pm diameter). At this density, the femtoliter wells are spaced at a distance of approx. 7.4 pm from each other. In some examples, the nanowell array may be fabricated to have individual wells with a diameter of 10 nm to 10,000 nm. For example, the array of nano-wells may be array of femtoliter wells, array of nanoliter wells, or array of microliter wells. In certain embodiments, the nano-wells in an array may all have substantially the same volume. The array of nano-wells may have a volume up to 100 pl, e.g., about 0.1 femtoliter, 1 femtoliter, 10 femtoliter, 25 femtoliter, 50 femtoliter, 100 femtoliter, 0.1 pL, 1 pL, 10 pL, 25 pL, 50 pL, 100 pL, 0.1 nL, 1 nL, 10 nL, 25 nL, 50 nL, 100 nL, 0.1 microliter, 1 microliter, 10 microliter, 25 microliter, 50 microliter, or 100 microliter.The placement of single capture particle per microwell allows for either a digital readout or analog readout. For example, for a low number of positive wells (<~70% positive) Poisson statistics can be used to quantitate the analyte concentration in a digital format; for high numbers of positive wells (>~70%) the relative intensities of signal-bearing wells are compared to the signal intensity generated from a single capture particle, respectively, and used to generate an analog signal. A digital signal may be used for lower analyte concentrations, whereas an analog signal may be used for higher analyte concentrations. A combination of digital and analog quantitation may be used, which may expand the linear dynamic range.The wells may be any of a variety of shapes, such as, cylindrical with a flat bottom surface, cylindrical with a rounded bottom surface, cubical, cuboidal, frustoconical, inverted frustoconical, or conical. In certain cases, the wells may include a sidewall that may be oriented to facilitate the receiving and retaining of capture particles that have been moved over the nano-well array.In some embodiments, the micro-chambers are micro-droplets. Digital microfluidics comprising an array of electrodes to manipulate droplets in a microfluidics device, e.g., move droplets, split droplets, merge droplets, etc. in a small space may be used for analysis of the droplets. As used herein, the terms “droplet(s)” and “fluidic droplet(s)” are used interchangeably to refer to a discreet volume of liquid that is roughly spherical in shape and is bounded on at least one side by a wall or substrate of a microfluidics device. Roughly spherical in the context of the droplet refers to shapes such as spherical, partially flattened sphere, e.g., disc shaped, slug shaped, truncated sphere, ellipsoid, hemispherical, or ovoid. The volume of the nano-droplets may range from about 10 pL to about 5 pL, such as, 10 pL - 1 pL, 7.5 pL -10 pL, 5 pL -1 nL, 2.5 pL - 10 nL, or 1 pL - 100 nL, e.g., 10 pL, 5 pL, 1 pL, 800 nL, 500 nL, or lesser.The micro-chambers, micro-wells, and micro-droplets described herein may have a diameter between about 0. 1 nm and about 10 microns, between about 50 nm and about 5 microns, between about 100 nm and about 1 micron, between about 0.1 nm and about 700 nm, between about 500 nm and about 10 microns, between about 500 nm and about 5 microns, between about 500 nm and about 3 microns, between about 100 nm and 700 nm, or between about 500 nm and 700 nm.In some embodiments, the first Anorogenic substrate is pyranine-O-phosphate, the second Auorogenic substrate is bis-pyranine-O-phosphate and the Auorogenic enzymatic product is pyranine.In some embodiments, each capture particle has up to 10 molecules of the analyte immobilized on the capture particles and wherein one molecule of the phosphatase enzyme associated is associated with one analyte molecule.In some embodiments, each capture particle has up to 5, 3, 2, 1 or 0 molecule of the analyte immobilized thereon. In some embodiments, each capture particle has 5, 3, 2, or 0 copy of the analyte immobilized thereon.In some embodiments, the detecting step is performed within 5 minutes after the contacting step. In some embodiments, the detecting step is performed within 1 minute to 5 minutes after the contacting step.In some embodiments, the analyte of interest is a peptide or protein, and the capture particles comprise a first binding member that specifically binds to the peptide or protein and wherein the peptide or protein is associated with the phosphatase enzyme via a second binding member that specifically binds to the peptide or protein and wherein the second binding member is conjugated to the phosphatase enzyme.In some embodiments, the analyte of interest is a nucleic acid, and the capture particles comprise a first binding member that specifically binds to the nucleic acid and wherein the nucleic acid is associated with the phosphatase enzyme via a second binding member that specifically binds to the nucleic acid and wherein the second binding member is conjugated to the phosphatase enzyme.In some embodiments, the nano-chambers are micro-wells. In some embodiments, the nano-chambers are micro-droplets. In some embodiments, the nano-chambers are nano-wells. In some embodiments, the nano-chambers are nano-droplets.In some embodiments, the phosphatase enzyme is an alkaline phosphatase enzyme. In some embodiments, the first Auorogenic substrate and the second Auorogenic substrate are presentat a ratio of 1:1. In some embodiments, the concentration of each of the first and second Anorogenic substrate is between 1 to 50 ,uM.Provided herein are methods for sample analysis using a phosphodiesterase enzyme and a Anorogenic substrate of formula (II):(II) wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation.The method for sample analysis may include detecting presence of an analyte of interest on capture particles, the method comprising contacting the capture particles present in microchambers with the Auorogenic substrate of Formula (II), where the micro-chambers are sized to contain only one capture particle per micro-chamber, the analyte of interest is associated with a phosphodiesterase enzyme, the phosphodiesterase enzyme converts the Auorogenic substrate into a Auorogenic enzymatic product, the Auorogenic substrate emits a first fluorescent signal and the Auorogenic enzymatic product emits a second Auorescent signal distinct from the first fluorescent signal; and detecting presence or absence of the second Auorescent signal in the micro-chambers, wherein presence of the second Auorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers. The Auorogenic enzymatic product may be pyranine. Each capture particle may have up to 10 molecules of the analyte immobilized on the capture particles and one molecule of the phosphodiesterase enzyme associated may be associated with one analyte molecule. Each capture particle may have up to 1 or 0 molecule of the analyte immobilized thereon. Each capture particle may have 1 or 0 copy of the analyte immobilized on the capture particle. The detecting step may be performed within 5 minutes after the contacting step. The detecting step may be performed within 10 seconds to 2minutes after the contacting step. The analyte of interest may be a peptide or protein, and the capture particles comprise a first binding member that specifically binds to the peptide or protein and wherein the peptide or protein is associated with the phosphodiesterase enzyme via a second binding member that specifically binds to the peptide or protein and wherein the second binding member is conjugated to the phosphodiesterase enzyme. The analyte of interest may be a nucleic acid, and the capture particles comprise a first binding member that specifically binds to the nucleic acid and wherein the nucleic acid is associated with the phosphodiesterase enzyme via a second binding member that specifically binds to the nucleic acid and wherein the second binding member is conjugated to the phosphodiesterase enzyme. The micro-chambers may be microwells. The micro-chambers may be micro-droplets.Also provided herein is a method of preparing pyranine-O-phosphate, comprising: reacting pyranine with a mono-reactive phosphate source to obtain pyranine-O-phosphate.In some embodiments, the mono-reactive phosphate source is diethyl chlorophosphate.In some embodiments, pyranine-O-phosphate is of formula (lb):wherein: X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation, Y is a phosphate. In some embodiments, pyranine-O-phosphate has a molecular weight in the range of 100 to 1000 Daltons.Also provided herein is a method of preparing pyranine-methyl-phosphate, comprising: reacting pyranine with a mono-reactive phosphate source to obtain pyranine-methyl-phosphate. In some embodiments, the mono-reactive phosphate source is di-tertbutyl chloromethylphosphate.In some embodiments, pyranine-O-phosphate is of formula (la):wherein: X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation.Also provided herein is a method of preparing pyranine-di-phosphate, comprising: reacting pyranine with a mono-reactive phosphate source to obtain pyranine-di-phosphate, further reacting with tetrabutyl ammonium phosphate to form pyranine-di-phosphate. In some embodiments, the mono-reactive phosphate source is phosphoryl chloride.In some embodiments, pyranine-di-phosphate is of formula (lb):wherein: X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation, Y is a diphosphate.Also provided herein is a method of preparing pyranine-tri-phosphate, comprising: reacting pyranine with a mono-reactive phosphate source to obtain pyranine-tri-phosphate, further reacting with tetrabutylammonium phosphate to form pyranine-tri-phosphate. In some embodiments, the mono-reactive phosphate source is phosphoryl chloride.In some embodiments, pyranine-di-phosphate is of formula (lb):wherein: X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation, Y is a triphosphate.Also provided herein is a method of preparing dipyranine phosphate, comprising: reacting 2 equivalents of pyranine with a di-reactive phosphate source to obtain dipyranine phosphate. In some embodiments, the di-reactive phosphate source is ethyl dichlorophosphate.In some embodiments, dipyranine phosphate is a compound of formula (II):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation.Exemplary Assay FormatsPreparation of capture particles comprising analytes of interest may be conducted using any suitable method. A number of immunoassay formats may be used. In some embodiments, a sample droplet containing or suspected of containing the target analyte may be merged with a droplet containing magnetic capture particles on which a first binding member that specifically binds to the target analyte present in the sample is attached. Merging creates a single droplet which may be incubated for a time sufficient to allow binding of the first binding member to an analyte present in the sample droplet. Optionally, the single droplet may be agitated to facilitate mixing of the sample with the first binding member. Mixing may be achieved by moving the single droplet back and forth, moving the single droplet around over a plurality of electrodes, splitting a droplet and then merging the droplets, or using SAWs, and the like. Next, the single droplet may be subjected to a magnetic force to retain the beads at a location in the device while the droplet may be moved away to a waste chamber or pad and replaced with a droplet containing a second binding member. The second binding member may be associated with a phosphatase enzyme. An optionalwash step may be performed, prior to adding the second binding member, by moving a droplet of wash buffer to the location at which the beads are retained using the magnetic force. The magnetic capture particles may or may not be resuspended in the wash buffer; a magnetic force is applied to the magnetic beads and the wash buffer is transported to a waste location. After a period of time sufficient for the second binding member to bind the analyte bound to the first binding member, the droplet containing the second binding member may be moved away while the beads are retained at the location. The beads may be washed using a droplet of wash buffer. Following the wash step, the magnetic force may be removed and a droplet containing the labeled beads which has a complex of the first binding member, analyte and the second binding member may be moved over an array of nano-wells or may be fused to a droplet comprising the Anorogenic substrate. The labeled beads may be allowed to settle into the array of wells. The beads may settle using gravitational force or by applying electric or magnetic force. Following a wash step to remove any beads not located inside the wells, the wells may contacted with the Anorogenic substrate, optionally be sealed by using a hydrophobic liquid. Alternatively, the droplet containing the labeled beads which has a complex of the first binding member, analyte and the second binding member may be fused to a droplet comprising the Auorogenic substrate.Exemplary Target AnalytesAs will be appreciated by those in the art, any analyte that can be specifically bound by a first binding member and a second binding member may be detected and, optionally, quantified using methods and devices of the present disclosure.In some embodiments, the analyte may be a biomolecule. Non- limiting examples of biomolecules include macromolecules such as, proteins, lipids, and carbohydrates. In certain instances, the analyte may be hormones, antibodies, growth factors, cytokines, enzymes, receptors (e.g., neural, hormonal, nutrient, and cell surface receptors) or their ligands, cancer markers (e.g., PSA, TNF-alpha), markers of myocardial infarction (e.g., troponin, creatine kinase, and the like), toxins, drugs (e.g., drugs of addiction), metabolic agents (e.g., including vitamins), and the like. Non-limiting embodiments of protein analytes include peptides, polypeptides, protein fragments, protein complexes, fusion proteins, recombinant proteins, phosphoproteins, glycoproteins, lipoproteins, or the like.In certain embodiments, the analyte may be a post-translationally modified protein (e.g., phosphorylated, methylated, glycosylated protein) and the first or the second binding member may be an antibody specific to a post-translational modification. A modified protein may be bound to a first binding member immobilized on a solid support where the first binding member binds tothe modified protein but not the unmodified protein. In other embodiments, the first binding member may bind to both the unmodified and the modified protein, and the second binding member may be specific to the post-translationally modified protein.In some embodiments, the analyte may be a cell, such as, circulating tumor cell, pathogenic bacteria, viruses (including retroviruses, herpesviruses, adenoviruses, lentiviruses, Filoviruses (Ebola), hepatitis viruses (e.g., A, B, C, D, and E); HPV, etc.; spores, etc.A non-limiting list of analytes that may be analyzed by the methods presented herein include B-type natriuretic peptide (BNP), N-terminal pro B-type natriuretic peptide (proBNP), Ap42 amyloid beta-protein, fetuin-A, tau, secretogranin II , prion protein, Alpha-synuclein, tau protein, neurofilament light chain, parkin, PTEN induced putative kinase I, DI-1, leucine-rich repeat kinase 2, mutated ATP13A2, Apo H, ceruloplasmin, Peroxisome proliferator-activated receptor gamma coactivator- 1 alpha (PGC-la), transthyretin, Vitamin D-binding Protein, proapoptotic kinase R (PKR) and its phosphorylated PKR (pPKR), CXCL13, IL- 12p40, CXCL13, IL-8, Dkk-3 (semen), p!4 endocan fragment, Serum, ACE2, autoantibody to CD25, hTERT, CAI25 (MUC 16), VEGF, sIL-2, Osteopontin, Human epididymis protein 4 (HE4), Alpha- Fetoprotein , Albumin, albuminuria, microalbuminuria, neutrophil gelatinase-associated lipocalin (NGAL) , interleukin 18 (IL- 18) , Kidney Injury Molecule -1 (KIM-1) , Liver Fatty Acid Binding Protein (L-FABP) , LMP1, BARF1, IL-8, carcinoembryonic antigen (CEA), BRAF, CCNI, EGRF, FGF19, FRS2, GREB 1, and LZTS1, alpha-amylase, carcinoembryonic antigen, CA 125, IL8 , thioredoxin, beta-2 microglobulin levels - monitor activity of the virus, tumor necrosis factoralpha receptors - monitor activity of the virus, CAI 5-3, follicle-stimulating hormone (FSH), leutinizing hormone (LH), T-cell lymphoma invasion and metastasis 1 (TIAM1), N-cadherin, EC39, amphiregulin, dUTPase, secretory gelsolin (pGSN), PSA (prostate specific antigen), thymosin 315 , insulin, plasma C-peptide, glycosylated hemoglobin (HBAlc), C-Reactive Protein (CRP), Interleukin-6 (IL-6), ARHGDIB (Rho GDP-dissociation inhibitor 2), CFL1 (Cofilin- 1), PFN1 (profilin-1), GSTP1 (Glutathione S-transferase P), S100A11 (Protein S100- Al l), PRDX6 (Peroxiredoxin-6), HSPE1 (10 kDa heat shock protein, mitochondrial), LYZ (Lysozyme C precursor), GPI (Glucose-6-phosphate isomerase), HIST2H2AA (Histone H2A type 2-A), GAPDH (Glyceraldehyde-3- phosphate dehydrogenase), HSPG2 (Basement membrane-specific heparan sulfate proteoglycan core protein precursor), LGALS3BP (Galectin-3-binding protein precursor), CTSD (Cathepsin D precursor), APOE (Apolipoprotein E precursor), IQGAP1 (Ras GTPase-activating-like protein IQGAP1), CP (Ceruloplasmin precursor), and IGLC2 (IGLC1 protein), PCDGF / GP88, EGFR, HER2, MUC4, IGF-IR, p27(kipl ), Akt, HER3, HER4, PTEN, PIK3CA, SHIP, Grb2, Gab2, PDK-1 (3 -phosphoinositide dependent protein kinase-1), TSC1,TSC2, mTOR, MIG-6 (ERBB receptor feedback inhibitor 1), S6K, src, KRAS, MEK mitogen- activated protein kinase 1, cMYC, TOPO II topoisomerase (DNA) II alpha 170 kDa, FRAP1, NRG1, ESRI, ESR2, PGR, CDKN1B, MAP2K1, NEDD4-1, FOXO3A, PPP1R1B, PXN, ELA2, CTNNB1, AR, EPHB2, KLF6, ANXA7, NKX3-1, PITX2, MKI67, PHLPP, adiponectin (ADIPOQ), fibrinogen alpha chain (FGA), leptin (LEP), advanced glycosylation end productspecific receptor (AGER aka RAGE), alpha-2-HS-glycoprotein (AHSG), angiogenin (ANG), CD14 molecule (CD14), ferritin (FTH1), insulin-like growth factor binding protein 1 (IGFBP1), interleukin 2 receptor, alpha (IL2RA), vascular cell adhesion molecule 1 (VCAM1) and Von Willebrand factor (VWF), myeloperoxidase (MPO), ILla, TNFa, perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA), lactoferrin, calprotectin, Wilm’s Tumor- 1 protein, Aquaporin- 1, MLL3, AMBP, VDAC1, E. coli enterotoxins (heat-labile exotoxin, heat-stable enterotoxin), influenza HA antigen, tetanus toxin, diphtheria toxin, botulinum toxins, Shiga toxin, Shiga-like toxin I, Shiga- like toxin II, Clostridium difficile toxins A and B, etc.Exemplary targets of nucleic acid aptamers that may be measured in a sample such as an environmental sample, a biological sample obtained from a patient or subject in need using the subject methods and devices include: drugs of abuse (e.g. cocaine), protein biomarkers (including, but not limited to, Nucleolin, nuclear factor-kB essential modulator (NEMO), CD-30, protein tyrosine kinase 7 (PTK7), vascular endothelial growth factor (VEGF), MUC1 glycoform, immunoglobulin p Heavy Chains (IGHM), Immunoglobulin E, avP3 integrin, a- thrombin, HIV gp!20, NF-KB, E2F transcription factor, HER3, Plasminogen activator inhibitor , Tenascin C,CXCL12 / SDF-1, prostate specific membrane antigen (PSMA), gastric cancer cells, HGC-27); cells (including, but not limited to, non-small cell lung cancer (NSCLC), colorectal cancer cells, (DLD-1), H23 lung adenocarcinoma cells, Ramos cells, T-cell acute lymphoblastic leukemia (T- ALL) cells, CCRF-CEM, acute myeloid leukemia (AML) cells (HL60), small-cell lung cancer (SCLC) cells, NCIH69, human glioblastoma cells, U118-MG, PC-3 cells, HER-2-overexpressing human breast cancer cells, SK-BR-3, pancreatic cancer cell line (Mia-PaCa-2)); and infectious agents (including, but not limited to, Mycobacterium tuberculosis, Staphylococcus aureus, Shigella dysenteriae, Escherichia coli O157:H7, Campylobacter jejuni, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella 08, Salmonella enteritidis).Exemplary targets of protein or peptide aptamers that may be measured in a sample obtained from a patient or subject in need using the subject methods and devices include, but are not limited to: HBV core capsid protein, CDK2, E2F transcription factor, Thymidylate synthase, Ras, EB1 , and Receptor for Advanced Glycated End products (RAGE). Aptamers, and use and methods of production thereof are reviewed in e.g., Shum et al., J Cancer Ther. 2013 4:872; Zhanget al., Curr Med Chem. 2011 ; 18:4185; Zhu et al., Chem Commun (Camb). 2012 48:10472; Crawford et al., Brief Funct Genomic Proteomic. 2003 2:72; Reverdatto et al., PLoS One. 2013 8:e65180.In some cases, the analyte may be present in a sample, such as, a liquid, fluent particulate solid, or fluid suspension of solid particles. In some cases, the sample may be processed prior to the analysis described herein. For example, the analyte may be separated or purified from its source prior to analysis; however, in certain embodiments, an unprocessed analyte may be assayed directly. The source of the analyte molecule may be synthetic (e.g., produced in a laboratory), the environment (e.g., air, soil, fluid samples, e.g., water supplies, etc.), an animal, e.g., a mammal, a plant, or any combination thereof. In a particular example, the source of an analyte is a human bodily substance (e.g., bodily fluid, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, lacrimal fluid, lymph fluid, amniotic fluid, interstitial fluid, lung lavage, cerebrospinal fluid, feces, tissue, organ, or the like). Tissues may include, but are not limited to skeletal muscle tissue, liver tissue, lung tissue, kidney tissue, myocardial tissue, brain tissue, bone marrow, cervix tissue, skin, etc. In certain cases, the source of the analyte may be an organ or tissue, such as a biopsy sample, which may be solubilized by tissue disintegration / cell lysis.A wide range of volumes of the fluid analyte may be analyzed. In a few exemplary embodiments, the analyte volume may be about 0.5 nL, about 1 nL, about 3 nL, about 0.01 pL, about 0.1 pL, about 1 pL, about 5 pL, about 10 pL, about 100 pL, about 1 mL, about 5 mL, about 10 mL, or the like. In some cases, the volume of the fluid analyte is between about 0.01 pL and about 10 mL, between about 0.01 pL and about 1 mL, between about 0.01 pL and about 100 pL, or between about 0.1 pL and about 10 pL.In some cases, the fluid analyte may be diluted prior to use in an assay. For example, in embodiments where the source of an analyte molecule is a human body fluid (e.g., blood, serum), the fluid may be diluted with an appropriate solvent (e.g., a buffer such as PBS buffer). A fluid sample may be diluted about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or greater, prior to use.In some cases, the analyte may undergo pre-analytical processing. Pre-analytical processing may offer additional functionality such as nonspecific protein removal and / or effective yet cheaply implementable mixing functionality. General methods of pre-analytical processing may include the use of electrokinetic trapping, AC electrokinetics, surface acoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, or other pre-concentration techniques known in the art. In some cases, the fluid analyte may be concentrated prior to use in an assay. Forexample, in embodiments where the source of an analyte molecule is a human body fluid (e.g., blood, serum), the fluid may be concentrated by precipitation, evaporation, filtration, centrifugation, or a combination thereof. A fluid sample may be concentrated about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or greater, prior to use.In certain embodiments, the analyte is not amplified (i.e., the copy number of the analyte is not increased) prior to the measurement of the analyte. For example, in cases where the analyte is DNA or RNA, the analyte is not replicated to increase copy numbers of the analyte. In certain cases, the analyte is a protein or a small molecule.Binding MembersAs will be appreciated by those in the art, the binding members will be determined by the analyte to be analyzed. Binding members for a wide variety of target molecules are known or can be readily found or developed using known techniques. For example, when the target analyte is a protein, the binding members may include proteins, particularly antibodies or fragments thereof (e.g., antigen-binding fragments (Fabs), Fab' fragments, F(ab')2 fragments, recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, such as variable heavy chain domains (“VHH”; also known as “VHH fragments”) derived from animals in the Camelidae family (VHH and methods of making them are described in Gottlin et al., Journal of Biomolecular Screening, 14:77-85 (2009)), recombinant VHH singledomain antibodies, and VNAR fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti- id”) antibodies, and functionally active epitope -binding fragments of any of the above, full-length polyclonal or monoclonal antibodies, antibody-like fragments, etc.), other proteins, such as receptor proteins, Protein A, Protein C, or the like. In case where the analyte is a small molecule, such as, steroids, bilins, retinoids, and lipids, the first and / or the second binding member may be a scaffold protein (e.g., lipocalins) or a receptor. In some cases, binding member for protein analytes may be a peptide. For example, when the target analyte is an enzyme, suitable binding members may include enzyme substrates and / or enzyme inhibitors which may be a peptide, a small molecule and the like. In some cases, when the target analyte is a phosphorylated species, the binding members may comprise a phosphate-binding agent. For example, the phosphate- binding agent may comprise metal-ion affinity media such as those describe in U.S. Pat. No. 7,070,921 and U.S. Patent Application No. 20060121544.In certain cases, at least one of the binding members may be an aptamer, such as those described i5,705,337. Nucleic acid aptamers (e.g., single-stranded DNA molecules or single-stranded RNA molecules) may be developed for capturing virtually any target molecule. Aptamers bind target molecules in a highly specific, conformation-dependent manner, typically with very high affinity, although aptamers with lower binding affinity can be selected. Aptamers may distinguish between target analyte molecules based on very small structural differences such as the presence or absence of a methyl or hydroxyl group and certain aptamers can distinguish between D- and L-enantiomers and diastereomers. Aptamers may bind small molecular targets, including drugs, metal ions, and organic dyes, peptides, biotin, and proteins. Aptamers can retain functional activity after biotinylation, fluorescein labeling, and when attached to glass surfaces and microspheres.Nucleic acid aptamers are oligonucleotides that may be single stranded oligodeoxynucleotides, oligoribonucleotides, or modified oligodeoxynucleotide or oligoribonucleotides. “Modified” encompasses nucleotides with a covalently modified base and / or sugar. For example, modified nucleotides include nucleotides having sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Thus, modified nucleotides may also include 2' substituted sugars such as 2'-O-methyl-; 2-O-alkyl; 2-O-allyl; 2'-S-alkyl; 2'-S-allyl; 2'- fluoro-; 2'-halo or 2-azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose.Peptide aptamers may be designed to interfere with protein interactions. Peptide aptamers may be based on a protein scaffold onto which a variable peptide loop is attached, thereby constraining the conformation of the aptamer. In some cases, the scaffold portion of the peptide aptamer is derived from Bacterial Thioredoxin A (TrxA).When the target analyte is a carbohydrate, potentially suitable capture particles (as defined herein) include, for example, antibodies, lectins, and selectins. As will be appreciated by those of ordinary skill in the art, any molecule that can specifically associate with a target molecule of interest may potentially be used as a binding member.For certain embodiments, suitable target analyte / binding member complexes can include, but are not limited to, antibodies / antigens, antigens / antibodies, receptors / ligands, ligands / receptors, proteins / nucleic acid, enzymes / substrates and / or inhibitors, carbohydrates (including glycoproteins and glycolipids) / lectins and / or selectins, proteins / proteins, proteins / small molecules, etc.In a particular embodiment, the first binding member may be attached to a capture particle via a linkage, which may comprise any moiety, functionalization, or modification of the capture particle and / or binding member that facilitates the attachment of the binding member to thecapture particle. The linkage between the binding member and the capture particle may include one or more chemical or physical (e.g., non-specific attachment via van der Waals forces, hydrogen bonding, electrostatic interactions, hydrophobic / hydrophilic interactions; etc.) bonds and / or chemical spacers providing such bond(s).In certain embodiments, a capture particle may also comprise a protective, blocking, or passivating layer that can eliminate or minimize non-specific attachment of non-capture components (e.g., analyte molecules, binding members) to the binding surface during the assay which may lead to false positive signals during detection or to loss of signal. Examples of materials that may be utilized in certain embodiments to form passivating layers include, but are not limited to: polymers, such as poly(ethylene glycol), that repel the non-specific binding of proteins; naturally occurring proteins with this property, such as serum albumin and casein; surfactants, e.g., zwitterionic surfactants, such as sulfobetaines; naturally occurring long-chain lipids; polymer brushes, and nucleic acids, such as salmon sperm DNA.Certain embodiments utilize binding members that are proteins or polypeptides. As is known in the art, any number of techniques may be used to attach a polypeptide to a wide variety of solid supports. A wide variety of techniques are known to add reactive moieties to proteins, for example, the method outlined in U.S. Pat. No. 5,620,850. Further, methods for attachment of proteins to surfaces are known, for example, see Heller, Acc. Chem. Res. 23:128 (1990).As explained herein, binding between the binding members and the analyte, is specific, e.g., as when the binding member and the analyte are complementary parts of a binding pair. In certain embodiments, the binding member binds specifically to the analyte. By "specifically bind" or "binding specificity," it is meant that the binding member binds the analyte molecule with specificity sufficient to differentiate between the analyte molecule and other components or contaminants of the test sample. For example, the binding member, according to one embodiment, may be an antibody that binds specifically to an epitope on an analyte. The antibody, according to one embodiment, can be any antibody capable of binding specifically to an analyte of interest. For example, appropriate antibodies include, but are not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies (dAbs) (e.g., such as described in Holt et al. (2014) Trends in Biotechnology 21:484-490), and including single domain antibodies sdAbs that are naturally occurring, e.g., as in cartilaginous fishes and camelid, or which are synthetic, e.g., nanobodies, VHH, or other domain structure), synthetic antibodies (sometimes referred to as antibody mimetics), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and fragments of each, respectively. As another example, the analyte molecule may be an antibody and the first binding member may be an antigen and thesecond binding member may be a secondary antibody that specifically binds to the target antibody or the first binding member may be a secondary antibody that specifically binds to the target antibody and the second binding member may be an antigenIn some embodiments, the binding member may be chemically programmed antibodies (cpAbs) (described in Rader (2014) Trends in Biotechnology 32: 186-197), bispecific cpAbs, antibody-recruiting molecules (ARMs) (described in McEnaney et al. (2012) ACS Chem. Biol. 7:1139-1151), branched capture agents, such as a triligand capture agent (described in Millward et al. (2011) J. Am. Chem. Soc. 133:18280-18288), engineered binding proteins derived from nonantibody scaffolds, such as monobodies (derived from the tenth fibronectin type III domain of human fibronectin), affibodies (derived from the immunoglobulin binding protein A), DARPins (based on Ankyrin repeat modules), anticalins (derived from the lipocalins bilin-binding protein and human lipocalin 2), and cysteine knot peptides (knottins) (described in Gilbreth and Koide, (2012) Current Opinion in Structural Biology 22:1-8; Banta et al. (2013) Annu. Rev. Biomed. Eng. 15:93-113), WW domains (described in Patel et al. (2013) Protein Engineering, Design & Selection 26(4):307-314), repurposed receptor ligands, affitins (described in Behar et al. (2013) 26:267-275), and / or Adhirons (described in Tiede et al. (2014) Protein Engineering, Design & Selection 27:145-155).In some embodiments, the binding affinity between analyte molecules and binding members should be sufficient to remain bound under the conditions of the assay, including wash steps to remove molecules or particles that are non-specifically bound. In some cases, for example in the detection of certain biomolecules, the binding constant of the analyte molecule to its complementary binding member may be between at least about 104and about 106M1, at least about 105and about 109M1, at least about 107and about 109M1, greater than about 109M1, or greater.SYSTEMSProvided herein are systems for sample analysis using a phosphatase enzyme and a single type of fluorogenic substrate or a mixture of two types of fluorogenic substrates. A single type of fluorogenic substrate, such as, pyranine-O-phosphate may be used for detection of an analyte present at low concentrations in a sample in a digital detection method. A mixture of two types of fluorogenic substrates, such as, may be used for detection of an analyte present at medium to high concentrations in a sample in an analog detection method or a combined digital and analog detection method.In certain embodiments, the system for detecting presence of an analyte of interest on capture particles, may comprise a processor; a cartridge comprising micro-chambers; the microchambers comprising the capture particles and a fluorogenic substrate of formula (I):wherein: X is H+, K+, Ca2+, tris, triethylammonium, or any other cation, Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate, the microchambers are sized to contain only one capture particle per micro-chamber, the analyte of interest is associated with a phosphatase enzyme, the phosphatase enzyme converts the fluorogenic substrate into a fluorogenic enzymatic product, the fluorogenic substrate emits a first fluorescent signal and the fluorogenic enzymatic product emits a second fluorescent signal distinct from the first fluorescent signal, the processor is programmed to execute instructions for detecting presence or absence of the second fluorescent signal in the micro-chambers, wherein presence of the second fluorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers.Tn certain embodiments, the instructions for detecting presence or absence of the second fluorescent signal in the micro-chambers comprise instructions for directing an excitation wavelength to the micro-chambers and detecting an emission wavelength corresponding to the second fluorescent signal.In some embodiments, the processor is programmed to execute instructions for contacting the capture particles present in micro-chambers with the fluorogenic substrate prior to the detecting step.The systems of the present disclosure may be operated manually or automatically or semiautomatically. In certain cases, the systems may be operated by a processor that runs a program for carrying out the steps required for generating an analyte related signal and detecting the signal. As used hereon, the phrase “analyte related signal” or “analyte associated signal” or “fluorescent signal” refers to a signal that is indicative of presence of an analyte and is proportional to the amount of the analyte in a sample. The signal may be fluorescence, chemiluminescence, colorimetric, turbidimetric, etc. In certain cases, the read out may be digital, for example, thenumber of positive counts (e.g., wells) is compared to the number of negative counts (e.g., wells) to obtain a digital count.In some embodiments, the fluorogenic substrate molecule is pyranine-O-phosphate and the fluorogenic enzymatic product is pyranine.In some embodiments, the phosphatase enzyme is an alkaline phosphatase enzyme.In some embodiments, each capture particle has up to 10 molecules of the analyte immobilized on the capture particles and wherein one molecule of the phosphatase enzyme associated is associated with one analyte molecule.In some embodiments, each capture particle has up to 1 or 0 molecule of the analyte immobilized thereon.In some embodiments, each capture particle has 1 or 0 copy of the analyte immobilized on the capture particle.In some embodiments, the detecting step is performed within 5 minutes after the contacting step.In some embodiments, the detecting step is performed within 10 seconds to 2 minutes after the contacting step.In some embodiments, the analyte of interest is a peptide or protein, and the capture particles comprise a first binding member that specifically binds to the peptide or protein and wherein the peptide or protein is associated with the phosphatase enzyme via a second binding member that specifically binds to the peptide or protein and wherein the second binding member is conjugated to the phosphatase enzyme.In some embodiments, the analyte of interest is a nucleic acid, and the capture particles comprise a first binding member that specifically binds to the nucleic acid and wherein the nucleic acid is associated with the phosphatase enzyme via a second binding member that specifically binds to the nucleic acid and wherein the second binding member is conjugated to the phosphatase enzyme.In some embodiments, the micro-chambers are micro-wells. In some embodiments, the micro-chambers are micro-droplets.Also provided herein is a system of detecting presence of an analyte of interest on capture particles, the system comprising a processor; a cartridge comprising micro-chambers; the microchambers comprising the capture particles, a first fluorogenic substrate and a second fluorogenic substrate, wherein the first fluorogenic substrate has the structure of formula (la):and the second fluorogenic substrate has the structure of formula (lb):wherein:X is H+, K+, Ca2+, tris, triethylammonium, or any other cation, Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate, the micro-chambers are sized to contain only one capture particle per microchamber, the analyte of interest is associated with a phosphatase enzyme, the phosphatase enzyme converts the first and second fluorogenic substrates into a fluorogenic enzymatic product, the first and second fluorogenic substrate emit a first fluorescent signal and the fluorogenic enzymatic product emits a second fluorescent signal distinct from the first fluorescent signal; the processor is programmed to execute instructions for detecting presence or absence of the second fluorescent signal in the microchambers, wherein presence of the second fluorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers.In some embodiments, the fluorogenic enzymatic product is pyranine. In some embodiments, each capture particle has up to 10 molecules of the analyte immobilized on the capture particles and wherein one molecule of the phosphatase enzyme associated is associated with one analyte molecule.In some embodiments, each capture particle has up to 5, 3, 2, 1 or 0 molecule of the analyte immobilized thereon. In some embodiments, each capture particle has 5, 3, 2, or 0 copy of the analyte immobilized thereon.In some embodiments, the detecting step is performed within 5 minutes after the contacting step. In some embodiments, the detecting step is performed within 1 minute to 5 minutes after the contacting step.In some embodiments, the analyte of interest is a peptide or protein, and the capture particles comprise a first binding member that specifically binds to the peptide or protein and wherein the peptide or protein is associated with the phosphatase enzyme via a second binding member that specifically binds to the peptide or protein and wherein the second binding member is conjugated to the phosphatase enzyme.In some embodiments, the analyte of interest is a nucleic acid, and the capture particles comprise a first binding member that specifically binds to the nucleic acid and wherein the nucleic acid is associated with the phosphatase enzyme via a second binding member that specifically binds to the nucleic acid and wherein the second binding member is conjugated to the phosphatase enzyme.In some embodiments, the micro-chambers are micro-wells. In some embodiments, the micro-chambers are micro-droplets. In some embodiments, the phosphatase enzyme is an alkaline phosphatase enzyme.In some embodiments, the first fluorogenic substrate and the second Anorogenic substrate are present at a ratio of 1: 1. In some embodiments, the concentration of each of the first and second fluorogenic substrate is between 1 to 50 M.In certain embodiments, the system for detecting presence of an analyte of interest on capture particles, may comprise a processor; a cartridge comprising nano-chambers; the nanochambers comprising the capture particles and a fluorogenic substrate of formula (I):wherein: X is H+, K+, Ca2+, tris, triethylammonium, or any other cation, Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate, the nanochambers are sized to contain only one capture particle per nano-chamber, the analyte of interest is associated with a phosphatase enzyme, the phosphatase enzyme converts the fluorogenic substrate into a Auorogenic enzymatic product, the Auorogenic substrate emits a first fluorescentsignal and the Anorogenic enzymatic product emits a second Auorescent signal distinct from the first Auorescent signal, the processor is programmed to execute instructions for detecting presence or absence of the second Auorescent signal in the nano-chambers, wherein presence of the second Auorescent signal in the nano-chambers indicates presence of an analyte of interest on capture particles present in the nano-chambers.Also provided herein is a system of detecting presence of an analyte of interest on capture particles, the system comprising a processor; a cartridge comprising nano-chambers; the nanochambers comprising the capture particles, a first Auorogenic substrate and a second Auorogenic substrate, wherein the first Auorogenic substrate has the structure of formula (la):and the second Auorogenic substrate has the structure of formula (lb):wherein:X is H+, K+, Ca2+, tris, triethylammonium, or any other cation, Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate, the nano-chambers are sized to contain only one capture particle per nano-chamber, the analyte of interest is associated with a phosphatase enzyme, the phosphatase enzyme converts the first and second Auorogenic substrates into a Auorogenic enzymatic product, the first and second Auorogenic substrate emit a first Auorescent signal and the Auorogenic enzymatic product emits a second Auorescent signal distinct from the first Auorescent signal; the processor is programmed to execute instructions for detecting presence or absence of the second Auorescent signal in the nano-chambers, wherein presence of the second fluorescent signal in the nano-chambers indicates presence of an analyte of interest on capture particles present in the nano-chambers.EXAMPLESThe following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure and are not intended to limit the scope of what the inventors regard as their disclosure nor are they intended to represent that the experiments below are all or the only experiments performed.Materials & Abbreviations:Pyranine (from SIGMA-ALDRICH) / DiEA = diisopropylethylamine (from SIGMA- ALDRICH) I DCM = dichloromethane (from SIGMA-ALDRICH) I 15C5 = 15-Crown-5 ether (from TCI) I DECP = Diethylchlorophosphate (SIGMA- ALDRICH) I Et2O = Diethyl ether (from WAKO) / TMSBr = bromotrimethylsilane (from TCI) I methanol (from SIGMA-ALDRICH) / ethanol (from WAKO) / tBuOK = potassium tert-butoxide (SIGMA-ALDRICH).Example 1: Preparation of pyranine-O-phosphateIn a IL Erlenmeyer flask equipped with a stir bar, 20 grams of HPTS was suspended in 400 mL of DCM and was stirred at room temperature. To this was added in order 20 mL of DiEA, 100 mL of 15-crown-5-ether, and 18 mL of DECl-Phos. This was allowed to stir for 1 hour at room temperature, during which time the solution became homogenous, and the color changed from yellow to light yellow. The mixture was then filtered through a Buchner funnel into a 2L filter flask. To the filtrate solution was added 400 mL of MTBE and allowed to sit for 30 minutes, causing a gummy precipitate to form in the flask. The semi-solid was decanted and an additional 200 mL of MTBE was added to the gummy precipitate and the flask was swirled several times before decanting the semi-solid. To the flask was added 400 mL of DCM and a stir bar followed by 35 mL of 15-crown-5-ether followed by a portion-wise addition of 85 mL of TMSBr. This mixture was allowed to stir overnight (18 hours) wrapped in foil to protect from light. Next, thereaction mixture was diluted with 700 mL of ethanol before adding 1080 mL of a previously prepared 50 mg / mL solution of tBuOK / ethanol, which caused a precipitate to form. The precipitate was collected by vacuum filtration (4L filter flask) over a Buchner funnel and Whatman filter paper. The crude solid was stirred in ethanol (500 mL), dried by vacuum filtration, and rinsed (3xl50mL) with ethanol and MTBE (IxlOOmL). The solid was dried under high vacuum being stored at -20°C overnight. A yield of ~40 grams crude solid was obtained.Example 2: Preparation of Pyranine-O-Phosphate TetraTris SaltThe product was purified using 1.9 kg C18Aq column. The column was previously equilibrated with 5L of water. Separately, a 100g Solid Sample Cartridge was loaded with 45g of Dowex 500WX4-100 ion exchange resin and was washed with 500 mL of water. Next, 5 g of crude pyranine phosphate was dissolved in 15mL of water and was loaded on the Solid Sample cartridge with IEX resin, which was then placed in line with the 1.9 kg C18Aq column. An isocratic (100% water) eluent was used, and the product was collected. Fractions were checked for purity using UV-vis by diluting an aliquot of the fractions in pH 9 buffer solution. Fractions containing high purity pyranine phosphate were combined. The concentration of pyranine phosphate was determined using an extinction coefficient 18,560 cm-1M-1and then 4 equivalents of Tris(hydroxymethyl)aminomethane was added to the solution before shell freezing and lyophilizing to dryness. Yield = 2g yellow powder.'H NMR (400 MHz, D2O): chemical shift / ppm, 3.61 (24H, s), 8.83 (1H, s), 8.92 (1H, d, J=9.4Hz), 9.03 (1H, d, J=9.9Hz), 9.06 (1H, d, J=9.5Hz), 9.19 (2H, m).ESI-MS: calcd. for CI6HIOOI3PS3= 536.90; found=537.19Example 3: Purification of Pyranine-O-Phosphate Tetrakis Triethylammonium SaltThe crude product was purified using 1.9 kg C18Aq column. The column was previously equilibrated with 5L of 50 mM TEAA buffer. Then 14.2 grams of crude pyranine phosphate was dissolved in 50 mL of IM TEAA and was loaded on to the column with a 50 mL syringe. An isocratic (100% 50 mM TEAA) eluent was initially used and flow rate raised from 50 mL / min to 150 ml / min. Next a gradient with increasing concentration of methanol to 100% over the course of 45 minutes was used. Fractions were checked for purity using UV-vis by diluting an aliquot of the fractions in pH 9 buffer solution. Fractions containing high purity pyranine phosphate were combined before shell freezing and lyophilizing to dryness. Yield = 5.6g white powder.’H NMR (400 MHz, D2O): chemical shift / ppm, 1.15 (36H, t, J=7.8Hz), 3.04 (24H, q, J=7.4Hz), 8.77 (1H, s), 8.85 (1H, d, J=10.0Hz), 9.08 (1H, d, J=10.2Hz), 9.11 (1H, d, J=9.3Hz), 9.21 (2H, m).ESI-MS: calcd. for Ci6HioOi3PS3= 536.90; found=537.19Example 4: Preparation of Pyranine-Methyl-PhosphateIn a 50 mL round bottom flask, 1 g of HPTS was suspended in 10 mL DMSO. To this was added 1.4 mL (1.47 g, 5eq) of DBU followed by 2g (4eq) of di-tertbutyl chloromethyl-phosphate. The solution was allowed to stir overnight. Next, 30 mL of a 1: 1 mixture of acetonitrile and ethyl ether was added to the solution causing a precipitate to form. The organic layer was isolated by decanting and a majority of the organic solvent was removed by rotovap leaving the crude product in the remaining DMSO. The DMSO solution with the crude intermediate was purified by flash purification system by injection on a 275g C18Aq column using a MeCN / fFO gradient with 0% MeCN to 100% MeCN, where a mixture of di-tertbutyl and mono-tertbutyl phosphate esters were collected.Fractions containing the two crude intermediates were lyophilized to dryness. Next the crude material was dissolved in 2.5 mL of water and 2.5 mL of TFA was added. The solution was stirred for 30 minutes before being injected on a weak anion exchange 30g amine Gold column (activated by 5% acetic acid in water) and eluting with 1:1 MeCN / HiO (solvent A) and IMNH4HCO3 (solvent B). Gradient of solvent B from 0% to 100%. Fractions containing product were buffered with Tris and lyophilized to dryness.Yield - 360 mg of light yellow powder’H NMR (400 MHz, D2O): chemical shift / ppm, 3.64 (24H, s), 5.97 (2H, d, J=11.3Hz), 8.62 (1H, s), 8.93 (1H, d, J=10.3Hz), 9.02 (1H, d, J=9.7Hz), 9.07 (1H, d, J=9.7Hz), 9.19 (2H, m).ES1-MS: calcd. for Ci7Hi20i4PS3 = 566.91; found=567.29Example 5: Preparation of Pyranine-diphosphateIn a 20 mL reaction vial, 0.2 g of HPTS was suspended in 4 mL DCM and 1 mL of crown ether. To this was added 0.05 mL POCh and the mixture was allowed to stir for 40 mins. Next, 6 mL of ethyl ether was added to the solution causing a precipitate to form. The solvent was decanted and the gummy precipitate was dissolved in acetonitrile. To this was added tetrabutyl ammonium phosphate and DiEA and the reaction was stirred overnight. Product was purified by injection on a weak anion exchange 5g amine Gold column (activated by 5% acetic acid in water) and eluting with water (solvent A) and IM NH4HCO3 (solvent B). Gradient of solvent B from 0% to 100%. Fractions containing product were lyophilized to dryness.Yield - 70 mg of light yellow powder’H NMR (400 MHz, D2O): chemical shift / ppm, 3.64 (24H, s), 8.78 (1H, s), 8.96 (1H, d, J=9.4Hz), 9.08 (1H, d, J=9.6Hz), 9.11 (1H, d, J=9.6Hz), 9.21 (2H, m).ESI-MS: calcd. for Ci6HnOi6P2S = 616.87; found=617.19Example 5: Preparation of Pyranine-TriphosphateIn a 20 mL reaction vial, 0.2 g of HPTS was suspended in 4 mL DCM and 1 mL of crown ether. To this was added 0.05 mL POCI3 and the mixture was allowed to stir for 40 mins. Next, 6 mL of ethyl ether was added to the solution causing a precipitate to form. The solvent was decantedand the gummy precipitate was dissolved in acetonitrile. To this was added tributylammonium pyrophosphate and DiEA and the reaction was stirred overnight. Product was purified by injection on a weak anion exchange 5g amine Gold column (activated by 5% acetic acid in water) and eluting with water (solvent A) and IM NH4HCO3 (solvent B). Gradient of solvent B from 0% to 100%. Fractions containing product were combined before adding Tris base for buffering and lyophilized to dryness. Yield - 55 mg of light yellow powder'H NMR (400 MHz, D2O): chemical shift / ppm, 3.64 (24H, s), 8.77 (1H, s), 8.92 (1H, d, J=9.9Hz), 9.09 (1H, d, J=9.6Hz), 9.13 (1H, d, J=9.4Hz), 9.21 (2H, m).ESI-MS: calcd. for CieH OigPsS ^ 696.84; found=697.11Example 6: Single enzyme detection using alkaline phosphatase (ALP) by familiar microscopy which is optimized for fluorescein fluorophore (FITC microscopy, with a 480- 490nm exciter).Single enzyme molecules of ALP were encapsulated into nano-chambers (diam.4pm x h4|im) with the Anorogenic substrate - fluorescein-diphosphate (FDP) or pyranine-O-phosphate. ALP enzymatically hydrolyzed the substrate into green Auorescent molecule Auorescein (from FDP) or pyranine (from pyranine-O-phosphate). Degree of the Auorescence accumulation was compared by visualization for the nano-chamber array (see figure 1). PyPhos system can give equivalent or brighter spots / nanochambers even in the microscopy optimized for fluorescein.FIG. 1 shows comparison of pyranine-O-phosphate and Auorescein diphosphate (FDP) using digital detection in the single enzyme molecule detection using the nano-chamber array by FITC microscopy. Pyranine-O-phosphate system can give equivalent or brighter nano-chambers than FDP system. CONDITIONS: [ALP]=0 or 20pM at a loading step / Buffer: IM DEA-HC1 (diethanol amine) pH 9.25 including ImM MgCF, 2 mM pyranine-O-phosphate and 0.1% Tween20 / Nanowell device: Flow-through device (capacity lOul) / Optical filter: FITC set of Olympus Microscope.Example 7: Single enzyme detection using alkaline phosphatase (ALP) by the custom made compact camera system (with a 450nm exciter and 515nm-long pass emitter).Fluorescent accumulation of pyranine-O-phosphate system is several times faster than that of FDP; pyranine-O-phosphate system can give the similar brightness of FDP-at-120min incubation after only 20min (see figure 2). Large Stokes shift* of pyranine may allow efficient Auorescence acquisition. (*Stokes shift / nm = Absorption peak - emission peak in wavelength / nm; 60nm for pyranine, 25nm for fluorescein).FIG. 2 shows comparison of pyranine-O-phosphate and fluorescein diphosphate (FDP) in the single enzyme molecule detection using the nano-chamber array by compact camera system with 450nm-blue light excitation. Pyranine-O-phosphate system can allow fast detection. Kinetics is several times (~6x) faster than that of FDP system. CONDITTIONS: [ALP]=0 or 20pM at a loading step / Buffer: IM DEA-HC1 (diethanol amine) pH 9.25 including ImM MgCh, 2 mM pyranine-O-phosphate and 0.1% Tween20 / Nanowell device: Flow-through device (capacity lOpl) I Optical filter: Ex450 and Em515 long-pass.Example 8: Performance as a Anorogenic substrate in the digital ELISA using HBsAg antigen.Pyranine-O-phosphate allows for more rapid detection. Within 5 min of the enzymatic reaction, an intense output (=signal%) is observed for positive samples. The output pattern for various inputs of antigen (O.l-lOfM HBsAg) was the same as that at 10 min incubation (see figure 3). This means the enzymatic amplification step is almost complete in less than 5 min* (*detectable within 30 sec under optimized microscopy condition. (See Example 11). It is evident that the amplification duration is shorter than the conventional immunoassay reaction time (18 min for HBsAg assay on ARCHITECT Abbott). The enzyme amplification step in the digital immunoassay is less time-consuming when pyranine-O-phosphate is used. The FDP system is slower and requires about 30min of amplification time to obtain a maximum signal output.FIG. 3 shows comparison of pyranine-O-phosphate and FDP (fluorescein diphosphate) in Digital ELISA for HBsAg. The fluorescent amplification using pyranine-O-phosphate allows rapid detection within 5min of enzymatic reaction. FDP system at 5min detection has been growing up (accumulating fluorescent molecule). CONDITIONS: [HBsAg (antigen)]=0, 0.1, 1, 10 fM. [Substrate]=300pM.Example 9: Application of dual emission property of pyranine-O-phosphate systemDual-color imaging was conducted for pyranine-O-phosphate system (see figure 4) to confirm expansion of dynamic range. In the green channel, more spots appeared for higher concentrations of ALP. However, the count-based signal output (digital signal) was saturated (-100%) at lambda=10. On the other hand, intensity of the blue channel showed a decrease at the high concentration region from 1 to 1,000 of lambda. Dark images of the blue channel indicate that pyranine-O-phosphate is consumed by enzymatic hydrolysis. When only a green channel for the digital counting is used in the analysis, it is difficult to quantify the higher concentration of enzyme (>10 of lambda). The secondary channel for the blue fluorescent nano-chambers canexpand the dynamic range at high concentration region (see figure 5). The dual-emission analysis can work even in high concentration up to 1,000 of lambda, the range expanded by approx. 100- hold.FIG.4 shows dual-color imaging for pyranine-O-phosphate system. Upper and bottom are fluorescent imaging channels for green emission (of pyranine, the enzymatic product) and for blue emission (of pyranine-O-phosphate, the substrate), respectively. Various concentrations of alkaline phosphatase (ALP) are entered (left to right in the figure). Values of “Lambda” on the images mean an average number of enzyme molecule in a single nano-chamber; lambda=l is corresponding to 33pM of ALP. In the green channel, more bright spots were observed in higher concentrations of ALP. However, the count-based signal (the digital signal) was saturated (-100%) at Lambda=10. On the other hand, analog intensity of the blue channel decreased at high concentration region, the change occured from 1 to 1,000 of Lambda. When pyranine-O- phosphate is consumed, the image become dark one. CONDITIONS: 22min of enzymatic reaction / [pyranine-O-phosphate]=500pM / Microscopy imaging with x40 objective, Nikon / [Ex / Em]=[340-380 / 435-485] for the blue channel, and [460-500 / 510-560] for the green channel.FIG. 5 shows dynamic range expansion using the dual-emission properties of pyranine- O-phosphate system. Only digital counting (green dots) cannot quantify the higher concentration of enzyme (>10 of Lambda, of which unit is average number of enzymes-per-nanochamber). The intensity of blue channel changes from 1 to 1,000 of Lambda. Therefore, the analysis using both of the blue and the green (Digital) channels shows wider dynamic range up to 1 ,000 of Lambda. The dynamic range can be expanded into the region approx. 100-times higher.Example 10: Monitoring of stability of the substrate solution at ambient temperature.Pyranine-O-phosphate can keep the high activity level (approx. 75%-active) for over 6 months in working solution in pH9.25 at room temperature (see figure 6). FDP is not robust; residual activity fraction was approx. 25% after 4 months storage at RT. The half- lives are estimated to 463 and 22 days for pyranine-O-phosphate and FDP storing at RT, respectively (see figure 7). Pyranine-O-phosphate is more suitable as an assay reagent than FDP because of the high stability in the solution state at RT.FIG. 6 shows monitoring of thermal stability of pyranine-O-phosphate and FDP. Fluorescent intensity growth of the bright single nano-chambers is monitored for 5 samples (i-v): pyranine-O-phosphate in buffer at (i) 25 °C, (ii) -20°C, and (iii) -80°C stored for 180 days, and FDP in buffer at (iv) 25°C and (v) -20°C stored for 120 days. Blue dots indicate the average brightness of bright single nano-chambers (averaged with more than 1,000 nano-chambers tomake one dot). The pyranine-O-phosphate solution at room temperature (RT) had kept high activity of 75% after 180 days. FDP had retained only 25%-active fraction after storing at RT for 120 days. [STORAGE CONDITION]: IM DEA-HC1 (diethanol amine) pH 9.25 including ImM MgCh and 0.1% Tween20. [ACTIVITY MEASUREMENT CONDITION]: [ALP]=4pM / Microscope: FITC imaging, Olympus.FIG. 7 shows half-life of the substrate in the storage solution at room temperature. (Blue dot and black square) residual activity to the reference (stored at -20 °C). Red lines are theoretical fitting curves of an exponential decay function. Half-lives for pyranine-O-phosphate and FDP systems are estimated to 463 and 23 days, respectively.Example 11: Fast detection using pyranine-O-phosphate under optimized conditions in chemistry and optical imaging.A RGP / beta-galactosidase system has achieved a 30sec-detection (RGP= resorufin-beta- D-galactopyranoside) (“Multiplexed single molecule immunoassays”; David C. Duffy et al.; Lab Chip 13, 2902-2911 (2013)). The said system used two fluorescent images at 0 and 30sec of amplification time, the differential image of the two images shows a fluorescent growth which was yielded by single enzymes in nanochambers (a 2-point time-lapse analysis). In present disclosure, pyranine-O-phosphate system was evaluated by the similar analysis method to the Quanterix RGP system. Pyranine-O-phosphate system has a faster detection; 15 seconds of amplification is enough to form bright signals (see figure 8).FIG. 8 shows rapid detection test using pyranine-O-phosphate. (A-C) Fluorescent images of the nano-chamber array containing single enzyme molecules of alkaline-phosphatase (ALP). Concentration of ALP was approx. 0.5 enzyme-per- nano-chamber. The pictures have been taken at (A) 0 (= first shot), (B) 15, and (C) 405 sec, respectively. The first shot IMG

[0000] was taken after 15 sec since the nano-chamber array formation by oil-sealing. (D) Differential image of the two images IMG

[0000] and IMG

[0015] . (E) Growth of bright nano-chamber fraction versus enzyme reaction times. The differential analysis by a 15-sec interval allows almost full-count detection. [pyranine-O-phosphate]: 500uM. [Microscopy]: Ex. filter: 452 / 45 nm; Dichroic mirror: 495nm; Em. Filter: 525 / 45nm. [Buffer]: 0.25 DEA pH9.25 containing ImM MgCh and 0.05% Tween20.Example 12: The fluorogenic substrate system of present disclosure was demonstrated in actual ELISA assay. HBsAg assay was used for the evaluation (see figure 9). Commercially available calibrator solutions (for Architect HBsAg assay, Abbott) were tested in full -automated Digital ELISA box (Abbott Japan). All calibrators are separated / distinguished at 160sec-enzyme reaction.After assay processing, obtained Nanowell image was analyzed by Digital counting mode and Analog Intensity mode. Digital counting mode for the low concentration showed similar result in the both cases of Only mono-PyPhos (also referred to as PyPhos and pyranine-O-phosphate) and the mixture. In the Analog intensity mode, signal of mono-PyPhos was saturated around 10,000 fM-antigen, however, the mixture system worked still over 10,000fM. Moreover, 20,000fM and 50,000fM was separatable.Theoretical fitting for HBsAg assay shows that the upper limit is around 150,0000 fM- antigen (FIG. 10). It is based on the result in example 9, “17-fold slower than mono-pyranine-O- phosphate”.Typically, our digital counting allows us to detect 0.3fM-antigen, so the 150,000fM-limit system means that it has a wide-dynamic range of 5.7-logs. Conventional way is 4.5-logs. By expansion using the substrate mixture (the fast and the slow) can cover the range of CAL-A to - F, - all calibrators of Abbott Architect.FIG. 9 shows output signal for the demonstration using an actual Digital ELISA assay. Reagents for HBsAg assay were applied. Measured panels (10fM-50,000fM) were based on Abbott ARCHITECT calibrators. After assay processing, obtained Nanowell image was analyzed by Digital counting mode (Top) and Analog Intensity mode (Bottom). Nanowell-Image was captured at 160-sec enzyme reaction time.FIG. 10 shows theoretical curve for the result of HBsAg assay. Fitting formula is based on sum of two first-order kinetics events: fast and slow hydrolysis.Example 13: Residual pyranine present in pyranine phosphate salts with either Tris or TEA counterion was measured by fluorescence spectroscopy. A 5 M solution of either pyranine phosphate tetratris or pyranine phosphate tetrakis triethylammonium salts were prepared and excited at 440 nm. Lower fluorescence in observed in the TEA salt, indicating a higher purity of this form of pyranine phosphate. 250 mM DEA, ImM MgCh, and 0.5% Tween pH 9.25 (see figure 11).Example 14: Solid form thermal stability of pyranine phosphate salts was tested by heating powder at 50°C for 72 hours. Purity was checked by UV-Vis upon dilution in pH 9 buffer solution. See table 1 below.Table 1:Example 15: A solution of pyranine phosphate was converted to pyranine using excess alkaline phosphatase. Pyranine phosphate will convert to equal moles of pyranine alkaline phosphatase. Therefore: Abs of AJP / e395=Abs of pyranine / e455, e455 of pyranine is known (21600M-1 cm - 1) 0.226 / e395=0.263 / 21600. Therefore, the extinction coefficient of pyranine phosphate is e395=l 8,560. 250 mM DEA, ImM MgCh, and 0.5% Tween pH 9.25 (see figure 12).Example 16: Enzyme kinetics of pyranine phosphate with calf intestinal alkaline phosphatase (BBI ALP21G) was measured and compared with the faster substrate pyranine methyl phosphate. See table 2 below.Table 2:Example 17: Enzymatic reaction rate of pyranine phosphate with calf intestinal alkaline phosphatase (BBI ALP21G) was measured and compared with slower substrates pyranine diphosphate and pyranine triphosphate measured by UV-vis at 455 nm. 250 mM DEA, ImM MgCh, and 0.5% Tween pH 9.25 (see figure 13).Example 18: Preparation of Dipyranine phosphateIn a 20 mL reaction vial equipped with a stir bar, 0.4 grams of 8-Hydroxypyrene- 1,3,6- trisulfonic acid (HPTS) was suspended in 5 mL of DCM and was stirred at room temperature. To this was added in order 0.2 mL of DiEA, 1.5 mL of 15-crown-5-ether, and 0.045 mL of ethyldichlorophosphate. This was allowed to stir overnight at room temperature. The mixture was then filtered through a 0.45 um nylon syringe filter. To this was added 5 mL of diethyl ether, causing a gummy precipitate to form in the flask. The solvent was poured out and 5 mL of DCM and a stir bar was added to the gummy precipitate. To this was added in order 0.5 mL of 15-crown- 5-ether followed by a portion-wise addition of 0.8 mL of TMSBr. This mixture was allowed to stir overnight (18 hours). Next, the reaction mixture was diluted with 1 mL ethanol and then slowly added 5 mL of a previously prepared solution of 50 mg / mL tBuOK / EtOH causing a precipitate to form. The solution was then centrifuged at 3000 rpm for 1 minute. The solid was dried under reduced pressure before being purified twice on an Amine 30g Gold column (activated by 5% acetic acid in water) using a gradient of 0% to 100% IM ammonium bicarbonate in water. Fractions containing the product were collected and lyophilized to dryness. Yield = 75 mg of lightyellow product.’H NMR (400 MHz, D2O): chemical shift / ppm, 8.71 (1H, d, J=9.5Hz), 8.93 (1H, s), 9.00 (1H, d, J=9.5Hz), 9.05 (1H, d, J=10.1Hz), 9.18 (2H, m).ESI-MS: calcd. for CsiHisG Se^ 976.84; found=977.07Example 19: Dipyranine phosphate was incubated with calf intestinal alkaline phosphatase (BBI ALP21G) for 18 hours. No reaction was observed between this molecule and alkaline phosphatase. 250 mM DEA, ImM MgCh, and 0.5% Tween pH 9.25 (see figure 14).In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should beinterpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized assufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.Accordingly, the preceding merely illustrates the principles of the disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the disclosure and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the disclosure and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the disclosure as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.The scope of the present disclosure, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present disclosure is embodied by the appended claims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. § 112(6) is not invoked.The following list of embodiments is envisaged:1. A compound of formula (I):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate. 2. The compound of embodiment 1, wherein the compound is of formula (la):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation.3. The compound of embodiment 1, wherein the compound is of formula (lb):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is selected from a group consisting of diphosphate, triphosphate, or phosphate.4. The compound of any one of embodiments 1-3, wherein the compound is selected from:1-75. A method of detecting presence of an analyte of interest on capture particles, the method comprising: contacting the capture particles present in micro-chambers with a Anorogenic substrate of formula (I):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate; the micro-chambers are sized to contain only one capture particle per micro-chamber, the analyte of interest is associated with a phosphatase enzyme, the phosphatase enzyme converts the Anorogenic substrate into a Auorogenic enzymatic product, the Auorogenic substrate emits a first fluorescent signal and the Anorogenic enzymatic product emits a second fluorescent signal distinct from the first Auorescent signal; detecting presence or absence of the second Auorescent signal in the micro-chambers, wherein presence of the second Auorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers.6. The method of embodiment 5, wherein the Auorogenic enzymatic product is pyranine.7. The method of embodiment 5 or 6, wherein the phosphatase enzyme is an alkaline phosphatase enzyme.8. The method of any one of embodiments 5-7, wherein each capture particle has up to 10 molecules of the analyte immobilized on the capture particles and wherein one molecule of the phosphatase enzyme associated is associated with one analyte molecule.9. The method of embodiment 8, wherein each capture particle has up to 1 or 0 molecule of the analyte immobilized thereon.10. The method of any one of embodiments 5-9, wherein each capture particle has 1 or 0 copy of the analyte immobilized on the capture particle.11. The method of any one of embodiments 5-9, wherein the detecting step is performed within 5 minutes after the contacting step.12. The method of any one of embodiments 5-11, wherein the detecting step is performed within 10 seconds to 2 minutes after the contacting step.13. The method of any one of embodiments 5-12, wherein the analyte of interest is a peptide or protein, and the capture particles comprise a first binding member that specifically binds to the peptide or protein and wherein the peptide or protein is associated with the phosphatase enzyme via a second binding member that specifically binds to the peptide or protein and wherein the second binding member is conjugated to the phosphatase enzyme.14. The method of any one of embodiments 5-12, wherein the analyte of interest is a nucleic acid, and the capture particles comprise a first binding member that specifically binds to the nucleic acid and wherein the nucleic acid is associated with the phosphatase enzyme via a second binding member that specifically binds to the nucleic acid and wherein the second binding member is conjugated to the phosphatase enzyme.15. The method of any one of embodiments 5-14, wherein the micro-chambers are microwells.16. The method of any one of embodiments 5-15, wherein the micro-chambers are microdroplets.17. A method of detecting presence of an analyte of interest on capture particles, the method comprising: contacting the capture particles deposited into micro-chambers with a first fluorogenic substrate and a second fluorogenic substrate, wherein the first fluorogenic substrate has the structure of formula (la):and the second fluorogenic substrate has the structure of formula (lb):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate,the micro-chambers are sized to contain only one capture particle per microchamber, the analyte of interest is associated with a phosphatase enzyme, the phosphatase enzyme converts the first and second Anorogenic substrates into a Auorogenic enzymatic product, the first and second Auorogenic substrate emit a first Auorescent signal and the Auorogenic enzymatic product emits a second Auorescent signal distinct from the first Auorescent signal; detecting presence or absence of the second Auorescent signal in the micro-chambers, wherein presence of the second Auorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers.18. The method of embodiment 17, wherein the Auorogenic enzymatic product is pyranine.19. The method of embodiments 17 or 18, wherein each capture particle has up to 10 molecules of the analyte immobilized on the capture particles and wherein one molecule of the phosphatase enzyme associated is associated with one analyte molecule.20. The method of embodiment 19, wherein each capture particle has up to 5, 3, 2, 1 or 0 molecule of the analyte immobilized thereon.21. The method of embodiment 19, wherein each capture particle has 5, 3, 2, or 0 copy of the analyte immobilized thereon.22. The method of any one of embodiments 17-21, wherein the detecting step is performed within 5 minutes after the contacting step.23. The method of any one of embodiments 17-22, wherein the detecting step is performed within 1 minute to 5 minutes after the contacting step.24. The method of any one of embodiments 17-23, wherein the analyte of interest is a peptide or protein, and the capture particles comprise a first binding member that specifically binds to the peptide or protein and wherein the peptide or protein is associated with the phosphatase enzyme via a second binding member that specifically binds to the peptide or protein and wherein the second binding member is conjugated to the phosphatase enzyme.25. The method of any one of embodiments 17-24, wherein the analyte of interest is a nucleic acid, and the capture particles comprise a first binding member that specifically binds to the nucleic acid and wherein the nucleic acid is associated with the phosphatase enzyme via a second binding member that specifically binds to the nucleic acid and wherein the second binding member is conjugated to the phosphatase enzyme.26. The method of any one of embodiments 17-25, wherein the micro-chambers are microwells.27. The method of any one of embodiments 17-25, wherein the micro-chambers are microdroplets.28. The method of any one of embodiments 17-27, wherein the phosphatase enzyme is an alkaline phosphatase enzyme.29. The method of any one of embodiments 17-28, wherein the first Anorogenic substrate and the second Anorogenic substrate are present at a ratio of 1 : 1.30. The method any one of embodiments 17-29, wherein the concentration of each of the first and second Anorogenic substrate is between 1 to 50 M.31. A method of preparing pyranine-O-phosphate, comprising: reacting pyranine with a mono-reactive phosphate source to obtain pyranine-O-phosphate; wherein pyranine-O-phosphate is a compound of formula (lb):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is a phosphate.32. The method according to embodiment 31, wherein the mono-reactive phosphate source is diethyl chlorophosphate.33. The method according to any one of embodiments 31-32, wherein pyranine-O-phosphate has a molecular weight in the range of 100 to 1000 Daltons.34. A method of preparing pyranine-methyl-phosphate, comprising: reacting pyranine with a mono-reactive phosphate source to obtain pyranine- methylphosphate; wherein pyranine- methyl-phosphate is a compound of formula (lb):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation.35. The method according to embodiment 34, wherein the mono-reactive phosphate source is di-tertbutyl chloromethyl-phosphate.36. A method of preparing pyranine-di-phosphate, comprising: reacting pyranine with a mono-reactive phosphate source to obtain pyranine-O-phosphate; further reacting with tetrabutylammonium phosphate to pyranine-di-phosphate; wherein pyranine-di-phosphate is a compound of formula (lb):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation; andY is a diphosphate.37. The method according to embodiment 36, wherein the mono-reactive phosphate source is phosphoryl chloride.38. A method of preparing pyranine-tri-phosphate, comprising: reacting pyranine with a mono-reactive phosphate source to obtain pyranine-O-phosphate; further reacting with tributylammonium phosphate to pyranine-tri-phosphate; wherein pyranine-tri-phosphate is a compound of formula (lb):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is a triphosphate.39. The method according to embodiment 38, wherein the mono-reactive phosphate source is phosphoryl chloride.40. A system for detecting presence of an analyte of interest on capture particles, the system comprising: a processor; a cartridge comprising micro-chambers; the micro-chambers comprising the capture particles and a fluorogenic substrate of formula (I):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate, the micro-chambers are sized to contain only one capture particle per micro-chamber, the analyte of interest is associated with a phosphatase enzyme,the phosphatase enzyme converts the Anorogenic substrate into a Anorogenic enzymatic product, the Anorogenic substrate emits a fir t Auorescent signal and the Anorogenic enzymatic product emits a second Auorescent signal distinct from the first Auorescent signal, the processor is programmed to execute instructions for detecting presence or absence of the second Auorescent signal in the micro-chambers, wherein presence of the second Auorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers.41. The system of embodiment 40, wherein the instructions for detecting presence or absence of the second Auorescent signal in the micro-chambers comprise instructions for directing an excitation wavelength to the micro-chambers and detecting an emission wavelength corresponding to the second Auorescent signal.42. The system of embodiment 40, wherein the processor is programmed to execute instructions for contacting the capture particles present in micro-chambers with the tluorogenic substrate prior to the detecting step.43. The system of embodiment 40, wherein the tluorogenic substrate molecule is pyranine-O- phosphate and the Auorogenic enzymatic product is pyranine.44. The system of embodiment 40 or 43, wherein the phosphatase enzyme is an alkaline phosphatase enzyme,45. The system of any one of embodiments 40-44, wherein each capture particle has up to 10 molecules of the analyte immobilized on the capture particles and wherein one molecule of the phosphatase enzyme associated is associated with one analyte molecule.46. The system of embodiment 45, wherein each capture particle has up to 1 or 0 molecule of the analyte immobilized thereon.47. The system of any one of embodiments 40-46, wherein each capture particle has 1 or 0 copy of the analyte immobilized on the capture particle.48. The system of any one of embodiments 40-47, wherein the detecting step is performed within 5 minutes after the contacting step.49. The system of any one of embodiments 40-47, wherein the detecting step is performed within 10 seconds to 2 minutes after the contacting step.50. The system of any one of embodiments 40-49, wherein the analyte of interest is a peptide or protein, and the capture particles comprise a first binding member that specifically binds to the peptide or protein and wherein the peptide or protein is associated with the phosphatase enzymevia a second binding member that specifically binds to the peptide or protein and wherein the second binding member is conjugated to the phosphatase enzyme.51. The system of any one of embodiments 40-50, wherein the analyte of interest is a nucleic acid, and the capture particles comprise a first binding member that specifically binds to the nucleic acid and wherein the nucleic acid is associated with the phosphatase enzyme via a second binding member that specifically binds to the nucleic acid and wherein the second binding member is conjugated to the phosphatase enzyme.52. The system of any one of embodiments 40-51, wherein the micro-chambers are microwells.53. The system of any one of embodiments 40-52, wherein the micro-chambers are microdroplets.54. A system of detecting presence of an analyte of interest on capture particles, the system comprising: a processor; a cartridge comprising micro-chambers; the micro-chambers comprising the capture particles, a first fluorogenic substrate and a second fluorogenic substrate, wherein the first fluorogenic substrate has the structure of formula (la):and the second fluorogenic substrate has the structure of formula (lb):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate, the micro-chambers are sized to contain only one capture particle per microchamber, the analyte of interest is associated with a phosphatase enzyme, the phosphatase enzyme converts the first and second Anorogenic substrates into a Auorogenic enzymatic product, the first and second Auorogenic substrate emit a first Auorescent signal and the Auorogenic enzymatic product emits a second Auorescent signal distinct from the first Auorescent signal; the processor is programmed to execute instructions for detecting presence or absence of the second Auorescent signal in the micro-chambers, wherein presence of the second Auorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers.55. The system of embodiment 54, wherein the Auorogenic enzymatic product is pyranine.56. The system of embodiments 54 or 55, wherein each capture particle has up to 10 molecules of the analyte immobilized on the capture particles and wherein one molecule of the phosphatase enzyme associated is associated with one analyte molecule.57. The system of embodiment 56, wherein each capture particle has up to 5, 3, 2, 1 or 0 molecule of the analyte immobilized thereon.58. The system of embodiment 56, wherein each capture particle has 5, 3, 2, or 0 copy of the analyte immobilized thereon.59. The system of any one of embodiments 54-58, wherein the detecting step is performed within 5 minutes after the contacting step.60. The system of any one of embodiments 54-59, wherein the detecting step is performed within 1 minute to 5 minutes after the contacting step.61. The system of any one of embodiments 54-60, wherein the analyte of interest is a peptide or protein, and the capture particles comprise a first binding member that specifically binds to the peptide or protein and wherein the peptide or protein is associated with the phosphatase enzyme via a second binding member that specifically binds to the peptide or protein and wherein the second binding member is conjugated to the phosphatase enzyme.62. The system of any one of embodiments 54-61 , wherein the analyte of interest is a nucleic acid, and the capture particles comprise a first binding member that specifically binds to thenucleic acid and wherein the nucleic acid is associated with the phosphatase enzyme via a second binding member that specifically binds to the nucleic acid and wherein the second binding member is conjugated to the phosphatase enzyme.63. The system of any one of embodiments 54-62, wherein the micro-chambers are microwells.64. The system of any one of embodiments 54-62, wherein the micro-chambers are microdroplets.65. The system of any one of embodiments 54-64, wherein the phosphatase enzyme is an alkaline phosphatase enzyme.66. The system of any one of embodiments 54-65, wherein the first fluorogenic substrate and the second fluorogenic substrate are present at a ratio of 1 : 1.67. The system any one of embodiments 54-66, wherein the concentration of each of the first and second fluorogenic substrate is between 1 to 50 pM.68. A compound of formula (II):(ID wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation.69. The compound of embodiment 61 , wherein the compound is:70. A method of detecting presence of an analyte of interest on capture particles, the method comprising: contacting the capture particles present in micro-chambers with a fluorogenic substrate of formula (II):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation; the micro-chambers are sized to contain only one capture particle per micro-chamber, the analyte of interest is associated with a phosphodiesterase enzyme, the phosphodiesterase enzyme converts the fluorogenic substrate into a fluorogenic enzymatic product, the fluorogenic substrate emits a first fluorescent signal and the fluorogenic enzymatic product emits a second fluorescent signal distinct from the first fluorescent signal; detecting presence or absence of the second fluorescent signal in the micro-chambers, wherein presence of the second fluorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers.71. The method of embodiment 70, wherein the fluorogenic enzymatic product is pyranine.72. The method of embodiment 70 or 71, wherein each capture particle has up to 10 molecules of the analyte immobilized on the capture particles and wherein one molecule of the phosphodiesterase enzyme associated is associated with one analyte molecule.73. The method of embodiment 72, wherein each capture particle has up to 1 or 0 molecule of the analyte immobilized thereon.74. The method of any one of embodiments 70-73, wherein each capture particle has 1 or 0 copy of the analyte immobilized on the capture particle.75. The method of any one of embodiments 70-74, wherein the detecting step is performed within 5 minutes after the contacting step.76. The method of any one of embodiments 70-75, wherein the detecting step is performed within 10 seconds to 2 minutes after the contacting step.77. The method of any one of embodiments 70-76, wherein the analyte of interest is a peptide or protein, and the capture particles comprise a first binding member that specifically binds to the peptide or protein and wherein the peptide or protein is associated with the phosphodiesterase enzyme via a second binding member that specifically binds to the peptide or protein and wherein the second binding member is conjugated to the phosphodiesterase enzyme.78. The method of any one of embodiments 70-77, wherein the analyte of interest is a nucleic acid, and the capture particles comprise a first binding member that specifically binds to the nucleic acid and wherein the nucleic acid is associated with the phosphodiesterase enzyme via a second binding member that specifically binds to the nucleic acid and wherein the second binding member is conjugated to the phosphodiesterase enzyme.79. The method of any one of embodiments 70-78, wherein the micro-chambers are microwells.80. The method of any one of embodiments 70-78, wherein the micro-chambers are microdroplets.81. A method of preparing dipyranine phosphate, comprising: reacting 2 equivalents of pyranine with a di-reactive phosphate source to obtain dipyranine phosphate; wherein dipyranine phosphate is a compound of formula (II):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation.82. The method according to embodiment 81, wherein the di-reactive phosphate source is ethyl dichlorophosphate.

Claims

CLAIMSWhat is claimed is:

1. A compound of formula (I):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate.

2. The compound of claim 1, wherein the compound is of formula (la):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation.

3. The compound of claim 1, wherein the compound is of formula (lb):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is selected from a group consisting of diphosphate, triphosphate, or phosphate.

4. The compound of any one of claims 1-3, wherein the compound is selected from:1-75. A method of detecting presence of an analyte of interest on capture particles, the method comprising: contacting the capture particles present in micro-chambers with a fluorogenic substrate of formula (I):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate; the micro-chambers are sized to contain only one capture particle per micro-chamber, the analyte of interest is associated with a phosphatase enzyme, the phosphatase enzyme converts the fluorogenic substrate into a Anorogenic enzymatic product, the Anorogenic substrate emits a first Auorescent signal and the fluorogenic enzymatic product emits a second Auorescent signal distinct from the first Auorescent signal; detecting presence or absence of the second Auorescent signal in the micro-chambers, wherein presence of the second Auorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers.

6. The method of claim 5, wherein the Auorogenic enzymatic product is pyranine.

7. The method of claim 5 or 6, wherein the phosphatase enzyme is an alkaline phosphatase enzyme.

8. The method of any one of claims 5-7, wherein each capture particle has up to 10 molecules of the analyte immobilized on the capture particles and wherein one molecule of the phosphatase enzyme associated is associated with one analyte molecule.

9. The method of claim 8, wherein each capture particle has up to 1 or 0 molecule of the analyte immobilized thereon.

10. The method of any one of claims 5-9, wherein each capture particle has 1 or 0 copy of the analyte immobilized on the capture particle.

11. The method of any one of claims 5-9, wherein the detecting step is performed within 5 minutes after the contacting step.

12. The method of any one of claims 5-11, wherein the detecting step is performed within 10 seconds to 2 minutes after the contacting step.

13. The method of any one of claims 5-12, wherein the analyte of interest is a peptide or protein, and the capture particles comprise a first binding member that specifically binds to the peptide or protein and wherein the peptide or protein is associated with the phosphatase enzyme via a second binding member that specifically binds to the peptide or protein and wherein the second binding member is conjugated to the phosphatase enzyme.

14. The method of any one of claims 5-12, wherein the analyte of interest is a nucleic acid, and the capture particles comprise a first binding member that specifically binds to the nucleic acid and wherein the nucleic acid is associated with the phosphatase enzyme via a second binding member that specifically binds to the nucleic acid and wherein the second binding member is conjugated to the phosphatase enzyme.

15. The method of any one of claims 5-14, wherein the micro-chambers are micro- wells.

16. The method of any one of claims 5-15, wherein the micro-chambers are micro-droplets.

17. A method of detecting presence of an analyte of interest on capture particles, the method comprising: contacting the capture particles deposited into micro-chambers with a first fluorogenic substrate and a second fluorogenic substrate, wherein the first fluorogenic substrate has the structure of formula (la):and the second fluorogenic substrate has the structure of formula (lb):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate, the micro-chambers are sized to contain only one capture particle per microchamber, the analyte of interest is associated with a phosphatase enzyme, the phosphatase enzyme converts the first and second Anorogenic substrates into a Auorogenic enzymatic product, the first and second Auorogenic substrate emit a first Auorescent signal and the Auorogenic enzymatic product emits a second Auorescent signal distinct from the first Auorescent signal; detecting presence or absence of the second Auorescent signal in the micro-chambers, wherein presence of the second Auorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers.

18. The method of claim 17, wherein the Auorogenic enzymatic product is pyranine.

19. The method of claims 17 or 18, wherein each capture particle has up to 10 molecules of the analyte immobilized on the capture particles and wherein one molecule of the phosphatase enzyme associated is associated with one analyte molecule.

20. The method of claim 19, wherein each capture particle has up to 5, 3, 2, 1 or 0 molecule of the analyte immobilized thereon.

21. The method of claim 19, wherein each capture particle has 5, 3, 2, or 0 copy of the analyte immobilized thereon.

22. The method of any one of claims 17-21, wherein the detecting step is performed within 5 minutes after the contacting step.

23. The method of any one of claims 17-22, wherein the detecting step is performed within 1 minute to 5 minutes after the contacting step.

24. The method of any one of claims 17-23, wherein the analyte of interest is a peptide or protein, and the capture particles comprise a first binding member that specifically binds to the peptide or protein and wherein the peptide or protein is associated with the phosphatase enzyme via a second binding member that specifically binds to the peptide or protein and wherein the second binding member is conjugated to the phosphatase enzyme.

25. The method of any one of claims 17-24, wherein the analyte of interest is a nucleic acid, and the capture particles comprise a first binding member that specifically binds to the nucleic acid and wherein the nucleic acid is associated with the phosphatase enzyme via a second bindingmember that specifically binds to the nucleic acid and wherein the second binding member is conjugated to the phosphatase enzyme.

26. The method of any one of claims 17-25, wherein the micro-chambers are micro-wells.

27. The method of any one of claims 17-25, wherein the micro-chambers are micro-droplets.

28. The method of any one of claims 17-27, wherein the phosphatase enzyme is an alkaline phosphatase enzyme.

29. The method of any one of claims 17-28, wherein the first Anorogenic substrate and the second Auorogenic substrate are present at a ratio of 1 : 1.

30. The method any one of claims 17-29, wherein the concentration of each of the first and second Auorogenic substrate is between 1 to 50 pM.

31. A method of preparing pyranine-O-phosphate, comprising: reacting pyranine with a mono-reactive phosphate source to obtain pyranine-O-phosphate; wherein pyranine-O-phosphate is a compound of formula (lb):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is a phosphate.

32. The method according to claim 31 , wherein the mono-reactive phosphate source is diethyl chlorophosphate.

33. The method according to any one of claims 31-32, wherein pyranine-O-phosphate has a molecular weight in the range of 100 to 1000 Daltons.

34. A method of preparing pyranine-methyl -phosphate, comprising: reacting pyranine with a mono-reactive phosphate source to obtain pyranine- methylphosphate; wherein pyranine- methyl-phosphate is a compound of formula (lb):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation.

35. The method according to claim 34, wherein the mono-reactive phosphate source is ditertbutyl chloromethyl-phosphate.

36. A method of preparing pyranine-di-phosphate, comprising: reacting pyranine with a mono-reactive phosphate source to obtain pyranine-O-phosphate; further reacting with tetrabutylammonium phosphate to pyranine-di-phosphate; wherein pyranine-di-phosphate is a compound of formula (lb):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is a diphosphate.

37. The method according to claim 36, wherein the mono-reactive phosphate source is phosphoryl chloride.

38. A method of preparing pyranine-tri-phosphate, comprising: reacting pyranine with a mono-reactive phosphate source to obtain pyranine-O-phosphate; further reacting with tributylammonium phosphate to pyranine-tri-phosphate; wherein pyranine-tri-phosphate is a compound of formula (lb):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is a triphosphate.

39. The method according to claim 38, wherein the mono-reactive phosphate source is phosphoryl chloride.

40. A system for detecting presence of an analyte of interest on capture particles, the system comprising: a processor; a cartridge comprising micro-chambers; the micro-chambers comprising the capture particles and a fluorogenic substrate of formula (I):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate, the micro-chambers are sized to contain only one capture particle per micro-chamber, the analyte of interest is associated with a phosphatase enzyme,the phosphatase enzyme converts the Anorogenic substrate into a Anorogenic enzymatic product, the Anorogenic substrate emits a fir t Auorescent signal and the Anorogenic enzymatic product emits a second Auorescent signal distinct from the first Auorescent signal, the processor is programmed to execute instructions for detecting presence or absence of the second Auorescent signal in the micro-chambers, wherein presence of the second Auorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers.

41. The system of claim 40, wherein the instructions for detecting presence or absence of the second Auorescent signal in the micro-chambers comprise instructions for directing an excitation wavelength to the micro-chambers and detecting an emission wavelength corresponding to the second Auorescent signal.

42. The system of claim 40, wherein the processor is programmed to execute instructions for contacting the capture particles present in micro-chambers with the Auorogenic substrate prior to the detecting step.

43. The system of claim 40, wherein the Auorogenic substrate molecule is pyranine-O- phosphate and the Auorogenic enzymatic product is pyranine.

44. The system of claim 40 or 43, wherein the phosphatase enzyme is an alkaline phosphatase enzyme.

45. The system of any one of claims 40-44, wherein each capture particle has up to 10 molecules of the analyte immobilized on the capture particles and wherein one molecule of the phosphatase enzyme associated is associated with one analyte molecule.

46. The system of claim 45, wherein each capture particle has up to 1 or 0 molecule of the analyte immobilized thereon.

47. The system of any one of claims 40-46, wherein each capture particle has 1 or 0 copy of the analyte immobilized on the capture particle.

48. The system of any one of claims 40-47, wherein the detecting step is performed within 5 minutes after the contacting step.

49. The system of any one of claims 40-47, wherein the detecting step is performed within 10 seconds to 2 minutes after the contacting step.

50. The system of any one of claims 40-49, wherein the analyte of interest is a peptide or protein, and the capture particles comprise a first binding member that specifically binds to the peptide or protein and wherein the peptide or protein is associated with the phosphatase enzymevia a second binding member that specifically binds to the peptide or protein and wherein the second binding member is conjugated to the phosphatase enzyme.

51. The system of any one of claims 40-50, wherein the analyte of interest is a nucleic acid, and the capture particles comprise a first binding member that specifically binds to the nucleic acid and wherein the nucleic acid is associated with the phosphatase enzyme via a second binding member that specifically binds to the nucleic acid and wherein the second binding member is conjugated to the phosphatase enzyme.

52. The system of any one of claims 40-51, wherein the micro-chambers are micro- wells.

53. The system of any one of claims 40-52, wherein the micro-chambers are micro-droplets.

54. A system of detecting presence of an analyte of interest on capture particles, the system comprising: a processor; a cartridge comprising micro-chambers; the micro-chambers comprising the capture particles, a first fluorogenic substrate and a second fluorogenic substrate, wherein the first fluorogenic substrate has the structure of formula (la):and the second fluorogenic substrate has the structure of formula (lb):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation;Y is selected from a group consisting of diphosphate, triphosphate, oxymethyl phosphate, or phosphate, the micro-chambers are sized to contain only one capture particle per microchamber, the analyte of interest is associated with a phosphatase enzyme, the phosphatase enzyme converts the first and second Anorogenic substrates into a Auorogenic enzymatic product, the first and second Auorogenic substrate emit a first Auorescent signal and the Auorogenic enzymatic product emits a second Auorescent signal distinct from the first Auorescent signal; the processor is programmed to execute instructions for detecting presence or absence of the second Auorescent signal in the micro-chambers, wherein presence of the second Auorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers.

55. The system of claim 54, wherein the Auorogenic enzymatic product is pyranine.

56. The system of claims 54 or 55, wherein each capture particle has up to 10 molecules of the analyte immobilized on the capture particles and wherein one molecule of the phosphatase enzyme associated is associated with one analyte molecule.

57. The system of claim 56, wherein each capture particle has up to 5, 3, 2, 1 or 0 molecule of the analyte immobilized thereon.

58. The system of claim 56, wherein each capture particle has 5, 3, 2, or 0 copy of the analyte immobilized thereon.

59. The system of any one of claims 54-58, wherein the detecting step is performed within 5 minutes after the contacting step.

60. The system of any one of claims 54-59, wherein the detecting step is performed within 1 minute to 5 minutes after the contacting step.

61. The system of any one of claims 54-60, wherein the analyte of interest is a peptide or protein, and the capture particles comprise a first binding member that specifically binds to the peptide or protein and wherein the peptide or protein is associated with the phosphatase enzyme via a second binding member that specifically binds to the peptide or protein and wherein the second binding member is conjugated to the phosphatase enzyme.

62. The system of any one of claims 54-61, wherein the analyte of interest is a nucleic acid, and the capture particles comprise a first binding member that specifically binds to the nucleic acid and wherein the nucleic acid is associated with the phosphatase enzyme via a second bindingmember that specifically binds to the nucleic acid and wherein the second binding member is conjugated to the phosphatase enzyme.

63. The system of any one of claims 54-62, wherein the micro-chambers are micro- wells.

64. The system of any one of claims 54-62, wherein the micro-chambers are micro-droplets.

65. The system of any one of claims 54-64, wherein the phosphatase enzyme is an alkaline phosphatase enzyme.

66. The system of any one of claims 54-65, wherein the first Anorogenic substrate and the second Anorogenic substrate are present at a ratio of 1 : 1.

67. The system any one of claims 54-66, wherein the concentration of each of the first and second Anorogenic substrate is between 1 to 50 pM.

68. A compound of formula (II):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation.

69. The compound of claim 61, wherein the compound is:

70. A method of detecting presence of an analyte of interest on capture particles, the method comprising:contacting the capture particles present in micro-chambers with a fluorogenic substrate of formula (II):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation; the micro-chambers are sized to contain only one capture particle per micro-chamber, the analyte of interest is associated with a phosphodiesterase enzyme, the phosphodiesterase enzyme converts the fluorogenic substrate into a fluorogenic enzymatic product, the fluorogenic substrate emits a first fluorescent signal and the fluorogenic enzymatic product emits a second fluorescent signal distinct from the first fluorescent signal; detecting presence or absence of the second fluorescent signal in the micro-chambers, wherein presence of the second fluorescent signal in the micro-chambers indicates presence of an analyte of interest on capture particles present in the micro-chambers.

71. The method of claim 70, wherein the fluorogenic enzymatic product is pyranine.

72. The method of claim 70 or 71, wherein each capture particle has up to 10 molecules of the analyte immobilized on the capture particles and wherein one molecule of the phosphodiesterase enzyme associated is associated with one analyte molecule.

73. The method of claim 72, wherein each capture particle has up to 1 or 0 molecule of the analyte immobilized thereon.

74. The method of any one of claims 70-73, wherein each capture particle has 1 or 0 copy of the analyte immobilized on the capture particle.

75. The method of any one of claims 70-74, wherein the detecting step is performed within 5 minutes after the contacting step.

76. The method of any one of claims 70-75, wherein the detecting step is performed within 10 seconds to 2 minutes after the contacting step.

77. The method of any one of claims 70-76, wherein the analyte of interest is a peptide or protein, and the capture particles comprise a first binding member that specifically binds to the peptide or protein and wherein the peptide or protein is associated with the phosphodiesterase enzyme via a second binding member that specifically binds to the peptide or protein and wherein the second binding member is conjugated to the phosphodiesterase enzyme.

78. The method of any one of claims 70-77, wherein the analyte of interest is a nucleic acid, and the capture particles comprise a first binding member that specifically binds to the nucleic acid and wherein the nucleic acid is associated with the phosphodiesterase enzyme via a second binding member that specifically binds to the nucleic acid and wherein the second binding member is conjugated to the phosphodiesterase enzyme.

79. The method of any one of claims 70-78, wherein the micro-chambers are micro-wells.

80. The method of any one of claims 70-78, wherein the micro-chambers are micro-droplets.

81. A method of preparing dipyranine phosphate, comprising: reacting 2 equivalents of pyranine with a di-reactive phosphate source to obtain dipyranine phosphate; wherein dipyranine phosphate is a compound of formula (II):wherein:X is selected from a group consisting of H+, K+, Ca2+, tris, triethylammonium, or any other cation.

82. The method according to claim 81, wherein the di-reactive phosphate source is ethyl dichlorophosphate.

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